This workshop is for faculty currently working at primarily undergraduate institutions (PUIs) or early career scientists who would like to get a job at a PUI. PUIs are defined as institutions that offer few or no PhDs in the sciences. While teaching is a large part of being a PUI faculty member, maintaining a successful research program is also critical for career advancement and for providing undergraduates with high-caliber research experiences. The workshop will include presentations and discussions on turning undergraduate research projects into publishable works. This will include presentations by a panel of editors from various scientific journals as well as opportunities for small-group discussion among participants. Workshop has a fee to attend and requires preregistration.

Many mathematical and statistical problems exist in plant biology. Plant biologists may feel intimidated by the large variety of techniques available, as well as the numerous programming languages utilized to execute them. Alternatively, communication between disciplines is not trivial, and skilled computational biologists may be unaware of interesting problems in the field. The session will consist of brief discussion of interdisciplinary collaboration, the ‘language’ of computational scientists, and example problem statements. Prior to the session, we ask that attendees complete a short survey describing current research interest. The organizer will analyze the responses to suggest potential collaborations between complementary attendees and similarly-inclined attendees. The remaining portion of the session will be a speed-dating style networking event between the matches. With consent, all attendee responses will be made available for attendees to self-select potential networking opportunities. **WORKSHOP FULL** To be added to the wait list please email PBregistration@aspb.org.

Network with other undergraduates and other plant science professionals and students as you think about your next steps in your career. If you have a poster, let people know your poster number and when you will be at your poster in the main poster hall. Light refreshments will be provided. *No posters will be at this session. Workshop requires preregistration.

Plant biologists revel in the diversity and complexity of form and function among the hundreds of thousands of documented photosynthetic organisms on earth. Technological and conceptual breakthroughs in recent decades provide opportunities to understand these themes and variations. We increasingly are harnessing the genetic and functional variation within the plant kingdom to inform approaches for making plants more productive and agriculture more environmentally friendly. Just as our science benefits from embracing biological complexity, our community is made stronger by inclusion of scientists who represent the breadth of human culture. This symposium will explore approaches to harness biological diversity and complexity in the systems that we study, and ways to strengthen our community by broadening participation.

Photosynthesis is a fundamental process in biology as it converts solar energy into chemical energy and thus, directly or indirectly, fuels all life on earth. The chemical energy is used to fix atmospheric CO2 and produce reduced carbon compounds in the Calvin-Benson-Bassham cycle. The key enzyme for this process in all photosynthetic organisms is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), which is responsible for the conversion of an estimated amount of ~1011 tons CO2 per annum into organic material. It is the most abundant enzyme in nature, owing in part to its low catalytic turnover rate and limited specificity for CO2 versus O2. Additional complexity comes from the fact that the multistep catalytic reaction of Rubisco is prone to processing errors. As a result, tightly binding ‘misfire’ products are produced that inhibit catalysis and need to be removed by the AAA+ protein Rubisco activase. Compartmentalization is a cellular strategy to regulate metabolic pathways and increase their efficiency. Thus, to overcome some of the shortcomings of Rubisco, cyanobacteria and green algae have evolved proteinaceous compartments containing densely packed Rubisco together with carbonic anhydrase, generating high concentrations of CO2 in direct proximity of Rubisco. Recent forecasts suggest that global food production will need to rise more than 25 % by 2050 to meet the ever increasing demand. Engineering a catalytically more efficient Rubisco enzyme and/or compartmentalizing Rubisco in higher plants could contribute to reaching that goal. However, the complex nature of Rubisco’s folding and assembly pathway has made these efforts exceedingly challenging. In my talk I will review recent progress in understanding the complex chaperone machineries that are necessary for the efficient biogenesis of this most abundant enzyme and its functional maintenance. I will also discuss the mechanisms that mediate the condensation of Rubisco into a three-dimensional network during carboxysome formation in cyanobacteria. Just as Rubisco function depends on a complex network of factors, as scientists we rely on individuals with different expertise and personalities to work together. It has been my privilege to work with a group of gifted and dedicated students and postdoctoral fellows. International collaborations have also been very valuable and have led to lasting friendships.

Plant metabolic networks are in a constant state of change due to tandem and whole genome duplications, enzyme multi-functionality and promiscuity, and expression divergence. These phenomena have led to the emergence of novel metabolite classes restricted to specific clades. In my talk, I will describe the evolutionary origins and diversification of acylsugars – a class of defense metabolites produced exclusively in the Solanaceae family but not in its sister Convolvulaceae family – and our ongoing work on structurally-analogous resin glycosides – known to be produced only in the Convolvulaceae but not the Solanaceae family. Enzyme families such as cytochrome p450s, methyltransferases, lipases, transferases play important roles in emergence of such new pathways, however, functional annotation of enzyme family members is challenging due to their frequent gene duplication-divergence and promiscuity. I will describe our ongoing work on understanding how new functions emerge in these enzyme families. This research has potential implications in studying metabolic pathways in non-model plant lineages, where much of the metabolic diversity still remains uncharacterized.

The introductory science experience is a critically important time for all students. If the student plans to major in science, then the introductory experience is when they can begin to learn how science is conducted and to build self-efficacy in science. And if the student plans to study in a non-STEM discipline, then the introductory experience is often the only opportunity they will have to gain a glimpse of the important role of science in how we think about the world. Unfortunately, at many places the introductory science experience is broken. Too often success in the introductory lecture courses depends on memorization of “facts,” and success in the introductory laboratory courses requires performing an exercise for which the solution is already known. This emphasis on finding the “right answer” is inconsistent with what is important in the scientific process—encouraging curiosity, embracing uncertainty, and the exploration of the unknown. Engaging undergraduates in authentic research can be an effective way to promote student self-efficacy in science. Course-based research experiences (CREs) can engage large numbers of students in research during the introductory phase of their education. The HHMI Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES; www.seaphages.org) project is an example of an inclusive research and education community (iREC; Hanauer et al., 2017, www.pnas.org/cgi/doi/10.1073/pnas.1718188115). SEA-PHAGES is designed for beginning undergraduates and has no prerequisites. Last year, more than 5,500 students at 118 colleges and universities participated—80% of the students were in their first or second year. The outputs from the SEA-PHAGES project account for the largest collection of genetically characterized bacteriophages, resulting in several important scientific insights. In the decade since the program began, more than 70 papers have been published, many of them with students as co-authors. The SEA-PHAGES students show strong gains correlated with persistence in science relative to students in traditional laboratory courses regardless of academic, ethnic, gender, and socioeconomic profiles. By providing course materials and instructor training, SEA-PHAGES can be successfully implemented at all types of institutions, including schools with little or no research activity.

Lessons that have emerged from investigating the specific ways in which organisms such as plants adapt their patterns of growth and development to fluctuations in external cues to increase their survival and productivity are translated to progressive mentoring and professional development interventions. These lessons are intended to inform practices that promote the broad success of participants from diverse backgrounds in academic sciences, as well as provide insights into the roles of mentors and leaders as environmental stewards. Both individual and community-based interventions will be discussed.

Is this your first time at Plant Biology? Come to a special meetup during the opening reception and make some new friends!

This career panel targets graduate students and post-docs who want to learn about careers at primarily undergraduate institutions (PUIs), as well as faculty at research institutions who want to pass on information to their students and post-docs. PUIs are defined by the NSF as "accredited colleges and universities (including two-year community colleges) that award Associate's degrees, Bachelor's degrees, and/or Master's degrees in NSF-supported fields, but have awarded 20 or fewer Ph.D./D.Sci. degrees in all NSF-supported fields during the combined previous two academic years." A panel of PUI faculty will represent a diversity of PUI institutions, career trajectories, and career stages. Panelists will participate in whole group Q&A as well as breakout conversations. Participants will have the opportunity to apply for a year-long mentoring arrangement with a member of the PUI section. WORKSHOP FULL To be added to the wait list please email PBregistration@aspb.org.

Plant survival requires an integration of developmental status with the nature of the surrounding environment. Cell surface receptors allow plant perception of both biotic and abiotic signals, as well as cell-to-cell coordination for the integration of developmental responses. The huge expansion of plasma membrane-associated receptors in the genomes of most plant species is testament to the diversity of signals that plants can recognise in their extracellular environment. Despite this diversity of receptors, there is much integration in the mechanisms of signal transduction. In this symposium, we will explore cell surface receptors that function in both plant-microbe interactions and in plant development, covering the commonalities and differences that exist in receptor signaling in plants.

Cyril Zipfela,b a The Sainsbury Laboratory, University of East Anglia, Norwich Research Park, Norwich, UK. b Institute of Plant and Microbial Biology, Zürich-Basel Plant Science Center, University of Zürich, CH-8008 Zürich, Switzerland. Plants genomes encode hundreds of cell surface-localized receptor kinases that control almost all aspects of plant life, ranging from reproduction, growth to responses to the external environment. Using receptor kinases that function as immune receptors by perceiving microbial elicitors, we are studying the molecular basis of plant immunity, but also more generally how plant receptor kinases work at the mechanistic level. Using the leucine-rich repeat receptor kinases FLS2 and EFR (which perceive bacterial flagellin and EF-Tu, respectively) as model systems, we are investigating how plant receptor kinases function as part of multimeric protein complexes at the plasma membrane – often in complex with other receptor kinases, which act as regulatory proteins. I will present our recent work that uncovered the importance of these regulatory receptor kinases and receptor kinase-associated proteins in controlling the assembly and activity of functional heteromeric receptor complexes.

Brassinosteroid (BR) receptor BR INSENSITIVE1 (BRI1) has become a paradigm for understanding the interplay between receptor-mediated endocytosis and signaling in plants. The BR signaling is largely determined by the plasma membrane pool of BRI1, whereas BRI1 endocytosis ensures signal attenuation. Hormone depletion was previously used to demonstrate that BRI1 endocytosis is ligand-independent. This finding subsequently brings up the question how receptor inactivation is regulated. Here, we took advantage of BR biosynthesis and BR-binding deficient receptor mutants to re-examine the endocytosis model of BRI1 and to resolve mechanisms for signal attenuation.

In flowering plants, sexual reproduction depends on the directional, long-distance growth of pollen tubes that deliver sperm cells to the female gametophytes. The polarized and rapid growth of the pollen tube cell depends on the highly dynamic remodeling of its cell wall. However, how plants can sense, and signal cell wall properties is still poorly understood. A family of Catharanthus roseus Receptor Kinase-1 (CrRLK1L) membrane receptors have been genetically implicated in the recognition of RALF signaling peptides to control pollen tube growth and integrity. Here we present crystal structures of the CrRLK1Ls ANXUR1 and 2 at high atomic resolution. Our structures reveal a novel fold of two malectin-like domains connected by a short β-hairpin linker and stabilized by calcium ions. The canonical carbohydrate interaction surfaces of related animal and bacterial carbohydrate binding modules are not conserved among plant CrRLK1Ls. Instead, CrRLK1Ls have evolved a protein-protein interface between their malectin domains, which forms a deep cleft which could resemble a potential binding site. Our structural and biochemical data reveal that CrRLK1Ls could represent signaling hubs with the capacity to recognize different signaling molecules to control cell wall integrity.

Plants associate with microorganisms in the rhizosphere, with potential for benefit or detriment. Two primary signal transduction pathways control the nature of engagement with microorganisms, with immunity signaling blocking and symbiosis signalling promoting microbial associations. It has long been thought that different elicitors activate these different pathways, however, we have found that elicitors present on the surface of fungi and bacteria activate both immunity and symbiosis signaling, with the potential to restrict or promote microbial associations as a result of this recognition. This implies that it is not elicitor perception alone that differentiates an immunogenic or symbiotic response. Plants activate beneficial symbiotic associations at times when nutrients are limited and we demonstrate that under nutrient starvation immunity signalling is suppressed, while symbioisis signalling is promoted. Hence, at times of nutrient deprivation plants take a significant risk in order to facilitate beneficial microbial associations. Lipochitooligosaccharides (LCO) are produced by nitrogen-fixing bacteria and arbuscular mycorrhizal fungi and these act only as elicitors of symbiosis signaling. We propose that under nutrient starvation plants lower the barrier of immunity signaling and utilise LCO perception to select and promote colonisation by beneficial microorganisms in the rhizophere. Our work highlights the dynamic nature of the signaling pathways that control microbial associations and this may facilitate better utilisation of beneficial microbial associations in agriculture.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

The Visual Communicators group on the Plantae community site is a place for people to work together to create stunning visuals and help others appreciate and understand plant biology. At this meetup we will have the chance to meet each other in person and share our ideas and resources. If you would like to know more about the visual communicators network and become involved please join us!

Are you getting the most out of your mentoring relationship? Join this discussion to improve your mentor-mentee relationships. Speakers will also discuss using an Individual Development Plan, as well as strategies for both mentors and mentees.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

In January 2019, members of the ASPB community convened a meeting to take a creative and fresh look at broadening participation in the plant sciences. The purpose of the meeting was to expand the participants’ understanding of inclusivity, diversity, and equity in the plant sciences, and ultimately to uncover impactful approaches and recommendations to measurably broaden participation in plant science. Join ASPB staff and workshop participants to discuss the workshop report and future activities to broaden participation.

An open gathering to discuss challenges related to mid-career faculty including initiating collaborations between institutions, apply for travel grants, and suggestions for building a great tenure package. The conversation will focus on career progression and resources available.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Learn more about how ASPB members can get involved with education and outreach.

This session is open to all who want to learn best practices for making your lab/classroom/work environment welcoming and affirming for your LGBTQ+ employees and colleagues. We also welcome those who are part of the LGBTQ+ community to come and share their thoughts.

Learn about opportunities to guide and support federal research programs through temporary program officer positions with US Federal agencies including the NSF and DOE. Speakers will include career program officers, as well as current and former "rotators".

Dr. Denneal Jamison-McClung is the ADVANCE Program Coordinator at UC Davis, Interim Director of the UC Davis Biotechnology Program, and Director of BioTech SYSTEM. The UC Davis ADVANCE program has been working since 2012 to promote diversity among STEM faculty through four overarching goals: 1) Build a vibrant, welcoming and diverse STEM research community through establishment of the Center for Advancing Multicultural Perspectives on Science (CAMPOS); 2) Establish an institution-wide, inclusive climate in STEM departments/colleges in which diversity is valued; 3) Promote equitable career advancement, achievement, and recognition among all STEM faculty; and 4) Understand barriers and catalysts for Latinas in STEM. An emergent theme throughout much of our work has been the importance of mentoring for underrepresented groups in STEM, especially for people at the intersection of gender and ethnicity. This talk will outline best practices, policy changes and pilot programs that will be institutionalized at UC Davis to support effective recruitment and mentoring of an inclusive community of STEM faculty, starting with CAMPOS Faculty Scholars. The work of UC Davis ADVANCE was undertaken through a cooperative agreement between the campus and the National Science Foundation (HRD 1209235). Workshop has a fee to attend and requires preregistration.

Are you interested in learning more about the world of commercialization in plant science, or do you have an idea you’d like to pitch? Come join the first ever ASPB “Growth Chamber” where selected participants will get the opportunity to pitch your idea and get feedback form a panel of scientists, IP specialists, investors, entrepreneurs and strategic partners. Afterwards, there will be a discussion and Q&A with the panel on all things entrepreneurial, IP, investment and partnership, and (hopefully) a prize for the best pitch. Workshop is also open for participants wanting to learn about commercialization who do not wish to pitch an idea. Interested in pitching your idea? Please reach out to Phil Taylor (phil.taylor@bayer.com) or Rishi Masalia (rishimasalia@gmail.com). Workshop requires preregistration.

We need to increase the impact of plant scientists on our policymaking process. This workshop will provide participants with tools to facilitate the local engagement of federal, state, and local policymakers. The goals of our engagement are to educate policymakers on the importance of plant science research locally and nationally; increase public-sector support of plant science research, education, and training; and promote science-based regulations concerning plant products produced using modern ag biotechnology. Workshop requires preregistration.

Learn techniques that will make you a better mentor to support student-led science investigations, especially in an asynchronous online setting. During this workshop, you'll learn more about the award-winning PlantingScience online mentoring program and results of NSF-funded research on the program's efficacy. You will also learn techniques to improve the effectiveness of your mentoring of student-led independent investigations. The workshop will cover classroom activities from several of our PlantingScience investigation themes as well as practice with using questioning techniques and scaffolding strategies helpful for anyone aiming to push student science thinking forward. Finally, we'll discuss the advantages and potential pitfalls to mentoring students asynchronously online and tips for working with teachers to make the most impact on students. This workshop is highly interactive and will provide lots of time for technique practice, analysis of scientist-student mentoring dialog, and a chance to hear from PlantingScience teachers and mentors about their experiences.

Navigating the path to disseminating your work can be challenging for scientists at all stages of their careers. When should you start talking about your work and in which venues? When, if any, is the right time to post a preprint? Is it better to publish a short paper in a sound science journal like Plant Direct or to wait to submit to Plant Physiology or the Plant Cell? Join the editors of our journals and other interested community members for an open discussion on these questions.

STEM educators need practical implementations of emerging digital media that excite their students to learn about plant biology. For these educators, web-based virtual reality (webVR) and extended reality (XR) offer exciting new avenues for education in under-addressed learning domains such as affective educational storytelling and physical manipulation of 3D models. VRplants is an interdisciplinary project from the North Carolina State University Department of Plant & Microbial Biology and the NC State Libraries to develop a suite of web-based plant biology learning modules for open utilization by educators. The project also supports a series of workshops to disseminate essential XR creation tools and increase the size of the extended reality maker community. In this presentation we will review the project’s central mission, describe our programs (available at the BLOOME booth), and provide a vision for VR makership as the prescription for plant blindness. VRPlants is made possible by the ASPB-BLOOME (Plant Biology Learning Objectives, Outreach Materials & Education) grant program, North Carolina State University Libraries, and the NC State Department of Plant & Microbial Biology.

Chlamydomonas reinhardtii is a micro-green alga that retains many of the features of the green plant and of the common ancestor of plants and animals. It is an elegant experimental model system for conducting plant biology, biomedical and bioenergy research. Chlamydomonas is currently an under-utilized teaching tool and has immense potential to be developed into a powerful popular teaching tool. I have designed simple, inexpensive, hands-on-activities based on published Chlamydomonas research for active learning in K-16 classrooms. These activities are part of my awarded 2018 Plant BLOOME grant and have been designed for students ranging from 4th graders to college undergraduates in financially disadvantaged schools/universities. Some of the Biology topics in the K-16 curriculum that can be taught using Chlamydomonas are sexual reproduction, cell division, genetics, structure and function of eukaryotic flagella and eye spot, Optogenetics, eukaryotic photosynthesis, high light stress responses, generation of ROS and its detoxification via anti-oxidants in plant cells, photosynthetic pigment metabolism and, biomass and bioenergy production. Labs are designed using simple plant physiology, molecular, biochemistry and bioinformatics tools and art and crafts supply. I will present four sets of designed hands-on-activities. These topics are: photosynthesis/photosynthetic pigment biosynthesis, eyespot and flagella. I will show how each activity topic can be customized for students at different grade levels and, demonstrate how concepts from the labs on photosynthesis, pigment biosynthesis, eye and flagella can be linked together for NGSS Biology core concept mapping. These teaching strategies will show the intra- and inter- disciplinary nature of Biology and will help to generate enthusiasm and respect for a “pond scum” among 21st century Biology students, especially to those who have the notion that allied health disciplines have no connection with plant biology.

One of the most fascinating characteristics of plants is that they display sophisticated movements, particularly in growing tissues. Creating time-lapse movies from plants has recently become inexpensive and easy with the proliferation of smart phones. To better understand plant movement, we have developed Plant Tracer, an NSF funded App designed to enable the quantification of gravitropism (movement towards or away from gravity) and circumnutation (the periodic regular swaying found in plant organs). As part of a crowd sourced method, Plant Tracer is being used by both students and researchers to detect mutant genes in Arabidopsis that are impaired in plant movement. Plant Tracer represents a new approach to draw young scientists into the field of plant biology through research and inquiry using technology

The Soil Microbiome Project at Contra Costa Community College (CCC) mimics the typical environment a student experiences a lab internship, in a format that is accessible to a large number of community college students. The majority of CCC students are from populations traditionally underrepresented in the science fields. Students participating in the project are in their first or second year of college, and are typically biotechnology, biology, and chemistry students, many who intend to transfer to a 4-year institution, but include some who seek technical training and plan to join the biotechnology workforce soon after earning a certificate. In two separate courses, students learn and employ the techniques of microbiology, plant care, phenotyping, and sampling, molecular biology, bioinformatics, and good laboratory practice. While student participation in the work is semester-by-semester, the project itself is a multi-year analysis of soil development at a nearby site where Urban Tilth, a non-profit organization, employs permaculture and other farming methods to improve 3 acres of land at the North Richmond Farm, an urban location which will serve as a food and community hub in what is currently a “food desert”. This presentation will discuss the innovative pedagogy and course design that allows relatively inexperienced students to contribute to an ongoing scientific investigation by contextualizing technical training to authentic research, and builds both awareness and practice of work skills and habits. Application of concepts and competencies common in the field of biotechnology operations and supply chain management to this multi-year, team-based investigation will be discussed, along with learning outcome results. Student-generated data showing how the soil microbiome at the North Richmond Farm has changed and affected plant growth during the first two years of Urban Tilth’s soil-building will also be presented.

Universities have been transforming STEM education by exposing undergraduates to the process of research for decades. One way this has been done is through Course-based Undergraduate Research Experiences (CUREs) where students work on a research project during a semester-long course. At the University of North Texas (UNT), students have been participating in an advanced research course focused on the areas of Molecular and Cell Biology and Biochemistry for several years. BIOL 3900, Advanced Research in Life Sciences, provides a research experience for up to 16 students, at one of the nation’s largest and most diverse universities where 42 % of undergraduates are first-generation students. During this course, students read scientific literature, conduct experiments, maintain a lab notebook, and evaluate and interpret their results. At the end of the semester, students are required to write a scientific paper describing their results in the submission format of a suitable scientific journal and to deliver an oral presentation summarizing their project and results. Recently, this course focused on identifying protein interactors of N-Acylethanolamines (NAEs), a class of fatty acid derivatives that play a role in plant growth and development. Students cloned coding sequences for proteins previously identified in a NAE-interactor screen and conducted yeast three-hybrid assays. In order to assess the benefits of the course, students were asked to take pre- and post-course surveys about their experience. After participating in this CURE, students at UNT reported gains in scientific skills and a clarification in career path that is similar or higher than students participating in CUREs across the country. In all, this CURE program provided an effective and efficient opportunity to broaden participation in life science research at an institution where demand for these experiences outpaces the supply. Supported in part by National Science Foundation grant- NSF-IOS 1656263.

Abscisic acid (ABA) caused massive plant protein abundance changes plays regulatory roles in various physiological processes throughout plant growth and development and abiotic stress response. However, how plant balance plant growth and abiotic stress response via ABA signaling remain unclear. In this study, by using knockout mutants of the Arabidopsis thaliana Gβ and GɤIII subunit, we provide precise evidences that plants differentially requires Gβ and GɤIII to modulate photosynthesis pathway in response to ABA and water stress response. Both mutants showed higher photosynthesis indicated by lower leaf temperature and higher Fv/Fm under normal growth condition than WT. However, only agb1 showed in higher leaf temperature than WT during dehydration condition. Besides, both mutants exhibited greater ROS accumulation than WT, suggesting Gβ and GɤIII function in redox metabolism. To investigate roles of Gβ and GɤIII proteins in ABA and drought stress response, we conducted the whole and redox proteome analysis. Data showed that ABA and/or Gβ or GɤIII control abundance and/or redox status changing proteins as well as biological processes. Interestingly, Gβ and GɤIII differentially and negatively regulate redox protein changes but positively redox protein changes in response to ABA. In addition, comparison of the whole and redox proteome demonstrates the correlation between oxidation state and abundance of proteins, including photosynthesis- and redox-related proteins. Reduced photosynthesis caused by ABA and/or Gβ or GɤIII may attribute to downregulated abundance of light reaction- and electron transport-related proteins, which result from their increased oxidation state. Finally, our results provided basic important information of protein changes caused by ABA and G-proteins and their combination could be potentially used for further functional analysis.

Suberin lamella forms protective barrier in plant tissues against biotic and abiotic stresses. This hydrophobic structure assembles widely in various cell types during plant development and in response to stress-induced hormones. However, it remains unclear how developmental programs interplay with stress responses to direct the spatiotemporal precision of suberin deposition. Here we provide evidence that SHORT-ROOT (SHR) mediates the regulatory network through a group of MYB transcription factors to direct the specific suberization in root endodermis. Both SHR and MYBs promote the ABA level in Arabidopsis, but SHR mediated regulation appears to be independent of ABA induction despite these two pathways could share overlapping downstream modules. Compared to the fast and transient induction by ABA, SHR mediates a slow readout of developmental program that promotes suberization. In addition, defective Casparian strip triggers a complementary enhanced suberization possibly via a SCHENGEN3 (SGN3) mediated sub-network in SHR pathway. Developmental regulation and environmental stimulus act in parallel but form an interacting framework to provide plasticity of plant suberization in response to endogenous and exogenous cues.

Internal cellular membranes must have their lipid composition remodeled for plants to survive low temperatures. One mechanism necessary for freezing tolerance of the chloroplast envelope membranes is well defined. An enzyme named “Sensitive to Freezing 2” (SFR2) changes monogalactolipid into oligogalactolipids at temperatures below freezing. Interestingly, SFR2 activity does not respond to initial cool temperatures, it only responds to barely tolerable freezing temperatures. Here, we show that SFR2 is post-translationally regulated by modifications and changes to cytosolic acidification. We show that freezing increases cytosolic acidification and that proton pumps at both the plasma and vacuolar membranes participate in maintaining the acidification during low temperatures. Finally, quantitative measurements of SFR2 activation in a large number of plant species with diverse phylogenetic backgrounds shows that SFR2 is likely responding to membrane damage in some, if not all species. We conclude that plant low temperature sensing and response is likely a continuum rather than a switch, and that internal cellular membranes have systems set up to respond to damage in a diverse set of abiotic stresses.

Mediterranean plants are well adapted to resist hot and dry summers and mild cold winters characteristic from this climate region. However, some of these plants are highly sensitive to extreme cold temperatures which also limits its altitudinal distribution. Here we aimed at unravelling adaptation mechanisms of the Mediterranean dioecious plant Pistacia lentiscus to winter photoinhibition. Male and female plants of this genera were selected from a natural population and followed for 12 months to evaluate photoinhibition degree and peroxidation damage, as well as chemical photoprotective mechanisms to restore redox balance. Moreover, in order to understand how cold limits their altitudinal distribution, we also selected three natural populations of Pistacia lentiscus at three altitudinal levels (360, 530, 730 m a.s.l) to determine if the degree of photoinhibition could be a crucial factor for its distribution. Our results show higher photoinhibition when temperatures decrease at winter both in males and females with values of maximum efficiency of photosystem II under 0.6 in February, which was the coldest month. This photoinhibition period is coupled to an increase of anthocyanin biosynthesis which counteracts photoinhibitiory damage and allows shoot recovery when temperatures warm up. Besides, the population at the highest altitude where this mastic tree could be found at 730m shows a higher photoinhibition degree than the other two populations. These results indicate that low temperatures are fundamental to determine Pistacia lentiscus distribution and the control of photoinhibitory damage through anthocyanin biosynthesis is essential to overcome cold periods.

Low temperatures often affect plant growth and crop productivity which causes significant crop loses. Early planted sorghum usually experienced cooler night temperatures, which may result in delayed growth, floral initiation, and infertile pollen. These responses limit sorghum production in high altitudes, latitudes, and in regions with sub-optimal temperatures during early growth stages. Genetic variability for cold tolerance in sorghum has been measured by characterizing germination, emergence, vigor, and seedling growth under sub-optimal temperatures. However, the compounded effect of early season cold stress on plant growth and development as it relates to the genetic variability in potential grain yield loses (yield penalties) has not been evaluated. The physiological and agro-morphological responses of sorghum grown at three planting dates (early; April 1st, mid-early; May 1st, and normal; June 1st) in West Texas were characterized from seedling to maturity (seed-to-seed) using diverse lines and hybrids with different sources (Chinese landraces and Ethiopian converted germplasm) of cold tolerance selected at the early vegetative stage. These were evaluated in comparison with standard commercial cold tolerant hybrids and cold susceptible checks. Variability for agro-physiological traits and yield penalties were observed across planting dates for genotypes and the two agro-ecological sources of cold tolerance during seedling, early developmental stages, and at maturity. All previously selected cold tolerant lines and hybrids performed better than the cold susceptible checks. Some hybrids and lines outperformed the standard commercial cold tolerant checks. Thus, the development of molecular markers to screen for cold tolerance should not be limited to early seedling characterization but also consider agronomic traits that may affect yield penalties depending on the sources of tolerance.

Plants need to take up CO2 for photosynthesis while avoiding excessive water loss through transpiration. This vital process is regulated by specialized pores on the surface of leaves, the stomata. High CO2 concentrations in leaves induce stomatal closure while low concentrations trigger stomatal opening. The atmospheric CO2 concentration has been continuously rising since the industrial revolution, impacting stomatal gas exchange. Investigation of stomatal physiology and movements is crucial for a better understanding of plant-environment interactions. In grasses, stomata are surrounded by two groups of cells: the dumbbell-shaped guard cells and the stomatal subsidiary cells. In combination, these cells respond to both external and internal stimuli, thus enabling rapid stomatal opening and closing. However, the molecular mechanisms mediating stomatal movements in grasses remain to a large degree unknown. Using the reference grass species Brachypodium distachyon, a forward genetic screen was performed. Over 1,000 mutagenized lines were screened for changes in their canopy leaf temperature using real-time infra-red imaging. Differences in leaf temperature may indicate defective stomatal development or responsiveness. Using this approach, 21 mutant lines with consistent canopy leaf temperature changes compared to wild-type (WT) were selected and are currently being characterized. Interestingly, two of these mutant lines named “chill1” and “chill7” have impaired responses to elevated CO2 concentration but retain an intact stomatal closing response to the hormone abscisic acid in intact leaf gas exchange experiments, suggesting specificity. Moreover, stomatal indices in chill1 and chill7 lines are similar to WT stomatal indices. Whole Genome Sequencing (WGS) data suggest that new genes in CO2-mediated stomatal movement in grasses will be identified in these mutants.

Anisotropic growth, more in one direction over another, is a feature of plant growth at many stages and in many organs. Here we examine how the young seedling grows anisotropically to emerge successfully from the soil. This directional growth of the seedling in Arabidopsis is due mainly to cell expansion. We will present our new model of anisotropic growth control by the cell wall which includes multiple tissues. Furthermore, we will examine a case where anisotropy is reduced - meaning the seedling grows out of the soil slower and is fatter - and examine the cell biology and signalling mechanisms behind this phenomenon. We also explore the functional consequences of reduced anisotropy for seedling emergence. Come and find out if it ever pays to be slow and fat! Within this work, we will also describe new insights into cell wall mechanics related to viscoelasticity and cell wall pectin biochemistry. This will include the introduction of a new technique: nano-creep! This miniaturization of a classical viscoelastic testing method can be applied to single cells and single cell walls in vivo.

Many plant cells begin life with an isotropic shape; establishing a “polar” growth pattern requires the actin cytoskeleton and the phytohormone auxin. Several models link specific actin arrays to cell growth, but no consensus prevails on whether longitudinal bundles inhibit or stimulate growth. Polarity requires auxin gradients formed by asymmetric flow of auxin, which relies on a functional actin cytoskeleton. Studies have examined actin reorganization after long-term auxin treatments, but plants respond to auxin in minutes. How short-term auxin–actin interactions affect growth, and the molecular players involved, are largely unknown. With quantitative tools, I correlated actin array organization with degree of axial cell expansion, establishing a baseline for actin organization in wildtype Arabidopsis root epidermal cells: cell length was highly predictive of actin array, and rapidly expanding cells had clearly different actin organization than slowly expanding cells. Within 20–30 minutes of growth-inhibitory doses of natural auxin and known root growth inhibitor, indole-3-acetic acid (IAA), actin filaments became more dense, parallel, and longitudinally oriented. Actin filament organization increased after a treatment to stop elongation, demonstrating there is no direct relationship between actin organization and cell expansion, and refuting the hypothesis that “more organized” actin correlates with rapidly growing root cells. AUXIN RESISTANT 1 (AUX1), a plasma membrane-bound auxin influx protein, binds IAA with high affinity and is responsible for 80% of IAA uptake by root hairs. aux1 mutant roots grow in the presence of IAA, but are growth-inhibited by the membrane permeable auxin 1-naphthylacetic acid (NAA). Actin arrays in aux1 mutants failed to reorganize in response to short-term IAA treatments, and reorganization was only partially restored by NAA. These are the first data to demonstrate that AUX1 is critical to auxin signaling to the actin cytoskeleton.

While plant cells respond to local mechanical and chemical signals from cell walls, it’s unclear how they mediate these cues. THESEUS1 (THE1) is one receptor-like kinase predicted to function as a cell wall sensor that regulates cell expansion in response to altered cellulose content; however, its role in dividing cells remains largely uncharacterized. Thus, we asked if THE1 functions in coordination with cell walls in dividing cells of Arabidopsis shoot apical meristems (SAMs). We found that loss of the cytoplasmic domain of THE1 (THE1Cyto; the1-4) reduced the number of epidermal cells and cell divisions. Loss of function in its putative extracellular domain (the1-1) had no effect on SAMs. This occurred independently of CLV/WUS and cytokinin signaling and without affecting epidermal cell expansion. Instead, loss of THE1Cyto increased the number of cells expressing nuclear CYCB1;1-GFP, without affecting the number of cells with CYCB1;1-GFP localized at the metaphase plate. Moreover, BMF1-GFP and MAD1-GFP localized ectopically along spindles during prometaphase, suggesting that loss of THE1Cyto impedes metaphase by activating the spindle assembly checkpoint. THE1 is not a global cell wall sensor in dividing cells. Loss of xyloglucan rescued SAM size in the1-4 plants while also restoring the number of cells expressing nuclear CYCB1;1-GFP; however, loss of β-1,4-galactan did not have the same effect in the1-4 SAMs. Mechanically increasing cell wall tension in SAMs lacking THE1Cyto repolarized PIN1-GFP and reoriented microtubules normally, further suggesting that THE1 is not a global cell wall mechanosensor. Together, these data suggest that THE1Cyto is required for dividing cells to properly progress to metaphase through specific coordination with xyloglucan in cell walls.

Many microalgae accumulate oil in the form of triacylglycerol in response to nutrient deprivation, but also enter a state of quiescence, which impedes biomass production. Therefore, the regulation of cell division in response to the metabolic status of the cell is of interest for the optimization of microalgal feedstocks for biofuel and natural compound production. Using Chlamydomonas as our model, we have identified a protein (COMPROMISED HYDROLYSIS of TRIACYLGLCYEROL, CHT7), that represses cell cycle genes during N deprivation. This conclusion is based on detailed cell biological analysis of the CHT7 loss-of-function mutant and global and targeted transcriptomic analysis of synchronized cultures of Chlamydomonas wild type and cht7 mutant grown under different conditions in bioreactors. The CHT7 protein belongs to a group of CXC domain proteins some of which are components of the DREAM complex, a large nuclear complex involved in the transcriptional co-regulation of extensive sets of genes. Several CXC domain proteins have been shown to bind DNA directly through their CXC domain. However, the CXC domain of CHT7 in Chlamydomonas is dispensable for function. Detailed deletion analysis of the CHT7 protein and testing of rescue of the cht7 mutant phenotypes by specific mutants identified a small, essential domain near the C-terminus, which is likely involved in protein-protein interactions affecting the abundance of the CHT7 protein complex. We are currently searching for other protein components of the complex and are studying the behavior of the complex during the cell cycle and in response to nutrient deprivation. Understanding how the CHT7 complex affects cell cycle gene activity in response to N deprivation will ultimately allow us to design novel strategies for the optimization of microalgal feedstocks.

The trans-Golgi network (TGN) is an essential tubular-vesicular organelle derived from the Golgi and functions as an independent sorting and trafficking hub within the cell. However, the molecular regulation of TGN biogenesis remains enigmatic. Here we identified an Arabidopsis mutant loss of TGN (lot) that is defective in TGN formation and sterile due to impaired pollen tube growth. The mutation leads to overstacking of the Golgi cisternae and significant reduction in the number of TGNs and vesicles surrounding the Golgi in pollen, which is corroborated by the dispersed cytosolic distribution of TGN-localized proteins. Consistently, deposition of extracellular pectin and plasma membrane localization of receptor kinases and phosphoinositide species are also impaired. Subcellular localization analysis suggests that LOT is localized on the periphery of the Golgi cisternae, but the mutation does not affect the localization of Golgi-resident proteins. Furthermore, the yeast complementation result suggests that LOT could functionally act as a component of the guanine nucleotide exchange factor (GEF) complex of small Rab GTPase Ypt6. These findings suggest that LOT is critical for TGN biogenesis in the plant lineage, distinct from the function of its non-plant organisms (Jia et al., PNAS, 2018). Study of the lot homolog plants suggests that LOT also regulate TNG biogenesis and Golgi structure in root and hypocotyl (Jia et al., 2019). Transmission electron microscopic examination suggests that LOT regulate the very early generation of Golgi-associated TGN formation from the Golgi cisterna. Using biochemical and genetic strategies, substrates of LOT and components in the related signaling pathways are investigated. Proteomic analysis was used to discriminate the cargos of TGN-dependent and –independent trafficking pathways. We also find that GA and BR hormone signaling pathways are disrupted which may contributes to the plant vegetative growth defect.

Proper positioning of the new cell wall during cell division is essential for plant patterning and development. TANGLED1 (TAN1) and AUXIN INDUCED IN ROOTS9 (AIR9) are microtubule-binding division site marker proteins. Single tan1 and air9 mutants have no discernable phenotypes in Arabidopsis. However, Arabidopsis tan1 air9 double mutants have a synergistic phenotype displaying altered cell file rotation, root growth, and cell division orientation. Transformation with either full length TAN1 or AIR9 is sufficient to rescue these mutant phenotypes. This suggests that TAN1 works in conjunction with AIR9 to properly orient the division plane and potentially influence the organization of cortical microtubules in nondividing cells. Surprisingly, amino acids 1-132 of TAN1 (TAN1(aa1-132)) are capable of significantly rescuing the double mutant, which suggests that this section of the TAN1 protein plays a crucial role in division plane orientation. The present study focuses on investigating TAN1(aa1-132) and TAN1(aa1-132) interacting proteins identified previously by yeast two-hybrid screening. After mutagenesis of TAN1(aa1-132), loss of interaction with known interactors will be screened for by yeast two-hybrid. The in vivo relevance of these disrupted interactions will then be assessed by transforming the tan1 air9 double mutant with the mutagenized versions of TAN1(aa1-132). Transformed double mutants will be examined for changes in protein localization as well as the ability of mutagenized TAN1(aa1-132) to rescue. By doing so, interactions required for TAN1 localization, function in division plane orientation, or both will be determined.

Many biomass crops are perennial, but the models we use to assess them often overlook the influence of plant age on seasonal plant phenology. For example, sapling trees leaf out earlier than conspecific adults to capture early season sun before the older stand closes canopy over them. Little information on age and phenology dynamics exists for perennial warm season grasses. We used a novel REplicated PLAnting Year (REPLAY) experimental design to study age-related phenology changes during the establishment phase (first three years) of Miscanthus × giganteus. We also considered the interactive effects of nitrogen (N) fertilization on phenology since N pools could be diluted in older, larger individuals. We found that two- and three-year-old (mature) stands produced ~30% more stems, with ~20% more leaves and nodes than one-year-old (young) stands. Faster developmental rates were usually seen in young stands, but they did not lead to more advanced developmental stages. Normalized over thermal time (growing degree days), older stands emerged ~3 months earlier than newly planted rhizomes. Nitrogen fertilization partially overrode age-related changes in emergence and senescence, and delayed flowering in mature stands thereby extending the growing season at least 10 days. We then used the process-based ecosystem model Agro-IBIS to understand how observed age-related phenology changes could influence ecosystem performance over the life time of a M. × giganteus stand. Over a 20-year crop lifespan, we found growing season length, leaf area, biomass production, and associated water cycling to be sensitive to the effects of plant age and nitrogen on grass phenology, and will discuss implications for crop and ecosystem assessment.

Pennycress (Thlaspi arvense) is an oilseed plant of the Brassicaceae family, native to Eurasia and naturalized to North America. Wild strains grow widespread throughout temperate regions of the world. Once commercialized, the oilseeds of elite pennycress varieties will provide additional income to farmers and agribusinesses thereby strengthening rural communities. Pennycress will also provide ecosystem services as a cover crop, reducing soil and nutrients runoff from otherwise vacant farmland. About two-thirds of the value of the pennycress seed is in the oil, which can be extracted by crushing and used as a biodiesel or biojet fuel feedstock. The remaining one-third value is the left-over seed meal, which can be used as a protein supplement in animal feed. However, the meal from wild pennycress strains is of relatively low quality due to the seed coat having high fiber content. Fiber is composed of lignin, cellulose, hemicellulose, and condensed tannins. To reduce pennycress seed coat fiber content, we used a forward genetics approach to screen through our EMS mutant populations for light-colored seeds indicative of reduced tannins, as well as a reverse genetics approach employing CRISPR genome editing to knock out function of the majority of genes in the flavonoid biosynthetic pathway responsible for producing condensed tannins (so-called TRANSPARENT TESTA genes). Here we will present data characterizing these mutants including identified effects on the agronomically-relevant traits seed coat fiber content, seed protein and oil content, and seed dormancy.

Diverse populations of bacteria live within plant tissues without causing disease. A number of these bacterial endophytes have been demonstrated to promote plant growth and development, and increase host plant resilience to multiple abiotic and biotic stresses. Nutrients and water are key resources limiting world agricultural production. Nitrogen fertilizers, provided by the energy-demanding Haber-Bosch process, have enhanced crop yields at huge environmental cost, whereas drought has traditionally been managed by irrigation, resulting in ground water depletion. Across the world, many farmers cannot afford to use fertilisers, and in developed countries, perennial energy crops such as Miscanthus are largely undomesticated and must produce high annual biomass yields on low-quality land without environmentally costly inputs such as water, fertiliser or pesticides. Developing tolerant plant genotypes involves lengthy and costly plant breeding programs; low-input sustainable alternatives are urgently needed.The Farrar lab has cultured and identified bacterial endophytes from plants growing under abiotic stresses such as salinity and heavy metal contamination, as well as from Miscanthus tissues. We have compared the capacity of novel bacterial endophytes to improve plant growth under saline, and limited water and nitrogen conditions, using the model plant Brachypodium distachyon in the National Plant Phenomics Centre, Aberystwyth, UK. We have identified plant growth promoting (PGP) strains outperforming the well-characterised PGP strain Azospirillum brasilense Cd. Multi -omics analyses are underway to determine the plant and bacterial factors involved in the beneficial plant-endophyte interaction. We aim to apply novel PGP endophyte strains harbouring beneficial traits to improve plant performance under biotic and abiotic stress conditions, including energy crop production and phytoremediation applications.

Engineering photosynthetic organisms is a long-standing goal of plant and algal biologists. Here we describe mutant photosynthetic organisms that have an attenuated CheY-like gene (CheY) encoding a two-component regulatory system with Regulator Receiver domain and myb-like DNA binding domain. The mutant organism with attenuated CheY-like gene exhibits a higher biomass productivity as a result of reduced chlorophyll content (primarily related to reduced light-harvesting antenna), increased rates of electron transport, and increased carbon fixation. These mutants are also characterized by ~20% increase in protein per TOC, which could yield an increase of 40-60% in areal protein productivity, when compounding increased percentage of protein with the increased biomass productivity. Under N- batch growth the mutants outperformed the wild type in both the biomass and lipid (FAME) accumulation (with up to 50% higher FAME production). To determine the effects of a CheY mutation in higher plants, we tested A. thaliana with mutations in CheY-like homologs. In the mutated organisms we observed a significant reduction in the amount of chlorophyll; more studies are needed to assess the effects of this mutation on biomass productivity. Overall, mutants with attenuated CheY provide a genetic means of increasing biomass, protein, and lipid content in photosynthetic organisms contributing towards the ongoing efforts towards more sustainable production of foods and chemicals.

The green alga Chlamydomonas reinhardtii does not synthesize high-value ketocarotenoids like canthaxanthin and astaxanthin, however, a β-carotene ketolase (CrBKT) can be found in its genome. CrBKT is poorly expressed, contains a long C-terminal extension not found in homologues and likely represents a pseudogene in this alga. Here, we used synthetic re-design of this gene to enable its constitutive overexpression from the nuclear genome of C. reinhardtii. Overexpression of the optimized CrBKT extended native carotenoid biosynthesis to generate ketocarotenoids in the algal host causing noticeable changes the green algal colour to a reddish-brown. We found that up to 50% of native carotenoids could be converted into astaxanthin and more than 70% into other ketocarotenoids by robust CrBKT overexpression. Modification of the carotenoid metabolism did not impair growth or biomass productivity of C. reinhardtii, even at high light intensities. Under different growth conditions, the best performing CrBKT overexpression strain was found to reach ketocarotenoid productivities up to 4.5 mg L-1 day-1. Astaxanthin productivity in engineered C. reinhardtii shown here is competitive with that reported for Haematococcus lacustris (formerly pluvialis) which is currently the main organism cultivated for industrial astaxanthin production. In addition, the extractability and bio-accessibility of these pigments was much higher in cell wall deficient C. reinhardtii than the resting cysts of H. lacustris. Engineered C. reinhardtii strains could thus be a promising alternative to natural astaxanthin producing algal strains and may open the possibility of other tailor-made pigments from this host.

Crassulacean acid metabolism (CAM) is a specialized type of photosynthetic CO2 fixation pathway that results in enhanced water-use efficiency (WUE) compared to C3 and C4 photosynthetic plants. Increasing frequencies and intensity of drought and other abiotic stresses including high salinity, extreme temperatures, and high light intensities are major constraints for global crop production. Notable progress has been made towards genetic engineering crop plants to improve tolerances to different abiotic stresses. One widely uses bioengineering approach is the overexpression of transcription factors (TFs) to modify complex traits including tolerance to abiotic stresses in crop plants. Here, we have used abiotic stress-responsive TFs from CAM plants to improve abiotic stress tolerance in Arabidopsis presumably by activating regulatory mechanisms that mediate stress tolerance adaptations as CAM plants are naturally adapted to drought and other abiotic stresses. Previously, we identified several candidate TFs as key regulators of either CAM or water-deficit response or both in the obligate CAM plant, Kalanchoe fedtschenkoi. Of these TFs, functions of the K. fedtschenkoi NAC83 (KfNAC83) and KfbZip TFs are not known in CAM or C3 photosynthesis plants, but their A. thaliana orthologues display potential roles in abiotic stress responses and development. We have functionally characterized these TFs via overexpression in A. thaliana to determine their roles in abiotic stress responses and development. Overexpression of KfNAC83 TF in A. thaliana enhanced the drought and salt tolerance of transgenic lines, as well as enhanced plant growth and development. Remarkably, KfNAC83 overexpression lines showed a significant increase in integrated WUE with increased biomass productivity up to 42% compared to wild-type plants. Results of the phenotyping and potential improvement of abiotic stress tolerance, WUE, and productivity of utilizing CAM TFs will be discussed.

Rapid activation of plant immune responses involves transient remodeling of phosphorylation states on diverse cellular targets through concerted kinase and phosphatase activities. Using quantitative phosphoproteomic analyses of both maize and Arabidopsis, we identified networks of proteins with altered patterns of phosphorylation minutes after treatment with immunoregulatory Plant Elicitor Peptide (Pep) hormones. While some observed proteins were already implicated in plant innate immunity, most were not. In both species, nucleic acid-binding proteins were highly enriched targets, and screening of insertional mutants in corresponding genes revealed numerous candidate regulators with altered immune phenotypes. Investigation of select candidates has demonstrated that not only do target proteins contribute to plant disease resistance, but that specific phosphorylation sites identified through our analyses are critical to regulatory function. An RNA-binding protein was found to mediate alternative splicing of transcripts encoding key defense signaling proteins, shifting ratios of splice variants that result in premature stop codons and truncated proteins with lost or modified function. Critically, recruitment of transcripts to the RNA-binding protein is dependent on the phosphorylation state of the protein at the site observed to change after Pep treatment. Similarly, a candidate DNA-binding protein was found to regulate gene expression changes through phosphorylation-dependent recruitment of interacting transcription factors to affect immune function. Together our studies demonstrate the utility of quantitative phosphoprotemics for identification of novel signaling proteins contributing to the plant immune response, and more importantly, for pinpointing precise molecular switches controlling regulatory activity.

There are few genetic mechanisms for controlling the invasive and crop-damaging whiteflies of the world. Potent whitefly-resistance mechanisms have been identified in highly heterozygous and tetraploid crops such as alfalfa (lucerne, Medicago sativa L) and cassava (Manihot esculentum). These resistance mechanisms are trichome independent, phloem mediated, primarily manifested as death of first- and second-instar nymphs and prevent whitefly population expansion. Therefore, these resistance genes and have immense potential to enhance crop productivity in regions with strong whitefly pressure. Both alfalfa’s and cassava’s whitefly resistances are multigenic and are likely mechanistically different as they have distinct gene complements within their resistance loci. While alfalfa displays a potent resistance to B. tabaci MEAM1, this resistance is not as effective against B. tabaci NW1 or MED1. Our ability to make CRISPR-Cas9 mutations in B. tabaci will allow us to dissect the genetics controlling alfalfa’s resistance in the whitefly, as well as the host plant. In contrast, cassava’s whitefly resistance is broad conferring resistance to three South American whitefly species and three SubSaharan Bemisia tabaci species. To understand the molecular mechanisms of cassava’s whitefly resistance, temporal responses of whitefly-resistant and -susceptible cassava genotypes to whiteflies, jasmonic acid and salicylic acid were determined using RNA-seq. These studies have revealed that the defense-hormone programming of cassava is profoundly different from the model plant Arabidopsis thaliana and suggest a master switch maybe active in controlling whitefly resistance in cassava.

Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte) is one of the major insect pests of corn in the United States. WCR larvae feeds on corn roots and are causing significant economic losses when unprotected. Exposure to insecticidal protein expressed in plants trigger development of resistance in insects to these proteins over time decreasing the potency of Biotech traits. Discovering novel insecticidal molecules representing new mechanism of actions (MOA) will enable sustainable insect control and complement the current biotech traits which are based on the expression of the Bacillus thuringiensis Cry3Bb and Cry34/35 proteins. The dsRNA was shown to have insecticidal activity when derived from essential rootworm genes and will be utilized in next generation product. Comparison of mechanism of action of insecticidal protein and dsRNA indicate utilization of very diverse processes, membrane pore formation and RNAi pathway induction, respectively which suggest extended durability of future corn varieties containing both MOAs. In addition, new proteins with good activity on WCR have been discovered. These new proteins have sufficient sequence and structural diversity compared with Cry3Bb to provide new MOAs for the control of WCR. Diet bioassay data with these new proteins indicate that these proteins can control both populations of fully susceptible WCR and those that shows tolerance to Cry3Bb. Transgenic corns expressing these new proteins have demonstrated superior root protection in greenhouse tests and field trials. These new active molecules provide improved tools for the effective control of WCR populations in corn crops.

Signal propagation and coordination of whole-organism responses in plants rely heavily on small molecules. Systemic acquired resistance (SAR) is one such process in which long-distance signaling activates immune responses in uninfected tissue as a way to limit the spread of a primary, localized infection. Despite the importance of defense priming, the identity of the mobile defense signal that moves systemically throughout plants to initiate SAR has remained elusive. In this work, we report the discovery of N-hydroxy-pipecolic acid (NHP), a metabolite that plays a key role in initiating and amplifying SAR signaling in Arabidopsis. We show that Arabidopsis FMO1 (FLAVIN-DEPENDENT MONOOXYGENASE 1) synthesizes NHP from pipecolic acid and exogenously applied NHP moves systemically in Arabidopsis plants. We also provide evidence that NHP is conserved across the plant kingdom and demonstrate a role for NHP in mediating SAR responses in important crop plants. We used heterologous expression in Nicotiana benthamiana to identify a minimal set of genes required for NHP biosynthesis. Expression of these genes in tomato is sufficient to trigger SAR. Our results suggest chemical application or engineering strategies to induce NHP-mediated SAR are promising routes to improve broad-spectrum pathogen resistance in crops.

Enhanced resistance to pests and pathogens, resulting from the additive effects of two sets of defensive genes, may provide a selection for polyploidy, which has arisen frequently in the course of plant evolution. The allotetraploid perennial soybean Glycine dolichocarpa has resistance to both Aphis glycines (soybean aphid) and Acyrthosiphon pisum (pea aphid), whereas its diploid progenitors, Glycine tomentella D3 and Glycine syndetika, show resistance to only A. glycines or A. pisum, respectively. Transcriptomic and metabolomic assays demonstrated species-specific variation in the responses of perennial soybeans to A. glycines and A. pisum infestation. Resistance to A. pisum feeding was associated with isoflavone accumulation, whereas resistance to A. glycines increased with flavone content. This observation was recapitulated in artificial diet assays, where isoflavones had a greater negative effect on A. pisum and flavones had a greater negative effect on A. glycines. Correlative analysis of gene expression and aphid resistance in the three perennial soybean species identified likely resistance (R) genes. The functions of two cysteine-rich receptor-like protein kinases were confirmed through overexpression and expression silencing. Together, the observed additive effects of flavonoids and R genes in aphid resistance support the hypothesis that allotetraploidy in perennial soybeans provides an evolutionary advantage through the combination of two plant defense systems.

Plants defend themselves against infectious pathogens through inducible defenses operating at the molecular level through immune receptors that are activated by pathogen elicitors. Two inducible responses to pathogens are termed extreme resistance (ER), in which the pathogen is eliminated without cell death, and the hypersensitive response (HR), which limits the pathogen to the infected area by local programmed cell death. Specific Nicotiana glutinosa accessions display either ER or HR depending on the member of the Polerovirus genus infecting the plant. N. glutinosa accession TW59 exhibits HR when infected with turnip yellows virus (TuYV) as well as potato leaf roll virus (PLRV), while accession TW61 exhibits HR only when infected by PLRV and exhibits ER when infected by TuYV. This variation allows us to compare the mechanisms through which ER and HR are executed. To study these outcomes at the transcript level, leaves of TW61 were agro-infiltrated with TuYV and PLRV infectious clones alongside leaves agro-infiltrated with the empty pBIN61 vector as a control. RNA was extracted to analyze the gene expression changes in response to each virus through Next Generation RNA-sequencing. We hypothesized that there would be specific changes in gene expression associated with ER, versus resistance accompanied by HR, as well as an overlap in differential gene expression for these two responses. We identified 484 significantly differentially expressed genes from the TW61 transcriptome and selected 15 as candidates based on high relative fold-change, and different patterns of expression. The differential expression of these candidate genes was further quantified through reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Our analysis has identified genes with potential roles in both ER and HR, or with unique functions in one defense pathway. These findings could contribute to a better understanding of these agriculturally relevant plant defense responses.

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Many plants make use of environmental cues, such as daylength and temperature, to synchronize reproductive development with favorable climatic conditions, thereby increasing their reproductive success. Although the genetic mechanisms that regulate reproductive development in response to seasonal cues are largely conserved across flowering plants, it is not known whether this conservation extends beyond angiosperms to other land plant lineages. We are using the model moss Physcomitrella patens to probe the evolutionary origin of seasonal regulation in land plants, with the long-term goal of determining whether a core mechanism evolved in the common ancestor of all land plants, or if convergent mechanisms arose separately in distinct land plant lineages. We are coupling transcriptomic and genomic analyses of a set of P. patens ecotypes that vary in reproductive timing in response to seasonal cues with traditional mutagenesis screens to identify and characterize the genetic networks that underpin seasonal regulation of sexual reproduction in this basal land plant. Using a combination of cross-population genome-wide measures of selection and differential co-expression network analysis comparing ecotypes across daylength and temperature conditions, we have narrowed in on candidate genes homologous to upstream components of angiosperm temperature and daylength induced flowering time pathways. We have generated CRISPR-Cas9 mutants in four gene families and are currently evaluating their phenotypes. In parallel, we designed a mutagenesis screen, which has yielded heritable mutants with striking differences in the timing of onset of reproductive development in response to seasonal cues.

In many plant species, flowering occurs at a particular time of year in response to the sensing of seasonal cues such as changes in day-length and temperature. Many plants adapted to temperate climates have a biennial life history strategy. These plants become established in the fall, overwinter, and flower rapidly in the spring. Essential to this adaptive strategy is that flowering does not occur prior to winter, during which flowering would not lead to successful reproduction. Thus, plants have evolved ways to prevent fall flowering and sense the passing of winter to establish competence to flower. The block to flowering can be alleviated by exposure to prolonged cold or exposure to prolonged period of short-days (SD) which occurs during winter. The process by which flowering is promoted by SD followed by growth in longer photoperiods is known as SD vernalization. Some B. distachyon accessions exhibit SD vernalization whereas others do not. From crosses between such accessions, we found that SD vernalization segregates as a single locus and the responsible gene is a paralog of FT (“florigen” mobile floral signal) referred to as FT-LIKE 9 (FTL9). Accessions with a functional allele of FTL9 have both the SD and cold responses, whereas accessions with loss-of-function alleles can only respond to cold. There is a striking geographic pattern to the allelic variation: active alleles are found in warmer climates perhaps because in such climates it is adaptive to be able to use SD as a reliable indicator of winter, whereas accessions from places with longer and variable winters have inactive alleles and cannot sense SD vernalization—perhaps because this might lead to premature flowering in conditions in which winter cold would damage delicate flowers. The cloning of FTL9 provides the first molecular insight into the SD vernalization phenomenon.

Plants have evolved many traits that integrate external signals with internal rhythms to synchronize growth with daily cycles of temperature, light, and other factors. Solar tracking by sunflower stems is a dramatic example of such a diurnal rhythm. The shoot apex continuously tracks the sun from east at dawn to west at dusk during the day. At night, the apex reorients back to an eastward orientation. Although long observed, the adaptive value of solar tracking and the molecular mechanisms by which external cues and internal rhythms coordinate these daily growth rhythms have remained largely unknown. By applying diverse approaches, we have gained new insights into the physiology, genetics, and ecology of solar tracking. Our manipulative experiments implicate the circadian clock as a driver of nocturnal reorientation. Moreover, through GWAS and QTL mapping studies of natural variation, we have characterized the genetic architecture of intraspecific diversity in solar tracking traits. For instance, by filming a panel of >280 re-sequenced cultivars, we have detected SNPs associated with the start of daytime tracking relative to dawn or the start of nocturnal reorientation relative to dusk in genes homologous to gene families involved in light signaling, hormone signaling, and cell expansion. Several of these genes are differentially expressed between east- and west-facing stem segments, validating that our approach identifies biologically meaningful candidates. Likewise, in an F4 panel derived from a cross between two wild sunflower parents, we have mapped QTLs containing genes homologous to key downstream integrators of growth signals from light and plant hormones, and these candidates also show differential expression between east- and west-facing stem segments at dusk or dawn. Finally, our findings demonstrate that solar tracking and the eastward orientation of mature sunflower disks are critical for biomass accumulation and reproductive fitness, respectively.

To improve food security in the developing world, the current trend in rice production is to shift from transplanting seedlings to direct sowing of seeds. Following heavy rains, direct-sowed seeds may need to germinate under flooded, anaerobic conditions, but most rice genotypes cannot survive these conditions. To identify complex trait loci associated to anaerobic germination (AG), we integrated phenotypic germination information with a 700,000 SNP data base from the rice 3,000 genome initiative for genome-wide association studies (GWAS). Using 109,440 seeds, we quantified AG% in 2,700 (wet season) and 1,500 (dry season) rice genotypes and performed GWAS, followed by post-GWAS analysis that encompassed a generalized SNP-to-gene set analysis, metanalysis and a network dense module search. We determined that transcription factors linked to ethylene responses or genes involved in several metabolic processes are significantly associated with AG. SNP-to-gene, meta- and dense module network GWAS analyses identified genes that have shown changes in gene expression in response to AG in previous experiments. We found two significant gene-sets involved in sphingolipids metabolism, whose function in AG has not been characterized. In our network-GWAS analysis we evaluated the top 100 network modules; these modules showed genes involved in a wide variety of metabolic processes and found a fatty acid desaturase that also was significant in the SNP-to-gene set analysis. We determined that anaerobic germination percentages are highest among indica subpopulations, and AG is a polygenic trait with complex physiological differences among rice genotypes. We selected several genes of interest that have not been linked to AG before to perform further functional genomics analyses. Currently we are characterizing these genes’ relationship to flooding in rice mutants by doing genetic, physiological and biochemical experiments.

Genomic prediction where genotype information is used to predict phenotypes has accelerated breeding processes and can provide mechanistic insights into phenotypes of interests. In switchgrass (Panicum virgatum L.), a polyploid perennial grass and biofuel feedstock, past efforts in genomic prediction treated tetraploid and octoploid individuals as if they had the same ploidy and utilized only Single Nucleotide Polymorphisms (SNPs) with three discrete levels of variation (i.e. AA, AT, or TT). In this study, we assess the predictive performance of models generated using both SNP and Insertion/Deletion (indel) variants to predict 20 traits in a switchgrass association panel with 510 individuals. In our model, we accounted for the greater variant complexity for polyploids (i.e. AAAA, AAAT, AATT, etc.), sequencing strategies (exome capture and genotyping by sequencing [GBS]), and genome assembly versions. Surprisingly, the models based on variants identified with the v5.1 assembly did not outperform models based on v1.1 derived variants for most traits. However, models built with exome capture SNPs tended to outperform those built with GBS SNPs, and combining both data types resulted in even better performance. Additionally, SNP based models performed better than indel based models, with no improvement when both were used. Overall, models were better at predicting traits in tetraploids than in octoploids, highlighting the importance of considering ploidy in genomic prediction problems. Finally, we probed the genetic basis underlying multiple target traits by studying the variants that had the greatest impact on model performance. Our study provides insights into the best practices for performing genome prediction in species with multiple ploidy levels that can be used for improving switchgrass agronomic traits through selective breeding.

Genome instability is a disrupting phenomenon that can result from natural or artificial causes. The molecular mechanisms that trigger genome instability are not well understood. At the same time, while the consequences of genome instability on plant breeding and evolution can be drastic, they remain poorly characterized. We characterize the extent and nature of genome instability after regeneration from tissue culture or after intraspecific haploid induction crosses of potato. Both processes resulted in chromosome remodeling at different frequencies, the outcomes of which are either truncated or shattered chromosomes. We show that regeneration from protoplasts without introduction of transgenes or genome editing tools was highly disruptive to genome integrity, whereas regeneration of transgenic potatoes using standardized procedures had a milder effect. In the context of haploid induction, we observed rare remodeling of single haploid inducer chromosomes that were retained in otherwise haploid plants, which is broadly consistent with other plant haploidization crosses. Overall, our results provide both a resource for investigating causes and outcomes of genome instability in plants and perspective on genomic instability incurred via routine experimental techniques in plant biology.

Plants have sophisticated mechanisms for sensing neighbor shade and responding through enhanced elongation and physiological changes to maximize their ability to compete for light. The shade avoidance response affects many different organs and growth stages, yet the signaling pathways underlying this response have mostly been studied in seedlings. To understand the signaling pathways operating in older plants, we analyzed a gene expression time course of adult shade avoidance in wild-type and shade avoidance mutants. With this data we established a signaling cascade of hormone action during wild-type response to shade and used mutants to determine how genetic perturbation affects the cascade. We found pervasive misregulation of salicylic acid genes in many mutants, suggesting salicylic acid signaling to be an important shade avoidance growth regulator. Supporting our hypothesis, several salicylic acid pathway mutants reduced shade-induced and basal growth. The effect of these mutants on shade avoidance was specific to petiole elongation; neither hypocotyl nor flowering time responses were altered, thereby defining important stage-specific differences in the downstream shade avoidance signaling pathway. Shade treatment did not change salicylic acid levels, indicating salicylic acid mediation of shade avoidance is not dependent on modulation of salicylic acid levels.

The phytochrome (phy) gene family consists of multiple genes encoding photoreceptors with which plants perceive environmental information used to guide developmental decisions, such as germination, photomorphogenesis and flowering. In tomato phytochromes are encoded by five genes: PHYA, B1, B2, E, and F. As a general rule, red light activates phytochromes, while far-red light deactivates them, however, phyA is responsive to both wavelengths. PhyA is involved in early developmental processes during the seedling’s transition from growing in the dark to growth in light. The role of phytochromes during the dark growth phase, also known as skotomorphogenesis, has been the subject of considerably fewer research studies than those investigating their role in the light. We performed transcriptomic profiling and co-expression network analysis of tomato seedlings during the transition from dark to light growth. Our data suggest that phyA plays a role in the regulation of enzymes involved in carbon flux through glycolysis, beta-oxidation, and the Krebs cycle. Our analysis also showed that phyA is involved in the regulation of several sucrose transporter SWEET genes before the plant is exposed to light. This coincided with slightly longer hypocotyls of dark grown phyA mutants compared to WT seedlings grown in the dark, and increased root growth in the mutants soon after the seedling reached light. Intriguingly, these data suggest that phyA might play a role in carbon distribution in the dark, possibly in anticipation of light growth.

As an essential energy source for photosynthesis, light regulates plant development in various aspects in the shoot and root. While the quality of light is sensed by photoreceptors and known to affect root branching, we have recently found that rapid reduction of light intensity, without changing the spectrum, also strongly affects lateral root emergence in Arabidopsis. This dynamic regulation of root growth is dependent on light perception by shoot, and likely to be a photomorphogenesis-independent phenomenon, as various photoreceptors mutants do not disrupt the response. Characterization of mutants that affect redox status in the chloroplast, cyclophillin 38 (cyp38) and thioredoxin reductase C (ntrc), as well as treatment of electron transport inhibitors, revealed that such perturbations have a very similar inhibitory effect on lateral root emergence as low light. Using a grafting approach, we found that CYP38 acts non-autonomously in the shoot to promote branching of roots, consistent with the the exclusive chloroplast localization pattern of CYP38 and NTRC. Metabolomic analyses showed that the level of various defense related molecules, for example, the precursor of Jasmonic acid, are lower when photosynthesis is repressed. Applying those compound exogenously partially rescues the lateral root suppression under low light conditions or in the cyp38 mutant. All together, our findings indicate the importance of light dependent energy production and redox status in chloroplast in modulating lateral root formation, through shoot to root communication.

Plant growth is regulated by environmental conditions, including UV-B radiation. UV-B inhibits leaf and root growth, and inhibition of plant growth is, in part, regulated by the E2Fe transcription factor. E2Fe is a target of regulation by two transcription factors from the same family, E2Fb and E2Fc. While E2Fc acts as a repressor, E2Fb is a transcriptional activator of E2Fe. Therefore, we investigated if the modulation of UV-B responses by E2Fe is through its regulation by E2Fb and/or E2Fc. At UV-B intensities that produce DNA damage, inhibition of cell proliferation is regulated by both E2Fc and E2Fb. E2Fc controls plant growth under UV-B conditions regulating DNA damage responses, as E2Fc deficient plants show decreased programmed cell death in the roots after exposure and altered SOG1 and ATR expression. In addition, E2Fc has an epistatic role over the miR396 pathway under UV-B, which also regulates leaf growth under these conditions. In contrast, while E2Fb also controls cell proliferation under UV-B conditions; it does not regulate programmed cell death in the roots after exposure. Interestingly, E2Fb deficient leaf cells have increased DNA ploidy levels after UV-B exposure, similarly as E2Fe deficient cells. Together, our results demonstrate that E2Fc is required for miR396 activity on cell proliferation under UV-B, and that its role is independent of E2Fe, probably modulating DNA damage responses through the regulation of SOG1 and ATR levels. On the contrary, the regulation of DNA ploidy in leaf cells under UV-B previously described in E2Fe deficient plants could be regulated by E2Fb activity.

Light regulates gene expression at all levels of central dogma during photomorphogenesis, including alternative splicing (AS). However, the accurate determination of full-length splicing variants was greatly hampered by the short-read nature of commonly used RNA-seq technologies. To combat this limitation, we adopted PacBio isoform sequencing (Iso-seq) that offers advantages in long-read sequencing for the identification of full-length AS variants. Normalized cDNA libraries prepared from 4-d-old etiolated seedling with or without 4-h white light treatment were used for Iso-seq to achieve comprehensive and effective identification of full-length AS variants. Our analyses revealed greater than 30,000 splicing variant models from ~16,000 gene loci and identified ~700 previously unannotated transcripts. Among them, 14,644 transcripts represented new gene models, and one-third of the loci producing AS variants contain two or more splicing events. Intron retention (IR) is most frequently observed, and some IR-containing AS variants show evidence of engagement in translation. Through tackling the biological functions of the splicing isoforms, our study showed the formation of heterodimers of transcription factors in their annotated and IR-containing AS variants. Moreover, transgenic plants overexpressing the IR-forms of two BBX family members exhibited light hypersensitive phenotypes, suggesting the regulatory roles of these IR isoforms in modulating optimal light responses during photomorphogenic development. Our results provide a new approach for identifying de novo synthesized AS variants that impose regulatory functions in de-etiolating Arabidopsis.

Light-activation of a LOV-histidine kinase in cells of Rhizobium leguminosarum increases the number of nodules and the number of intranodular bacteroids on pea (Pisum sativumL.) roots grown in hydroponics systems (Bonomi et al., Proc. Natl. Acad. Sci. 109: 12135, 2012). We have investigated whether a similar response might be demonstrated under normal greenhouse conditions with pea plants grown in soil to determine whether the finding might ultimately prove beneficial in agriculture. We have also extended the experiments to measure the effects both of light treatment of the bacteria and timing of inoculation on final bean yield. Pre-irradiation of cells of Rhizobium leguminosarum with blue light induces an increase in the number of functional nodules (those containing leghemoglobin) and ultimately seed yield both when the inoculation takes place with the onset of imbibition and four days after the onset when primary roots have emerged. However, inoculation four days after the onset of imbibition and in the presence of primary roots greatly increases both the number of functional nodules and seed yield compared to inoculation at the start of imbibition with or without light treatment of the bacteria. We have measured several growth and developmental parameters, including numbers of flowers per plant per week, pod weight and numbers of peas per pod. Our findings show photoactivated bacterium suppresses floral abortion, and significantly increases pea yield. We are currently carrying out field tests to determine whether light treatment and the timing of inoculation might lead to increased yield in an agricultural context.

In Arabidopsis thaliana the two major populations of stem cells are located in the shoot apical meristem (SAM) and root apical meristem (RAM). These two SAMs are responsible for all post-embryonic growth. A family of five transcription factors known as the CLASS III HOMEODOMAIN-LEUCINE ZIPPERs (HD-ZIP III) has been shown to play pivotal roles in maintaining, regulating and patterning shoot stem populations as well as patterning differentiating tissues (stems, leaves and flowers). Loss-of-function, gain-of-function, and mis-expression studies indicate that HD-ZIP III members have overlapping and antagonistic roles in these processes, suggesting a regulation of both shared and unique target genes. Using a glucocorticoid inducible system coupled with RNA-seq, ChIP-seq, and reporter gene analyses we have analyzed the transcriptional response downstream of each HD-ZIP III member. Our data suggests HD-ZIP III members exhibit specific early and late transcriptional responses, and share a suprisingly small number of overlapping target genes. Among these differentially regulated genes, several gene families appear to be temporally co-regulated by HD-ZIP IIIs. ChIP-seq with each HD-ZIP III member suggests this transcriptional regulation is direct. Previous reports on these co-regulated families have implicated them in embryo development, meristem regulation and leaf patterning. Additionally, our transcriptome data also indicates that HD-ZIP IIIs may have the ability to promote and repress transcription. Taken together, these data may account for the redundancy and antagonism displayed in past genetic studies. Our approaches and findings are allowing us to shed light on a how closely related family of genes can evolve to exhibit specific roles in gene regulation and is advancing our understanding of the mechanisms required for SAM regulation and tissue patterning

Stomatal guard cells are some of the most dynamic cells in plants due to their ability to expand and contract to control the size of stomatal pores. However, our understanding of how the walls of sister guard cells separate to form stomatal pores, and how they imbue guard cells with the strength and elasticity required to repeatedly inflate and deflate over the lifetime of a leaf, is limited. We applied molecular genetics, cell biology, and mechanical modeling to probe stomatal development, function, and mechanical aging in Arabidopsis thaliana. Using time-lapse microscopy we determined the contributions of wall degradation and mechanical pressure to stomatal pore formation. Our results indicate that pectin degradation is the primary driver of pore initiation, whereas both pectin degradation and cell presurization contribute to pore enlargement. We also discovered that cellulose, xyloglucan, and pectin contribute to stomatal function in distinct ways: mutants with reduced cellulose have enlarged guard cells, display reduced relative increases in pore width during stomatal opening, and, counterintuitively, are modeled as having stiffer walls than wild type, whereas xyloglucan-deficient mutants have smaller guard cells that display normal relative opening, but are modeled as having softer cell walls than wild type. In a mutant with reduced pectin molecular mass, stomata appeared normal in the closed state, but opened much wider than wild type despite a modeled increase in the longitudinal stiffness of the wall. Finally, we found that manipulating the expression of different endogenous pectinases has differential effects on stomatal development and function, implying that pectin autodegradation has complex effects on guard cell size, stomatal pore formation, and the elastic behaviors of guard cells over developmental time. This research will inform efforts to generate crop plants with enhanced control of stomatal dynamics and improved water use efficiency.

Developmental outcomes are shaped by the interplay between intrinsic and external factors. The production of stomata—essential pores for gas exchange in plants—is extremely plastic and offers an excellent system to study this interplay at the cell lineage level. For plants, light is a key external cue, and it promotes stomatal development and the accumulation of the master regulator SPEECHLESS (SPCH), which initiates the stomatal lineage. However, how light signals are relayed to influence SPCH remains unknown. Here, we show that the light- regulated transcription factor ELONGATED HYPOCOTYL 5 (HY5), a critical regulator for photomorphogenic growth, is present in inner mesophylls and directly binds and activates STOMAGEN. STOMAGEN, the mesophyll-derived secreted peptide, in turn stabilizes SPCH on the epidermis, leading to enhanced stomatal production. Our work identifies a molecular link between light signaling and stomatal development that spans two tissue layers and highlights how an environmental signaling factor may coordinate growth across tissue types.

Ensuring a dynamic interface between the roots and the soil is essential for plant survival. Plant cell walls sit at the cornerstone of this interface, acting as the contact point through which communication and exchange with the environment occurs. The matrix that comprises the plant cell wall varies not only across cell types but also through the developmental progress of each cell. Some cell layers form additional diffusion barriers via the deposition of polymers such as suberin. A well-studied example is the endodermis, which forms such barriers to control the entry to the plant vasculature. However, many plant species also contain an additional “sister cell type” right underneath the outer epidermis known as the exodermis. Exodermal differentiation and its ability to dynamically adapt to external conditions have not been formally characterized, nor the genetic and molecular mechanisms that govern them. Deposition of suberin is a complex process coordinated by several transcription factors and biosynthetic enzymes. While hundreds of genes potentially associated with biosynthesis and polymerization of suberin have been annotated, only subsets of these will participate in cell types that form barriers. In order to identify potential candidates, we leveraged cross-species root cell type-resolution transcriptomic, phylogenetic, and gene network analyses. We then coupled the ability of Agrobacterium rhizogenes to induce stable transgenic (“hairy”) roots in tomato (S. lycopersicum), with the efficiency of CRISPR-Cas9 technology to rapidly generate mutants of these candidates. Finally, we used histochemical and transcriptional analyses to functionally validate the genes that form the exodermal diffusion barrier. Understanding how these processes are regulated is critical to a plant’s ability to adapt to changes in water or nutrient availability. Ultimately, determining barrier-relevant genes will enable the breeding of crops with higher resistance to abiotic stresses.

Gravity is a constant force that guides plant organs’ growth, allowing roots to take up water and nutrients from the soil, and shoots to grow above ground where they can access light for photosynthesis. In this study, we took advantage of the natural variation existing between accessions of Brachypodium distachyon, a monocot model, to investigate root-growth behavior in response to gravistimulation. When Brachypodium seedlings are reoriented within the gravity field, their roots display a biphasic response. The root tip initially shows a strong downward gravitropic curvature, followed by a slower downward response that is accompanied by tip oscillations. Curvatures associated with both phases occur at the distal elongation zone. To quantify features related to both phases, we developed a mathematical model that simulates the kinetics of root-tip angle, using (1) a sigmoid function to represent the rapid bending phase, and (2) a sinusoidal function that recapitulates the oscillatory component of the second phase along with a linear trend that simulates the progressive bending toward gravity. Fast-Fourier Transform analysis was used to evaluate the periodicity (P) of root-tip oscillations at the end of a gravitropic response. Equation fitting led to determination of quantitative parameters that explain distinct characteristics of the behavior, including: speed of initial curvature (MRBR), transition angle between response phases (TA), and amplitude (A) of oscillations. For each of these traits, average value and standard deviation were included as separate parameters in genome-wide association studies to identify associated polymorphisms likely to contribute to the behavior. In our results, three of eight parameters showed association peaks, including amplitude, standard deviation of amplitude, and MRBR. Multiple candidate genes were identified using this approach, whose molecular characterization will be discussed. This work is supported by a grant from NASA.

Organ size control is fundamental in biology. However, the mechanisms that determine final organ size in multicellular organisms are not fully understood. We found and characterized a novel gene, CHIQUITA1 (CHIQ1), which might be a key to elucidating organ size control mechanisms. Mature leaves of plants harboring the chiq1-1 null allele are smaller than wild type with fewer and smaller cells. Cell cycle marker studies indicated that cell proliferation ends prematurely in chiq1-1 leaves; and most chiq1-1 pavement cells do not enter endoreduplication after exiting the mitotic cell cycle. In addition, chiq1-1 pavement cells stop expanding prematurely. Surprisingly, 4D imaging studies on leaves in the proliferating phase indicated that meristematic cells divide and expand faster in chiq1-1 leaves and proliferating leaves are bigger. We hypothesize that an early onset of differentiation is triggered in chiq1-1 leaves because of a defect in attenuating division and/or growth during proliferation, which results in smaller adult plants. This work uncovers a genetic basis that connects cell proliferation, differentiation, and organ size in plants.

Deep analysis of protein networks using proximity labeling mass spectrometry

Multiprotein complexes are fundamental to molecular processes and pathways in an organism. Critical to understanding how these complexes function in biological processes is identifying the underlying proteins and their interacting partners. The yeast two-hybrid (Y2H) assay is widely used for the identification of protein-protein interactions (PPIs) and is highly scalable however acquiring protein interaction data on a proteome-wide scale can be expensive and time-consuming. To alleviate costs while maximizing interrogation space, Cre-lox recombination and yeast two-hybrid assay based CrY2H-seq was developed. After en masse mating, cells with Y2H interacting proteins induce reporter gene expression of Cre Recombinase. Y2H plasmids engineered with mutant loxP sequences then undergo Cre-recombination. The irreversible double mutant loxP linkage of each protein’s corresponding coding sequence allows the identification of protein interactions after analysis of Illumina paired-end sequencing. We applied CrY2H-seq screening to a collection of ~2000 Arabidopsis transcription factors (1) in ten all-by-all replicate experiments. The resulting Arabidopsis thaliana Transcription Factor Interaction Network 1 (AtTFIN1) is made up of 8577 PPIs (2) and shows high recall rate and literature overlap (http://signal.salk.edu/interactome/AtTFIN-1.html). We are now applying an improved Cry2H-seq2 method to the Arabidopsis Gateway ORFeome collection containing an unbiased set of ~12,000 clones. CrY2H-seq2 has the potential to be universally applied, e.g. for screening cDNA libraries generated from plants under attack by pathogens to any plant species where there are no ORF collections available. Integrating protein interaction networks with other omics data can lead to the production of beneficial plant phenotypic outcomes.(1) Pruneda-Paz JL et al. Cell Rep (2014):622-32(2) Trigg SA et al. Nat Methods (2017):819-825

Transcription factors (TFs) are fundamental components of biological regulation, facilitating the basal and differential gene expression necessary for life. TFs exert transcriptional regulation through interactions with both DNA and other TFs, ultimately influencing the action of RNA polymerase at a genomic locus. Current approaches are proficient at identification of binding site requirements for individual TFs, but few methods have been adapted to study oligomeric TF complexes. Further, many approaches that have been turned toward understanding DNA binding of TF complexes, such as electrophoretic mobility shift assays, require protein purification steps that can be burdensome or scope-limiting when considering more exhaustive experimental design. In order to address these shortfalls and to facilitate a more streamlined approach to understanding DNA binding by TF complexes, we developed the DIMR (Dynamic, Interdependent TF binding Molecular Reporter) system, a modular, synthetic yeast-based transcriptional activity reporter. As a proof of concept, we focused on the NUCLEAR FACTOR-Y (NF-Y) family of obligate heterotrimeric TFs. The DIMR system was able to reproduce the strict DNA-binding requirements of the NF-Y complex with high fidelity, including recapitulation of previously-characterized mutations in complex subunits that break either subunit interactions or DNA binding. With this model firmly established, we can directly test the effects DNA and amino acid changes have on NF-Y complex function, and have begun to address the DNA-binding impacts of an atypical conserved linker domain within a particular NF-YA subunit of Arabidopsis thaliana. The DIMR system provides a powerful, easy-to-use approach to address these and similar types of questions concerned with the binding of both monomeric and oligomeric TFs to DNA.

mRNA translation is an essential step in gene expression, and manipulating mRNA translation enables new opportunities to improve crop performance. To understand the translational landscape and its underlying regulation in plants, we compared global mRNA translation in Arabidopsis and tomato using an enhanced high-coverage ribosome profiling method. Although the two dicot plants diverged approximately 100 million years ago, many regulatory features controlling translation efficiency, such as upstream ORFs (uORFs), microRNAs, and Kozak sequences, are well preserved. Taking advantage of our high-quality data, we also observed conserved ribosome stalling in some uORFs, suggesting a shared mechanism for repressing the translation of downstream main ORFs and reducing mRNA stability. Non-AUG translation start sites and translation-associated trans-acting siRNA (tasi-RNA) production are also conserved. Besides annotated genes, ribosome profiling detected unannotated small ORFs (sORFs) with predicted secretion signals. In addition to unique sORFs, the conserved sORFs are actively translated in Arabidopsis and Tomato, implying their critical biological functions throughout evolution. In summary, our approach provides a high-throughput method to discover unannotated ORFs, elucidates evolutionarily conserved and distinct translational features, and identifies regulatory mechanisms hidden in plant genomes.

Transcription factors (TFs) are fundamental components of biological regulation, facilitating the basal and differential gene expression necessary for life. TFs exert transcriptional regulation through interactions with both DNA and other TFs, ultimately influencing the action of RNA polymerase at a genomic locus. Current approaches are proficient at identification of binding site requirements for individual TFs, but few methods have been adapted to study oligomeric TF complexes. Further, many approaches that have been turned toward understanding DNA binding of TF complexes, such as electrophoretic mobility shift assays, require protein purification steps that can be burdensome or scope-limiting when considering more exhaustive experimental design. In order to address these shortfalls and to facilitate a more streamlined approach to understanding DNA binding by TF complexes, we developed the DIMR (Dynamic, Interdependent TF binding Molecular Reporter) system, a modular, synthetic yeast-based transcriptional activity reporter. As a proof of concept, we focused on the NUCLEAR FACTOR-Y (NF-Y) family of obligate heterotrimeric TFs. The DIMR system was able to reproduce the strict DNA-binding requirements of the NF-Y complex with high fidelity, including recapitulation of previously-characterized mutations in complex subunits that break either subunit interactions or DNA binding. With this model firmly established, we can directly test the effects DNA and amino acid changes have on NF-Y complex function, and have begun to address the DNA-binding impacts of an atypical conserved linker domain within a particular NF-YA subunit of Arabidopsis thaliana. The DIMR system provides a powerful, easy-to-use approach to address these and similar types of questions concerned with the binding of both monomeric and oligomeric TFs to DNA.

Approximately one-third of global CO2 fixation is performed by eukaryotic algae. Nearly all algae enhance their carbon assimilation by operating a CO2 concentrating mechanism (CCM) built around an organelle called the pyrenoid, whose protein composition is largely unknown. Here, we developed tools in the model alga Chlamydomonas reinhardtii to determine the localizations of 135 candidate CCM proteins, and physical interactors of 38 of these proteins. Our data reveal the identity of 89 pyrenoid proteins, including Rubisco-interacting proteins, photosystem I assembly factor candidates and inorganic carbon flux components. We identify three previously un-described protein layers of the pyrenoid: a plate-like layer, a mesh layer and a punctate layer. We find that the carbonic anhydrase CAH6 is in the flagella, not in the stroma that surrounds the pyrenoid as in current models. These results provide an overview of proteins operating in the eukaryotic algal CCM, a key process that drives global carbon fixation.

Metabolic processes in eukaryotic cells are highly compartmentalized into organelles. In plants, unique reactions localized in organelles carry out specific metabolic functions, such as photosynthesis and starch metabolism in the chloroplast and oxidative phosphorylation in the mitochondria. Databases such as SUBA4 and CropPAL contains protein compartmentalization data from thousands of publications on Arabidopsis thaliana and several commodity crop species, which enabled training of classifiers to predict protein subcellular localization that can guide further research. Many classifiers have been built using various machine learning algorithms, but these methods rely on protein sequences as input. Although sequence-derived features, including target signals, have been successfully identified and utilized in sequence-based classification methods, recent studies have shown that many well-accepted prediction algorithms perform poorly for chloroplast and mitochondria-targeted proteins, possibly due to limitations in sequence-derived data. Considering that each organelle can perform specific metabolic functions, our study explored the possibility of using metabolic networks to infer subcellular localization of enzymatic reactions. Our algorithm, LocPred, utilizes graph mining techniques to train and predict reaction subcellular locations with features extracted from metabolic networks. LocPred showed surprisingly high performance, with average AUC-ROC in cross-validation around 0.79, and F1, precision, and recall, that doubled the baseline. Results from LocPred indicate that metabolic network are useful in inferring subcellular localization of reactions. Based on its high accuracy in predicting mitochondrial and plastidic reactions, 0.8 and 0.81 AUC-ROC respectively, LocPred may be a novel solution to circumvent the challenges of sequence-based methods.

To tackle the challenge of producing more food and fuel with fewer inputs a variety of strategies to improve and sustain crop yields will need to be explored. These strategies may include: mining natural variation of wild crop relatives to breed crops that require less water; increasing crop temperature tolerance to expand the geographical range in which they grow; and altering the architecture of crops so they can maintain productivity while being grown more densely. These research objectives can be achieved with a variety of methodologies, but they will require both high-throughput DNA sequencing and phenotyping technologies. A major bottleneck in plant science is the ability to efficiently and non-destructively quantify plant traits (phenotypes) through time. PlantCV (http://plantcv.danforthcenter.org/) is an open-source and open development suite of image processing and analysis tools that analyzes images from visible, near-infrared, and fluorescent cameras. Here we present new PlantCV analysis tools, which includes interactive documentation, color correction, and the development of thermal and hyperspectral imaging tools aimed at the identification of early abiotic stress response.

Great advances in the field of rapid non-destructive plant phenotyping have been made recently for aerial plant parts. However, few options exist for rapid non-destructive monitoring root growth and their phenotypic characteristics under control environments or in the field. We tested the application of ground penetrating radar (GPR) to non-destructively monitor the growth of cassava storage roots. The objective of the study was to test if GPR has sufficient to estimate growing storage root biomass. Because we tested the GPR in the Cassava by Free Air CO2 Enrichment experiment (CassFACE), we expected to see differences in root biomass due to the elevated CO2 concentration treatment. In total, we screened the roots of three African cassava cultivars during 2017 and 2018 field seasons. In both years, we collected data at three different root developmental stages. Improvements in the methodology a near doubling of data collection in 2018 compared to 2017. In 2018 the above-ground portion of the plant was harvested before the GPR screening allowing the GPR antennae to be run directly over the center of plant thereby improving resolution. Destructive harvests of the storage roots were performed directly after GPR scans to calibrate the GPR determinations. Fresh and dry weight were determined, digital pictures collected, and the average density of the roots determined. Preliminary analysis, show that the GPR can detect maturing storage roots of cassava and 3D reconstructions of the roots can be rendered. Preliminary results showed strong correlations between the estimations of the weight biomass calculated with the GPR data and with the destructive harvest data. The results of this research support the application of the GPR as a useful tool to non-destructively phenotype storage roots and tubers in the field.

Grafting is an ancient agricultural practice that has been used to improve crop performance for over two-thousand years. In tomato, the grafting of elite fruit producing shoots (scions) onto vigorous, interspecific hybrid root systems significantly increases yield.  We refer to this phenomenon as “grafting-induced vigor.” Here, we present data showing that grafting-induced vigor can be reciprocally transferred between the root and shoot systems of an interspecific hybrid genotype, called Maxifort, and domesticated tomato. Using molecular (RNA-sequencing), ionomomic, and physiological profiling of reciprocally grafted hybrid and domesticated genotypes we examine the effect that grafting-induced vigor has on whole plant responses. Here, we present data showing that grafting with hybrid Maxifort has a profound effect on the gene expression patterns, macro and micro nutrient profiles, and photosynthetic efficiency of domesticated tomato root and shoot systems. Moreover, we show that many of these phenotypes that are associated with grafting-induced vigor are emergent traits that are not expressed in either of the self-grafted “parents.” By integrating these multiple levels of molecular, nutritional, and physiological characterization, we present a model for how grafting-induced vigor is manifest on the organismal scale.

The earth is at risk to enter "Hothouse Earth" conditions - a climate stabilizing at a global average of 4-5°C higher than pre-industrial temperatures with a sea level 10-60 m higher than today. Avoiding a "Hothouse Earth" requires not only reduction of carbon dioxide but also enhancement biological carbon stores. One good solution is deeper maize root systems that converts atmospheric carbon to carbon stores into the soil. For example, the 91 million acres of maize grown in US provide an estimated one gigaton of stored carbon into the soil every year. The key to deeper maize root systems is its traits hidden underground. However, genetic studies on maize root traits are hampered by the difficulty to measure the dense and occluded root architecture. Therefore, the discovery of genes associated with deeper maize rooting requires advanced root phenotyping methods. We developed an optical 3D root phenotyping system that can automatically scan the maize roots in 6 minutes Our software can reconstruct 3D root model and measure root traits, such as root system density, distributions of diameters, number of whorls, crown roots, brace roots, 1st order lateral roots, and individual root traits like root curvatures, angles and lengths can also be obtained. We validate our method on 16 maize genotypes with six replicates for each genotype. Our system paves a promising way to access previously inaccessible traits that directly relate to deeper rooting. It contributes to increase soil carbon sequestration and carbon stores, and it will help to avoid our earth entering to "Hothouse Earth" conditions.

Large-scale phenotyping experiments are conducted by collaborative teams containing individuals with knowledge and skills that sit at points along a continuum between the biological system under study and complementary disciplines offering approaches useful for studying it. Common gaps in this continuum represent places where the training pipeline can be improved. The DIVAS project (Digital Imaging and Vision Applications in Science) is an NSF-funded effort to build an ‘on-ramp’ for preparing natural scientists to engage in computational tasks, which are essential to the success of phenotyping studies. Using image data as a hook, preliminary results indicate that program interventions which include a week-long coding workshop in Python using OpenCV libraries, two-week pair programming projects, and one-credit professional development and special topics seminars are effective in improving self efficacy toward computing as well as increasing interest in pursuing additional computational skills within the participants’ careers. DIVAS program elements have been utilized by individuals ranging from high school students through faculty members. The majority of participants are undergraduate natural science majors. This training pipeline has facilitated development of a phenotyping platform used to characterize spatial variation in root exudate production in corn. Surface compounds of seedling roots are absorbed onto polyethersulfone (PES) sheets. A chemical indicator printed onto the PES surface reacts with specific adsorbed compounds to produce an observable color change. Printed standards are detected and analyzed using automated image processing routines in Python using OpenCV libraries and their intensity values used to construct a standard curve for each sheet. This standard curve is used to quantify adsorbed compounds. Images of developed PES sheets are overlaid onto seedling images to localize signals to specific root structures.

While cultivated rice (Oryza sativa) provides the primary source of nutrition for more than one-third of the world’s population, relatively little use has been made of the vast genetic diversity found in the wild species of Oryza worldwide for resistance to abiotic stresses. Salinity limits rice growth and yield, modern rice cultivars are highly sensitive to salinity, especially during early vegetative and reproductive stages. In an effort to address this problem, we evaluated accessions of O. australiansis and O. meridionalis endemic to the savannah of northern Australia. Plants were assessed at the seedling stage for their growth at sodium chloride concentrations up to 80 mM. Multiple accessions were compared with O. sativa genotypes ranging from salt sensitive (IR29) to tolerant (Pokkali). An initial greenhouse-based screening revealed substantial salt tolerance in some but not all native accessions. To validate this, non-destructive image-based phenotyping was performed at the Plant Accelerator, an Australian national plant phenotyping facility. The combination of our two screening experiments uncovered striking levels of salt tolerance diversity among the Australian wild rice accessions tested and enabled analysis of their growth responses to a range of salt levels. With the aim of understanding the mechanism underlying this tolerance, we further investigated the tolerant and sensitive accessions through protein mass spectrometry (MS). Extracted proteins were quantified by tandem mass tags and two-stage MS. Over 3000 proteins were quantified, proteins significantly differentially expressed as compared with the control treatment and between accessions. A few transporters were found to be over-expressed in the tolerant line when compared to the sensitive line. The expression of some proteins were validated using RT-qPCR. Our results highlight the potential of exotic germplasm to provide new genetic variation for rice salinity tolerance.

Calling all LGBTQ+ community members and allies - Let's meet up and decide on an offsite location to go out to, so we can get to know each other!

Here’s your chance to expand your network! Students and post-docs will have the opportunity to talk one-on-one with professors, department chairs, industry scientists, journal editors, ASPB staff, and many others. Bring questions to ask and business cards to swap. Workshop has a fee to attend and requires preregistration.

This session will include a panel and activities to address assessment on multiple levels; eg: writing exam questions, evaluating student work beyond exams, how to look at outreach events, broader impacts, curricular outcomes and student research progress. WORKSHOP FULL To be added to the wait list please email PBregistration@aspb.org.

Want to be a better mentor? This workshop will provide you with ideas and tools to improve your mentoring skills. Panel will include people from academia and industry. Workshop has a fee to attend and requires preregistration.

The workshop will feature talks on Plant related funding opportunities from each agency by Program staff and a panel discussion with Program staff at the end of all the talks. In addition to the workshop, there will be ample time to meet the Program staff at the Joint USDA, DOE and NSF Booth to discuss the funding opportunities offered by the respective agencies. Lunch may be purchased in the exhibit hall or the underground mall. No preregistration is required for this workshop. Seating is first-come, first-served.

This workshop is intended for researchers at all levels but especially those relatively new to Plant Biology who would like to learn more about the variety of tools and resources available on the web. There will be (1) an overview of different categories of resources and their uses, (2) a primer on good data management practices that facilitate easy data reuse and adherence to FAIR principles, and then (3) a series of five speakers who will illustrate use of specific online resource using real world examples. •Overview of resources (including those not giving specific talks in the workshop), FAIR data practices, community contribution opportunities : Organizers •TreeGenes, Citrus, GDR, Hardwood Genomics : Dorrie Main, Washington State University •SGN/BrAPI/Nicotiana Database : Lukas Mueller/Hartmut Foerster, Boyce Thompson Institute •Gramene : Josh Stein, CSHL •Phylogenes/TAIR : Peifen Zhang, Phoenix Bioinformatics •The Bio-Analytic Resource for Plant Biology (BAR) : Asher Pasha, University of Toronto •Plant Metabolic Network : Charles Hawkins. WORKSHOP FULL To be added to the wait list please email PBregistration@aspb.org.

This is the one opportunity for the members and all interested in the research area of Environmental and Ecological Plant Physiology (EEPP) to meet together, and share ideas about future activities of the section. Workshop requires preregistration.

Governments play a major role in supporting the scientific enterprise. For example, federal agencies award grants and contracts to researchers and educators and set policy with regard to issues such as training, scholarly Publishing, and the establishment scientific priorities. As a result, there are a diversity of career opportunities for scientists interested in policy and advocacy. Come and learn about some of those opportunities and steps you can take to pursue a career in science policy.

Join Sabeeha Merchant, Mike Blatt and Ivan Baxter for a meet & greet!

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Curious about what it's like to work beyond academia? This informal discussion will include plant scientists working outside academia. Discussion topics will include transitioning between academia and industry, international opportunities in academia and industry, and how to write a CV for an industry job.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Connect with plant phenomics researchers, learn how to join the Plant Phenomics network on Plantae, and pitch your ideas for Phenome 2020!

Come and meet your fellow AFEs from ASPB's scientific journals. Also open to any researchers interested in the Publishing process to ask questions!

Interested in plant science related startups? Come hear about the new technologies being explored by PB19's Innovation AveNEW startup companies!

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Did you miss the Commercialization in Plant Science Workshop? Or did you attend, but want to chat more? Join us for a follow-up open conversation about this hot topic.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Learn about the many ways that you can get involved with ASPB: ASPB Ambassadors, Conviron Scholars programs, ASPB committees and governance, regional sections, mentoring and other volunteer opportunities.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Open discussion for faculty members that run existing programs or those who are interested in starting a training grant program at their institution. Join us to discuss how to start a successful training program and learn more about grants, existing initatives, and T-training opportunitites.

Follow-up to Saturday’s workshop to continue discussions about computational biology and collaborating across disciplines. Open to all PB19 attendees.

Are you a current or former Plantae Fellow? Let's get together for a meet & greet!

Wondering about the implications of Plan S and funder-mandated Open Access? ASPB will share the latest.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a (formerly Cpf1) has emerged as an effective genome editing tool in plants. Previously, FnCas12a and LbCas12a, recognizing TTV and TTTV protospacer-adjacent motifs (PAMs) respectively, were demonstrated as effective genome editing reagents in plants. To expand CRISPR-Cas12a tool box, we tested a battery of Cas12a orthologs originated from different bacteria for plant genome editing. We identified 8 Cas12a orthologs that can enable efficient genome editing in rice. To develop a very efficient multiplexing Cas12a genome editing system, we comprehensively compared 11 different strategies for multiplexing in stable transgenic rice plants. The top performing system results in simultaneous and biallelic editing of every target site in rice T0 lines, reaching 100% biallelic genome editing. The newly identified Cas12a orthologs and the efficient multiplexing systems have greatly expanded plant genome editing capability.

Non-green plastids are desirable for the expression of recombinant proteins in edible plant parts to enhance the nutritional value of tubers or fruits or to deliver pharmaceuticals. However, plastid transgenes are expressed at extremely low levels in the amyloplasts of storage organs such as tubers. Here we report a regulatory system consisting of a variant of the maize RNA binding protein PPR10 and a cognate binding site upstream of a plastid transgene encoding GFP. The binding site is not recognized by the resident potato PPR10 protein, restricting GFP protein accumulation to low levels in leaves. When the PPR10 variant is expressed from the tuber-specific patatin promoter, GFP accumulated up to 1.3% of total soluble protein, a 60-fold increase over 0.02%2, the maximum protein yield achieved to date in potato tuber. This regulatory system enables an increase in transgene expression in non-photosynthetic plastids without interfering with chloroplast gene expression in leaves.

Chemically-induced expression systems are useful both commercially and academically. The tetracycline repressor (TetR) is the basis for the most robust gene switch systems in eukaryotes. However, its use in plants is impractical since the ligands are antibiotics and light sensitive. TetR binds to the tet operator in a promoter, repressing expression of any gene regulated by this promoter. Binding of the ligand tetracycline to TetR creates a conformational change in the repressor, releasing it from the tet operator allowing transcription of the de-repressed gene to occur. Several rounds of gene shuffling of the tetracycline repressor (TetR) gene created mutants that bind sulfonylurea (SU) herbicides instead of tetracycline, creating an Ethametsulfuron Repressor (ESR). This ESR system was tested by de-repressing the fluorescent reporter DsRED in maize. ESR de-repression of transcription factors WUSCHEL and BABYBOOM in rice increased re-transformation and shoot formation frequencies. De-repression of WUSCHEL and BABYBOOM in maize led to differential expression of several genes involved in embryogenesis, cell cycle, and homologous recombination.

Access to elite, transformable germplasm is required to design and maintain transformation pipelines. Product pipelines typically use transformable germplasm to initially introduce the novel variant, which is then crossed into broad, diverse germplasm lines relevant to the geographies where the product will be grown. Effective transformation pipelines are valuable for product development in the Ag industry but are also important for serving the scientific community by enabling basic science research through gene and pathway discovery and characterization. Bayer Crop Science, in collaboration with NRGene and the University of Wisconsin, reports the release of the LH244 inbred maize transformation line germplasm and assembled reference genome to academic research communities. The germplasm will be released to public seed stock centers and the assembled, annotated genome and a physiological description of the line will be published, and resources for efficient transformation will be available to the University of Wisconsin Crop Innovation Center. LH244 is a commercially relevant inbred line that is readily transformable, thus making it a complete resource for genomic and genetic exploration. In this talk, we will share insights into the unique features of the LH244 genome, transformability and physiology that make it a foundation resource for the maize genetics community.

Here we report the implementation of a rapid and efficient Agrobacterium-mediated site-specific integration (SSI) system for maize product development. A total of 163 DNA constructs each containing 1-3 trait gene cassettes were inserted into SSI landing sites at the same genomic location in two elite maize inbreds. Over 7500 SSI events were regenerated to the plantlet stage with a transformation efficiency of ~7%. Characterization of the SSI event structure in these regenerated plants by qPCR identified 58% contained the desired single-copy DNA insertion at the targeted site and with no detectable vector backbone sequence transferred. The transformation efficiency across donor constructs with 1, 2 and 3 trait gene cassettes was similar. Through further process optimization on one inbred, the transformation efficiency was increased from 9.1% to 11.7% when the transformation procedure was truncated from three to two selection steps. This reduced the duration of transformation from embryo infection to the transfer of a plantlet to soil from 115 days to 96 days. In summary, this project has demonstrated reliable Agrobacterium-mediated site-specific integration transformation technology in maize inbreds.

Genetic screens with traditional mutagens such as EMS, fast neutrons, or T-DNA insertions have been used successfully for decades. However, these methods are limited by the time needed for self- and cross-pollinations, additional mutations caused by the mutagen and the laborious process of generating higher-order mutants. With the advance of CRISPR genome editing, the production of targeted gene knockouts is specific, scalable, and can be easily multiplexed. This has enabled us and others to perform in planta pooled CRISPR screens in tomato and rice. We are now actively developing and optimizing this technology, taking advantage of the multiplexing capability of CRISPR, to perform combinatorial knockout screens using Arabidopsis as a model system. This approach allows us to rapidly generate targeted mutant collections in practically any genotype or marker line of interest and potentially overcome genetic redundancy by targeting multiple members of a gene family or pathway. Here, we report the generation and use of a knockout mutant collection targeting ~20% of the proteases in Arabidopsis. For relatively small gene families (~20-40 genes) targeted by six gRNAs per vector, we can recover mutants in each individual gene and pairwise-gene combinations as well as a majority of the three-gene combinations with only a few hundred T1 plants. In the T1 and T2 generations, we consistently observed phenotypes such as leaf bleaching, dwarfism, seedling lethality and other developmental defects and rapidly associated these phenotypes with the incorporated gRNAs and mutated genes. Further refinements of this technology will utilize automated phenotyping platforms, generate even higher order combinations, and streamline the bioinformatics analysis.

Plants continually face a multitude of stresses that can severely compromise their ability to survive through the growing season. To mitigate the effects of a harsh environment, plants have evolved various developmental and physiological strategies through which they can effectively balance growth and defense against stressors. Arabidopsis XERICO (XER) is a stress-responsive putative RING E3 ubiquitin ligase that increases intracellular abscisic acid levels and promotes drought tolerance. To elucidate the molecular mechanism of XER induction by stress and better understand its role in regulating growth and stress responses, we began by analyzing its gene expression pattern and determined its protein subcellular localization. XER transcript is expressed throughout developing seedlings and its protein localizes to the endoplasmic reticulum in vivo. To isolate potential XER upstream regulators involved in stress signaling, we conducted a yeast one-hybrid screen. Our results demonstrate that one of the regulators identified from the screen, a member of the DREB class of transcription factors, is particularly important for repressing XER expression during osmotic stress. Lastly, we have shown that transgenic Arabidopsis plants with altered XER and XER RING-inactive variant levels exhibit different degrees of sensitivity to drought, and this may be linked to XER’s previously unknown role in modulating stomatal density. Taken together, we propose that XER expression is tightly regulated following initial stress perception to control ABA accumulation and stomatal development, ultimately ensuring survival during unfavourable growth conditions such as drought.

As plant roots grow through the soil they are faced by a variety of external signals such as heterogeneous water and nutrient availability. Plants are able to sense these signals and respond by altering root development to ensure roots continue to grow into areas where water and nutrients are present. My PhD has aimed to improve our understanding of how roots sense and respond to changes in water availability in soil. Using X-ray computed tomography (CT) scanning I have been able to non-destructively visualise root systems within soil and have observed that changes in water availability alter the branching pattern of roots. If water availability is higher on one side of the primary root than the other then branches will only form towards the wet side, a response called hydropatterning, and if the root experiences a temporary water deficit branching will stop completely, a response called xerobranching. Both are striking responses that illustrate how sensitive root development is to variations in water in the soil. In order to understand how water flow into the root may signal these developmental changes Arabidopsis thaliana knock out lines in water channels have been tested. Knocking out plasmodesmata related proteins can disrupt the hydropatterning response, suggesting water movement through the plasmodesmata is a necessity to pattern root branching in response to water. We have also modeled water movement during hydropatterning using the Model of Explicit Cross-section Hydraulic Architecture (MECHA) which highlights the importance of asymmetrical water movement during this response. These results provide interesting insights as to how water could be sensed during root growth.

The National Center for Environmental Information reported a loss of 236.6 billion U.S. dollars due to drought disaster from 1980 to 2018. Total annual crop damage from drought in the U.S. has been estimated at $6 to $8 billion. Therefore, identification of genetic mechanisms for tolerating periods of drought is critical to sustaining crop production. This research was initiated in 2018 to develop a high-throughput protocol for phenotyping, using aquaporin inhibitor, soybeans for expression of the limited transpiration (TRlim) trait under high vapor pressure deficit (VPD) leading to identification drought tolerant soybeans in the upper mid-south. The advantage of the TRlim trait is that it allows plant water conservation to increase soil water availability for use during late-season drought. A soybean population of 92 recombinant inbred lines (RIL) derived from the mating of Jackson× KS4895 was tested through applying 200 μM silver nitrate to de-rooted soybean shoots under high VPD. Among 92 RILs, half of the population showed higher sensitivity to silver ion than KS4895 (a parent line). However, several lines showed no or limited changes in water loss with the silver treatment. These lines may be candidates for a direct measurement of plant water loss under different humidity levels. A high correlation (R2=0.44) between leaf temperatures (LT) and normalized decrease in transpiration rate (NDTR) was found for 64 lines out of 92 RILs. Furthermore, a preliminary genetic mapping for LT and NDTR, examining obtained controlled environmental data with an older genetic marker dataset (548 markers, 95 RILs) using the Jackson×KS4895 RIL population was performed. Even with an incomplete dataset (experiment in process), a previously reported major-effect QTL for the NDTR trait on chromosome 19 was identified. The RILs experssing low NDTR should be studied further under field conditions for evaluation as potential drought tolerant parents in a breeding effort.

Group VII ERFs play a pivotal role in plant response against submergence stress and are regulated by N-end rule proteolysis pathway which is believed to be the oxygen sensing mechanism. Arabidopsis ERFVIIs are degraded via N-end rule pathway under normoxia, but are stabilized under hypoxia to trigger downstream responses. In rice, Sub1A-1 is a member of the group VII ERFs and confers the majority of submergence tolerance to rice. However, despite having the canonical N-degron sequence, SUB1A-1 is able to evade N-end rule protease degradation under normoxia. Besides, Sub1A-1 is reported to response to several stresses, including dehydration or prolonged darkness. This raised an interesting question of how rice senses low oxygen stress from the others. We found that the other two ERFVIIs, ERF66 and ERF67, are regulated by the N-end rule pathway and that ERF66 and ERF67 genes are transcriptionally regulated by SUB1A-1 as well. Our studies showed that Sub1A-1 and ERF66/ERF67 form a regulatory cascade in response to submergence stress in rice. SUB1A-1 is the only known ERFVII protein escaping from the N-end rule pathway by far, so it is interesting to know how SUB1A-1 escapes the N-end rule proteolysis degradation. Our preliminary results suggest that the C-terminus of SUB1A-1 is important for escaping. With sequential truncation of C-terminus of SUB1A-1, we identify a segment of the C-terminus of SUB1A-1 that is crucial to the escaping.

EPICON research focuses on drought, important due to the increased frequency and severity of this abiotic stress with climate change. Both transcriptomic and epigenetic changes play major roles in regulating drought responses. EPICON’s cohesive, high-resolution transcriptomic study was on sorghum [Sorghum bicolor (L.) Moench], a C4 cereal crop, noted for drought tolerance. This large-scale, multi-year field experiment explores spatiotemporal responses under fully irrigated and two different drought stress regimes in replicate plots of two sorghum genotypes, differing in pre- and post-flowering drought responses. Drought was imposed in fields in California’s Central Valley, where rare summer rainfall permits controlled drought conditions. Leaf and root samples were taken weekly over the plant’s lifetime with the goal of understanding mechanisms functioning in acclimation to and recovery from pre- and post-flowering drought, using RNA-Seq, BS-Seq, proteomics, metabolomics, and histone profiling. A resulting data set of over 350 transcriptomes revealed 44% of expressed genes being significantly affected by drought. Roots showed greater transcriptional disruptions than leaves; samples from pre-flowering drought had more complex temporal changes than post-flowering drought; large differences were found between genotypes. To gain additional insights into drought responses, impacts of this abiotic stress were also studied in microbial populations, using shotgun metagenomics, metatranscriptomics, and metabolomics. Composition of fungal and bacterial communities, associated with these same plants, led to additional comprehensive data resources that will be available to the community to unravel the complex drought responses of plants and their field-associated microbial communities. Cumulative data is being used to devise models to better predict and control roles and interactions of transcriptional regulation, epigenetics and the microbiome in sorghum’s response to drought.

The plant hormone abscisic acid (ABA) plays a critical role in drought resistance responses. The core signaling components are snf1-related protein kinases (SnRK2s) which are activated by ABA-dependent inhibition of type 2C protein phosphatases (PP2Cs). Activation of SnRK2 protein kinases requires phosphorylation of the SnRK2 kinases themselves. It remains unclear whether the activation of SnRK2s is mediated by auto-phosphorylation or by other protein kinases and associated proteins in planta. Through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant protein kinases that phosphorylate and activate the OST1/SnRK2 kinases in Arabidopsis. Mass-spectrometry revealed a specific trans-phosphorylation site in OST1/SnRK2.6 that is targeted by these kinases and required for strong SnRK2 activation. Reconstitution of full ABA-induced OST1/SnRK2.6 activation and S-type anion channel activation require these kinases, suggesting that they are a new member of the early ABA signaling core. Higher-order knock-out plants show not only reduced sensitivity to ABA but also strongly impaired osmotic stress-induced SnRK2 activation. Our results demonstrate that these protein kinases are required for ABA- and osmotic stress signaling through activation of SnRK2 kinases.

Maize is an important genetic model for elucidating transcriptional networks. Haplotype phasing of genetic variants is important for interpretation of the maize genome, population genetic analysis and functional genomic analysis of allelic activity. Recently, full-length transcript sequencing using long read technology has enabled us to characterize alternative splicing events and improve the maize genome annotation. However, the general Iso-Seq algorithm ignores SNP-level information, focusing instead on identifying alternative splicing differences. Here, we present an algorithm called IsoPhase that post-processes Iso-Seq data for transcript-based haplotyping. For each gene, IsoPhase gathers the associated full-length reads, each representing a single transcribed molecule. It then calls SNPs and is able to infer the haplotype of the reads due to the full-length nature of the sequencing. We applied IsoPhase to a maize Iso-Seq dataset consisting of two homozygous parents (B73 and Ki11) and two F1 reciprocal hybrids (B73xKi11, Ki11xB73). We validated the majority of the SNPs called with IsoPhase against matching short read data and identified cases of allele-specific, gene-level and isoform-level expression. Our results show that maize parental lines and hybrid lines display different splicing activity, and 6,847 genes can be phased through IsoPhase in two reciprocal hybrids using embryo, endosperm and root tissues. Our study identified parental origin isoforms in maize hybrids, different novel isoforms between maize parent and hybrid lines, provides measures of haplotypic expression that increase power and accuracy in studies of allelic expression. It is the first study of phased full-length isoforms in maize, as well as in plants, which provides insights about maize and plant heterosis at allele-specific full-length transcriptional level. The approach used in this study also provide important information for many other phasing studies in different species.

Plants produce diverse metabolites in metabolic pathways important for not only plant survival but also human nutrition and medicine. Some genes in the same pathway can be identified based on their correlated expression profiles. However, pathway gene co-expression may be only under specific spatiotemporal and conditional contexts, and the coexpression relations can be non-linear. Here, we develop a supervised machine learning approach to maximize the utility of gene expression data for predicting pathway gene memberships by considering 656 combinations of datasets, and linear/non-linear co-expression measures, using tomato as a model. With each combination dataset, we established a multi-class model to predict whether a gene belongs to one of the 85 pathways (classes) and evaluated model performance with the F1 score (range from 0 to 1, where 1 indicates perfect predictions). Among 656 models, the best overall model (i.e. have highest average F1 across 85 pathways) has significantly improved performance in predicting pathway memberships (average F1=0.34) compared to random guess (F1=0.008). By identifying which of the 656 models has the best membership prediction for each pathway, we uncovered optimal pathway models that have an even higher average F1 of 0.83, where 26% pathways are predicted perfectly. The optimal pathway models also have much better performance compared to those based on the best cluster (from unsupervised learning) that maximize F1 for each of 85 pathways (average F1=0.45), indicating the importance of modeling training with supervised learning. Our study highlights the need to extensively explore expression features to build models that can maximize the utility of expression data for pinpointing pathway membership. Through this detailed exploration, novel connections between pathways and biological processes can also be identified based the optimal expression dataset used, improving our mechanistic understanding of the metabolic network.

Transposable elements (TEs) play a major role in genome evolution because of their abundance and ability to induce changes to the genome. While there is ample evidence that host organisms employ mechanisms to regulate TEs, we hypothesize that TEs also rely on self-imposed regulatory mechanisms to prevent uncontrolled transposition that would lead to lethal genome damage. Revealing the mechanisms of self-regulation is critical to understanding how TEs evade host silencing and maintain host survival. The mPing DNA element from rice is a nonautonomous member of the PIF/Harbinger transposable element superfamily and is mobilized by the ORF1 and TPase proteins encoded by the Ping element. Analysis of mPing’s transposition mechanism in a yeast assay suggests that it exhibits self-regulatory mechanisms. Alteration of ORF1, TPase, and the terminal inverted repeats (TIRs) required for mobilization results in hyperactive rates of transposition. This suggests that protein localization and binding characteristics have been selected for relatively low efficiency. Evidence for the importance of low mobility elements in the evolutionary history of TEs was recently provided by analysis of the mPing and Ping elements from 3000 rice cultivars and their wild ancestor Oryza rufipogon. This study suggested that the pairing of the highly mobile mPing element with an extremely low mobility version of Ping facilitated the burst of mPing copy number observed in some rice cultivars. Together this indicates that low mobility actually allows some elements to evade host silencing mechanisms, increasing their overall fitness for long term coevolution.

While prokaryotic pan-genomes have been shown to contain many more genes than any individual organism, the prevalence and functional significance of eukaryotic pan-genomes remain poorly understood. Whole-genome de novo assembly and annotation of 54 lines of the grass Brachypodium distachyon yielded a pan-genome containing nearly twice the number of genes found in any individual genome. Genes present in all lines are enriched for essential biological functions, while genes present in only some lines are enriched for conditionally beneficial functions (e.g. defense and development), display faster evolutionary rates and lie closer to transposable elements. Our data suggest that differentially present genes contribute substantially to phenotypic variation within eukaryotic species. In addition, the pan-genome provides a new lens though which we can examine genome evolution in polyploid species by enabling us to differentiate between polymorphisms that evolved after polyploidization from those that were part of the standing variation in the diploid progenitors. To explore this, we sequenced multiple lines of an allopolyploid B. hybridum and used a pan-genomic approach to study the sub-genome that was derived from B. distachyon (D subgenome). Surprisingly, the vast majority of whole gene presence/absence variation in B. hybridum was part of the standing variation in B. distachyon. Analysis of nuclear SNPs, plastomes and k-mers revealed two independent origins for B. hybridum, ~1.4 and ~0.14 million years ago, creating a natural timecourse of polyploid genome evolution. Our analysis is consistent with a gradual accumulation of genomic changes in the polyploid lineages and an absence of sudden changes in sequence or expression. Significantly, had we compared a single reference genome for each species rather than using a pan-genomic approach, we would have grossly overestimated post-polyploidization evolution.

Since 1999, The Arabidopsis Information Resource (www.arabidopsis.org) has been curating data about the Arabidopsis thaliana genome. Our primary focus is on extracting experimental gene function information from the literature and codifying it as Gene Ontology (GO) and Plant Ontology (PO) annotations. Our goal is to produce a fully annotated, ‘gold standard’ functional annotation set that reflects the current state of knowledge about Arabidopsis genome. We used the set of GO annotations as a metric to evaluate the historical and current state of functional annotation and determine: (1) how the annotation landscape has changed over the past 20 years, (2) what is currently known about Arabidopsis gene function, and (3) the set of ‘unknown’ genes. Within TAIR, the extent and accuracy of annotation coverage has improved over time as new data is curated and lower quality information is removed. This is significant because these changes can impact the analysis and interpretation of experimental data, including gene set enrichment using GO classifications, and the transfer of gene function annotations to other species from Arabidopsis. To assess progress towards achieving our gold standard annotation, we evaluated both GO annotation status and evidence. Overall, 74% of the genome has been annotated to at least one GO term. Of those loci, half have experimental support for one or more GO aspect (e.g. molecular function, process or component). Our work also sheds light on the smaller but still significant portion of the genome for which we have not yet identified any published experimental data and for which we have no functional annotation. We anticipate that drawing attention to this set of unknown genes will bring into focus to the gaps in our knowledge and potential sources of interesting and novel discoveries.

Biological research is becoming increasingly data-driven. Data from funded research, when made FAIR (Findable, Accessible, Interoperable, Reusable), often becomes invaluable for further research. Making data from all publicly funded research available, however, requires authentic, detailed, accurate and explicit communication between all parties involved in generating and delivering the scientific data. In addition, making data FAIR requires development of effective methods, tools and resources, a goal made more achievable when biological database resources collaborate. The AgBioData consortium (https://www.agbiodata.org) formed in 2015 consists of over 150 database scientists from 30 plus genomic, genetic and breeding (GGB) databases and allied resources. Collectively, the AgBioData member databases served 27 million pages and 950,000 users in 2017, and between 2012-2017, they were cited in over 24,000 publications. The databases cover an extensive range of crops, livestock and model organisms, including arabidopsis, corn, wheat, legumes, fruits and nuts, vegetables, insects, cattle, chicken, fish, horses, pigs, and sheep. To move closer to making every piece of biological data available to researchers through organized, easy-to-find and use resources, we need to continue to work together to adopt a common set of metadata, and associate more data with ontologies; make it easy to share data; share curation practices; and provide solutions for long-term funding for all genomic, genetic and breeding databases. AgBioData is a model for how databases can work together to be more resource-efficient and use a collective voice to lead efforts for better data management and database resource availability.

Fungi produce an array of structurally diverse specialized metabolites including terpenes. The fungus Sclerotinia homoeocarpa is a widespread fungal pathogen, notable for being the causal agent of dollar spot in turf grass. However, it is also known to produce nortetralabdane diterpenoids that exhibit antibacterial and insecticidal activity. The biological functions of the majority of the fungal metabolites are unknown but are assumed to be an adaptation of the fungus to its biological niche. Regardless of the role, they have a diverse and useful chemistry. Our objective is to evaluate the terpenome of S. homoeocarpa with an emphasis on deciphering the biosynthetic pathway of the nortetralabdane diterpenoids. Through transcriptome analysis, we identified three terpene synthases and associated downstream genes. Biochemical characterization reveals that ShTPS1 that produces C20 pimara-8, 14-diene scaffold that we perceive to be one of the precursor scaffolds to tetranorditerpenoids described in S. homoeocarpa. Interestingly, we have also identified a promiscuous sesterterpene- C25 chimeric cyclase (ShTPS2) that harbors both the prenyl and terpene synthase and produces six metabolites. We are carrying out structure function analysis of ShTPS2 to further understand the basis for this promiscuity. The third enzyme is yet to be characterized. Phylogenetic analysis of terpene synthase genes in S. homeocoarpa compared to related mold causing fungi Sclerotinia indicate that the fungi possess genes for the production of terpene synthases with varying numbers. This study highlights the enzymatic diversity of the terpene scaffolds in S. homoeocarpa and provides foundational knowledge in evaluating the phylogenetic conservation of terpene biosynthesis across this clade of fungal pathogens.

Plants produce diverse secondary metabolites. Although each metabolite is made through its own biosynthetic pathway, plants coordinate multiple biosynthetic pathways simultaneously. Recent studies have shown a crosstalk between glucosinolate metabolism and phenylpropanoid production. Glucosinolates are defense compounds made from various amino acids. Phenylpropanoids such as lignin are made from phenylalanine through the phenylpropanoid pathway. The study with Arabidopsis mutants having a defect in the indoleglucosinolate biosynthetic enzyme revealed that the accumulation of indole-3-acetaldoxime (IAOx) or its derivatives affects the phenylpropanoid production. The mechanism behind the crosstalk involves increased expression of genes encoding F-Box proteins responsible for the degradation of phenylalanine ammonia lyase (PAL) which functions at the entry point of the phenylpropanoid pathway. Given that aldoximes are precursors of various compounds in addition to glucosinolates and the phenylpropanoid pathway is present in most plants, it is possible that this mechanism is conserved throughout plant kingdom. We examined the impact of aldoxime metabolism on phenylpropanoid production in Camelina sativa by overexpressing Arabidopsis thaliana CYP79B2 which encodes IAOx producing enzyme. We found that overexpression of AtCYP79B2 affects plant growth and phenylpropanoid metabolism. The transgenic plants display characteristic high auxin morphology and reduced phenylpropanoid contents and PAL activity. From phylogenetic study, we identified a total of 459 non-redundant proteins containing kelch-motif(s) in Camelina sativa and found that the expression of a set of KFBs involving in PAL degradation is increased in the transgenic lines. The results suggest that the aldoxime accumulation negatively influences on the phenylpropanoid production through the transcriptional activation of KFBs responsible for PAL degradation in Camelina sativa.

Because of its importance for plant vascular function and stress responses, and the economics of the food, paper, pulp, and biorefining industries, the biosynthesis of the cell wall polymer lignin is one of the most intensively studied areas of plant biochemistry. Lignin biosynthesis is evolutionarily conserved among higher plants and features a critical 3-hydroxylation reaction involving phenolic esters. However, increasing evidence questions the involvement of a single pathway to control lignin formation in vascular plants. Here we describe an enzyme catalyzing the direct 3-hydroxylation of 4-coumarate to caffeate in lignin biosynthesis as a bifunctional peroxidase that oxidizes both ascorbate and 4-coumarate at comparable rates. A combination of biochemical and genetic evidence in the model plants Brachypodium distachyon and Arabidopsis thaliana supports a role for this coumarate 3-hydroxylase (C3H) in the early steps of lignin biosynthesis. The subsequent efficient O-methylation of caffeate to ferulate in grasses is substantiated by in vivo biochemical assays. Our results identify C3H as the only non-membrane bound hydroxylase in the lignin pathway and revise the currently accepted models of lignin biosynthesis, suggesting new gene targets to improve forage and bioenergy crops.

Glandular trichomes of the Solanaceae species produce protective acylsugars for insect defense. These specialized metabolites are mixtures of sugar aliphatic esters with acyl chains varying in carbon numbers and branching patterns. We observe phylogenetically-associated variation in acyl chain length across the family: Nicotiana, Petunia and Salpiglossis species accumulate acylsugars with short acyl chains (carbon number, C≤8), whereas species of Solanum and other close genera make acylsugars with long acyl chains (C≥10). We identified a gene cluster in tomato with tandem duplications of BAHD acyltransferase, acyl-CoA synthetase (ACS), and enoyl-CoA hydratase (ECH) genes. CRISPR-Cas9 ablation of two trichome expressed genes in the cluster (ACS30 or ECH80) affects long chain containing acylsugar in the cultivated tomato. This is likely by disrupting acyl-CoA metabolism: these are the donor substrates for acylsugar biosynthesis. ACS30 and ECH80 were co-opted from primary metabolism and are targeted to the mitochondria. This contrasts with the well-studied ACSs or ECHs, which generate long chain acyl-CoAs from lipid biosynthesis (mainly in chloroplasts) or fatty acid breakdown (mainly in peroxisomes). Syntenic analysis of this gene cluster in the tomato genome revealed a homologous region on chromosome 12, which encodes SlASAT1 – the core acylsugar biosynthetic enzyme. Comparative genomic analysis led to evolutionary reconstruction of the gene cluster across the family. Gene duplication, gene transposition, and pseudogenization facilitated emergence of this gene cluster in the Solanum. These events presumably shaped the phylogenetically-restricted distribution of long chain containing acylsugars in the Solanaceae. Analysis of this system is providing insights into evolution of specialized metabolism by co-option of primary metabolic enzymes, emergence of cell type specific gene expression and furthering our understanding of mechanisms by which gene clustering arises.

Duplication and divergence of essential primary pathway genes underlay the evolutionary expansion of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defense pathways often remain speculative. For example, the primary pathway precursor ent-kaurene is required for gibberellin biosynthesis and is likewise a proposed intermediate for maize kauralexin antibiotics. By integrating transcriptional co-regulation patterns, Genome Wide Association Mapping Studies, combinatorial enzyme assays, proteomics and targeted mutant analysis, we show that maize kauralexin biosynthesis instead proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous Cytochrome P450s and a novel steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required forFusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics that avoids large-scale production of growth hormone precursors during pathogen defense.

Plants deploy specialized metabolites to communicate with other organisms and cope with environmental challenges. For instance, diterpenoid metabolites serve as key components of biotic and abiotic defenses in major crops such as rice and maize. Here, we report the discovery and functional characterization of a novel group of bioactive diterpenoids, termed dolabralexins, that occur perhaps uniquely in maize. Patterns of inducible dolabralexin accumulation in maize roots exposed to fungal pathogens or abiotic stress suggest broad relevant of dolabralexins in below-ground defenses. Furthermore, we show maize root diterpenes to selectively alter the root microbiome composition. Combining pathway engineering and bioassays of purified compounds, we demonstrate that dolabralexins exhibit potent and species-specific antibiotic activity against major Fusarium pathogens. These results and gene resources can provide new targets for breeding and engineering of crop resistance traits in maize and other crops, as well as the manufacture of useful biocides.

There is increasing interest in the plant microbiome, both as it relates to plant health and how it can impact upon agricultural sustainability. One key unanswered question is whether we can develop a plant microbiome that would be stable against invasion after colonization of target plant hosts. To address this question, we set out to select for a well-adapted tomato phyllosphere microbiome using a multi-generational passaging experiment. Beginning with a highly diverse microbial community generated from field-grown tomato plants, we inoculated six replicate plants across five different plant genotypes for four eight-week long passages, sequencing the microbial community at each passage. We observed consistent shifts in both the bacterial (16S amplicon sequencing) and fungal (ITS amplicon sequencing) communities across replicates over time, as well as a general loss of diversity over the course of the experiment. However, using a ‘community crashing’ experiment, we found that bacterial communities from the end of the experiment were not invadable by bacterial communities from the start of the experiment. These results highlight that evolving a stable and well-adapted microbiome to a particular plant/environment is indeed possible, highlighting the great potential of this approach in agriculture.

Plants and animals recognise conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signalling is understood, we know little on the release of immunogenic fragments. Here we discovered that plant-secreted β-galactosidase-1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O-glycan. In counter defence, P. syringae pathovars evade host immunity by using BGAL1-resistant O-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.

The ascomycete fungus Fusarium graminearum not only infects wheat to cause Fusarium head blight and seedling blight, but also infects maize to cause Gibberella stalk rot. In addition, it can also grow in axenic media. To comprehensively understand how F. graminearum invades various hosts, various tissues and causes different diseases, we take an approach of cellular tracking and gene profiling of fungal infection process. We tracked F. graminearum growth inside host plants during disease development, and found fungal growth inside hosts has different paths: intercellularly and intracellularly. Using laser microdissection, we profiled in planta fungal gene expression during wheat seedling coleoptile infection and during maize stalk infection in a stage-specific manner, and started to elucidate its molecular strategies in confronting the host environments. In wheat infection, we identified that a nonribosomal peptide, as a product of secondary metabolite biosynthesis cluster of F. graminearum, facilitates cell-to-cell invasion in wheat, probably through manipulating plasmodesmal permeability and/or chloroplast activity. In maize stalk disease progression, we showed that F. graminearum remodels membrane lipids to overcome the phosphate limitation in the intercellular space of maize stalks.

Resistance (R) genes have principal functions in plant defense against pathogens and pests. The RESISTANCE AGAINST POWDERY MILDEW8 (RPW8) locus in Arabidopsis thaliana accession Moscow-0 (Ms-0) contains two non-canonical R genes RPW8.1 and RPW8.2 that confer resistance against powdery mildew disease caused by Golovinomyces spp. In the accession Columbia (Col-0), which is susceptible to powdery mildew, this locus contains the RESISTANT TO MYZUS PERSICAE (RMP) instead of RPW8.1 and RPW8.2. We find that RMP contributes to basal resistance against green peach aphid (GPA; Myzus persicae Sülzer), which is an important pest of a wide variety of plants from over 50 families. In no-choice assay, which monitors the combined effects of antixenosis and antibiosis, the GPA colonization was greater on the rmp mutant than on the wild type (WT). Aphid fecundity was significantly higher on the rmp mutant than on the WT plants. Artificial diet assays demonstrated that phloem sap-enriched petiole exudates collected from the rmp mutant accumulate lower levels of an antibiosis activity against the GPA, which is correlated with the increased fecundity of GPA on the rmp mutant compared to the WT plants. When given a choice GPA preferred the rmp mutant than the WT plants. Similarly, dispersal assays indicate that in comparison to the WT plants, emigration of GPA from rmp mutant was reduced, thus indicating that the insect prefers to stay on rmp plants compared to the WT plant. RMP is required for turning on premature senescence and cell-death in response to extracts derived from the GPA. We hypothesize that RMP is required for response to the aphid-derived elicitors of plant defense. Future efforts are directed towards understanding the mechanism underlying RMP’s function in defense against the GPA.

Generation of reactive oxygen species (ROS) is a critical component for innate immune responses against pathogen infection. The Arabidopsis NADPH oxidase RBOHD is a key ROS producing enzyme in response to pathogen perception. Excessive ROS production of can negatively impact host development and viability, thus ROS production must be tightly regulated at a resting state. Here we show that RBOHD’s stability is reduced by the receptor-like protein kinase PBL13. PBL13 phosphorylates the C-terminus of RBOHD at six conserved residues in vitro. Genetic and biochemical analyses revealed that two PBL13 phosphorylation sites are required for RBOHD activity and stability. Mimicking phosphorylation of RBOHD by PBL13 enhances ubiquitination of RBOHD in vivo. Using protein array technology, we identified a novel E3 ubiquitin ligase PIRE (PBL13 interacting RING domain E3 ligase) that interacts with both PBL13 and RBOHD. PIRE specifically ubiquitinates RBOHD but not PBL13. PIRE and PBL13 mutants display enhanced RBOHD abundance, elevated ROS production and enhanced resistance to bacterial infection. Taken together, our study provides valuable insights on post-translational mechanisms involved in ROS production through regulating the stability of a conserved NADPH oxidase during host-pathogen interaction.

Cyst nematodes are soil-dwelling parasites that substantially reduce yields of many crops. They establish feeding sites deep within the root vasculature tissue and divert nutrients from the host plant to serve their own needs. To better understand this host-parasite relationship, wheat roots were inoculated with cereal cyst nematodes (CCN, Heterodera avenae) and infected root tissue was examined using confocal microscopy. To support this, we developed methods to obtain high-quality three-dimensional images of thick (up to 150 μm) sections of root tissue. This provided unprecedentedly clear views of the feeding sites and surrounding tissues. Surprisingly, segments of the central metaxylem (cMX) vessels near the feeding sites looked very different from the expected narrow hollow tubes. In the atypical cMX segments, individual elements were short and plump rather than long, narrow and cylindrical. We determined that during a period of 15 days in which cMX vessel elements would normally elongate and then mature to form a hollow tube, cMX vessel elements near CCN infection sites do not elongate. Instead they grow radially, becoming plump. Their outer walls undergo secondary thickening and not all walls between elements degrade. It is still not clear whether this anatomical change benefits the parasite or is part of the host’s defense (or both). Our findings raise new questions about how these developmental changes are induced and how the host plant survives the blockage of what should be a major conduit for transporting water and nutrients from root to shoot.

Trehalose 6-phosphate (Tre6P) is an essential signaling metabolite that links plant growth and development to carbon status. Tre6P is synthesized from UDP-glucose and glucose 6-phosphate by trehalose 6-phosphate synthase (TPS) and then dephosphorylated to trehalose by trehalose 6-phosphate phosphatase (TPP). Under the current working model, Tre6P is thought to act as both a positive and negative feedback regulator of sucrose levels in plants. This hypothesis is encapsulated by the Tre6P-sucrose nexus model, which states that the Tre6P:sucrose ratio is highly adaptable to the specific needs of different tissues, developmental stages and environmental conditions experienced by the plant. Although it has been shown that the Tre6P:sucrose ratio is a critical parameter for plants, an understanding of how this ratio is established has yet to be elucidated. In comparison to the TPS protein family, in which TPS1 is the predominant Tre6P synthesizing enzyme, it was demonstrated that all ten TPP proteins in Arabidopsis have phosphatase activity. As the TPP gene family expanded exclusively through genome duplication events (with eight out of ten genes being paired paralogs), such a high degree of paralog retention could indicate a greater regulatory role of these proteins. In addition, TPPs exhibit diverse and specific spatio-temporal expression patterns and may be inhibited by sucrose. Combing these data, we hypothesize that the Tre6P:sucrose ratio is determined by the specific TPP isoform expressed. To test this hypothesis, we first performed kinetic analyses of heterologously expressed TPPs in E. coli and are currently attempting to alter the Tre6P:sucrose ratio in Arabidopsis tissues by replacing the natively expressed TPP protein with an alternative TPP that has different kinetic properties. Findings from this study could provide new insight into the molecular mechanisms of Tre6P signaling in plants and provide new targets to develop plants with an altered carbon economy.

Parasitic angiosperms directly attach to host plants using specialized organs known as haustoria, which function as physiological bridges to extract nutrients and water from their hosts. Cuscuta species (dodders) are common and agriculturally destructive flowering stem parasitic plants. Reports have shown a 50–72% reduction in tomato yield when attacked by dodders. The physiological connection between host plants and parasites makes traditional herbicides and control methods ineffective. The Heinz hybrid cultivars H9492 and H9553 exhibit resistance to dodders. The stem cortex in these lines responds with local lignification upon C. campestris attachment causing the C. campestris strand to fall off. To identify the key resistant genes, we focused on genes that have different expression patterns under C. campestris infestation in the resistant cultivars, compared to susceptible cultivars. Based on these criteria, we identified an AP2-like transcription factor, MYB55, and CC-NBS-LRR as key resistant genes. The transient overexpression of MYB55 and AP2-like induced stem lignification in the susceptible cultivar. These results suggest that MYB55 and AP2-like may directly regulate the biosynthesis of lignin in the cortex. Therefore, we termed this AP2-like protein as LRF1 (Lignin-based resistance factor 1). On the other hand, overexpression of this CC-NBS-LRR only induced lignification upon C. campestris attachments. This result indicates that this CC-NBS-LRR functions as a receptor for receiving C. campestris signals, thereby leading to the lignification-based resistance. Thus, we named it CuLiRR1 (Cuscuta-induced lignin-based resistance receptor). We also identified a transcription factor WRKY16 as a negative regulator of the lignin-based resistance. WRKY16 CRISPRed plants also induced lignification in the cortex and became more resistant to C. campestris. The results of this study provide the starting point for developing a parasitic plant-resistant system in crops.

Stomatal apertures regulate gas exchange and water loss in response to environmental cues in plants. Both elevated [CO2] and the plant hormone abscisic acid (ABA) rapidly induce stomatal closure. However it is unclear and has remained a matter of debate whether ABA signaling is involved in elevated [CO2]-triggered stomatal closure. We have combined genetics, in vivo ABA-reporter imaging, time-resolved gas exchange, guard cell patch clamp, and biochemical analyses to study the convergence mechanisms between ABA and CO2 signal transduction pathways in regulating stomatal closure. We found that guard cells of strong ABA synthesis mutants, nced3/5 and aba2-1, retain the rapid response to [CO2] elevation. While in AABA receptor sextuple mutant pyr1/pyl1/2/4/5/8, rapid elevated [CO2]-induced stomatal closure is not disrupted but delayed. Patch-clamp analyses indicated that slow-type anion channels in the guard cells of nced3/5 double mutant and pyr1/pyl1/2/4/5/8 ABA receptor sextuple mutant leaves can be robustly activated by [CO2] elevation. Using a real-time ABA FRET nano-reporter, ABAleon2.15, and an ABA-responsive promoter reporter, pRAB18-GFP, we found that ABA concentration and ABA signaling in guard cells are not obviously altered by [CO2] elevation. Unexpectedly, in gel kinase assays showed that elevated CO2 does not activate OST1 protein kinase activity in guard cells, even though ost1 mutants show an impaired CO2 response. Together our analyses show that primary CO2 signal transduction mechanisms do not signal via the early ABA signal transduction pathway. Furthermore, our findings indicate that basal ABA signal transduction can modulate and amplify CO2-induced stomatal closure. In addition, our study leads to a model that CO2 signal transduction triggers stomatal closure via an ABA-independent pathway downstream of OST1/SnRK2.6. New transcriptomic and additional data will be present that correlate with this model.

FY is known for its function in flowering time regulation. One of the mechanisms of such a regulation is through its role in alternative polyadenylation of pre-mRNA of other flowering time pathway genes. However, what role FY plays in polyadenylation is not understood. A crucial step for mRNA polyadenylation is poly(A) signal recognition by trans-acting factors. It was recently demonstrated that mammalian Cleavage and Polyadenylation Specificity Factor (CPSF30) and a WD40-domain repeat protein, WDR33, recognize canonical poly(A) signal (AAUAAA) for efficient polyadenylation. It turns out that FY is the homologous of WDR33 in Arabidopsis. To find how FY exercise this function of poly(A) signal recognition, we employed a poly(A) tag sequencing approach, we found that at least 60% polyadenylation events were altered by FY single or double mutants with AtCPSF30. In FY, the broadly conserved WD40-repeat domain has a stronger effect than the plant unique PPLPP domain. Mutations of FY disrupt AAUAAA or AAUAAA-like poly(A) signal recognition. Notably, adenosine-rich signal usage is suppressed in WD40-repeat mutation, while promoted in PPLPP mutations. However, neither WD40 repeat nor PPLPP mutations aggravate the alteration of signal usage caused by a null mutant of AtCPSF30. Furthermore, the WD40-repeat mutation shows a preference of 3’UTR shortening, but the PPLPP domain mutation shows a preference for lengthening. Interestingly, the WD40-repeat mutant exhibits shortens primary roots and confers late flowering with alteration on poly(A) sites usages of related genes. Importantly, the functions of different polyadenylated transcripts generated by FY mutation were tested in two genes, where the long transcripts of ARK2 and a zinc ion binding protein gene are shown to be related to salt and oxidative stress responses. Overall, these results reveal a conserved and specific role of FY in mRNA polyadenylation.

The defining characteristic of circadian rhythms is that they have a period of about 24 h in constant conditions. However, circadian period is not fixed, it is variable. Many signals regulate the speed of the circadian clock in a reversible manner, with the effect dependent on the time of the day that the stimulus is experienced in a process we have called dynamic plasticity (Webb et al., 2019 Nature Comms 10, 550). We have been investigating the mechanism and purpose of the dynamic plasticity of the circadian oscillator to sugar signals. We have previously demonstrated that sugars can speed up the circadian oscillator and identified three signalling pathways by which sugars regulate the circadian oscillator, including one dependent on the regulation of the expression of the circadian clock gene PSEUDO-RESPONSE REGULATOR 7 (PRR7) by the energy sensitive transcription factor bZIP63 (Frank et al., 2018 Current Biol. 28, 2597-2609). We are now investigating why the circadian oscillator responds to sugar signals. We will describe new data that demonstrates that the circadian oscillator responds to endogenous changes in sugars that affect the entrainment of the circadian oscillator to light intensity and photoperiod dependent on the correct functioning of PRR7. Experimentation and mathematical modelling demonstrate that responses of the circadian oscillator to responses to moderate changes in light intensity can be explained in terms of changes in sugar signalling associated with the management of transient starch reserves in the leaf. Our data suggest that response the circadian oscillator to endogenous sugar signals is required for the correct timing of internal events with respect to the environment.

In animals, 7-transmembrane G-protein coupled receptors (GPCRs) at the plasma membrane that activate G-protein coupled signaling become phosphorylated at their cytoplasmic C-terminal tail in a ligand-dependent manner by specific cytoplasmic kinases. This leads to decoupling of the phosphorylated GPCR from its cognate G protein complex resulting in de-sensitization toward that ligand. The well-studied adaptor called β–arrestin recognizes the phosphorylated tail and recruits the clathrin complex to initiate endocytosis of this GPCR. Instead of GPCRs, most plants have a 7 –transmembrane protein (AtRGS1 in Arabidopsis) that modulates the active state of a self-activating G protein complex. Analogous to the animal GPCR paradigm, endocytosis of AtRGS1 is phosphorylated at its C terminal tail by receptor-like kinases such as BAK1 and this leads to endocytosis. However, plants lack canonical β–arrestins. Therefore, we mined structures of plant proteins for homology to animal β–arrestins and discovered AtVPS26, a component of the retromer well known for trafficking vesicles from the endosomes to the trans Golgi network. In vivo analysis shows AtVPS26A and AtVPS26B form dimers and interact with AtRGS1, promoting clathrin-mediated endocytosis. We conclude that AtVPS26 and AtVPS26B serve as arrestin-like proteins in plants that are crucial for AtRGS1 internalization and subsequent trafficking to endosomal compartments.

Iron (Fe) is an essential micronutrient for all living organisms and plants are the major source of Fe for humans and livestock. Fe deficiency in humans has been described as the most common nutritional deficiency affecting nearly 30% of the world’s population. Fe deficiency also has a negative impact on plant development, crop yield, and seed quality. Understanding sensing and signaling regulation in plants will help in developing crops with higher nutritional value. In Arabidopsis, OPT3 has recently been identified as a component of the systemic network mediating Fe deficiency responses. opt3-2 mutants show a constitutive Fe-deficiency response and over-accumulate Fe in roots and leaves. Using RNA-seq, we demonstrated that opt3-2 roots display an activation of the major networks mediating Fe uptake. However, markers for Fe excess are exclusively induced in leaves, suggesting that Fe excess is properly sensed in leaves. In addition, we have found that the leaf vasculature responds more rapidly than roots to changes in Fe availability, suggesting that the vasculature is the primary site for sensing the Fe status of the whole plant. Our current experiments, including high-throughput protein-DNA interaction together with gene network analyses of leaf specific time-course RNA-seq data, are directed towards the identification of the transcriptional networks that coordinate the response to changes in Fe availability and the crosstalk to other nutrients.

Iron deficiency is a major nutritional problem for human populations throughout the developing world and the majority of people acquire iron primarily from plant sources. Additionally, iron bioavailability is a major limiting factor in about 30% of arable croplands worldwide. An improved understanding of iron uptake and homeostasis is necessary to help combat both issues. We are focused on understanding the role of a mitochondrially-localized ferric iron reductase (FRO3) in cellular iron dynamics and whole plant iron homeostasis. While FRO3 is expressed throughout the plant, its expression is greatest in the vasculature. Knockout of FRO3 causes a 50% reduction in mitochondrial iron content and also alters whole plant iron sensing. fro3 lines accumulate 1.2X as much iron as WT plants do, while showing an increased iron deficiency response compared to WT suggesting that while accumulating more total iron, they sense some level of iron deficiency. Furthermore, RNA-seq data suggests that fro3 lines have an altered genomic response to iron deficiency, and sense a greater iron deficiency than WT. These data show that loss of FRO3 disrupts Fe homeostasis and suggest that vascular mitochondrial iron content may play an important role in whole plant iron homeostasis.

Durum wheat (Triticum durum L. subsp. durum) accumulates a high level of cadmium if grown in Cd-polluted soils. To unravel the molecular mechanisms activated by durum wheat in response to cadmium, we employed two near-isogenic lines with opposite behavior to cadmium accumulation in grains and leaves. The NILs were subjected to transcriptome analysis by RNA-sequencing, and the results have highlighted the central role of the genes encoding nicotianamine synthase (NAS) and nicotianamine amino transferase (NAAT), suggesting the hypothesis that durum wheat produces the phytosiderophores nicotianamine (NA) and mugineic acid (MA) to contrast cadmium stress. Moreover, the biosynthesis of these chelators is sustained by the activation of the methionine salvage pathway that supply the S-adenosyl methionines (SAM) needed for NA synthesis. The transcriptome analysis has revealed also the up-regulation of several vacuolar transporters of NA and MA, suggesting the compartmentalization of chelated cadmium into the root vacuoles. To demonstrate this hypothesis, we have quantified at first, using liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS), NA and MA in roots and leaves of NIL plants treated and not-treated with cadmium. Significant differences were found between the treated and non-treated sample and between the two genotypes (low-cadmium genotype produced more NA than high-cd genotype). The differences were more evident in root tissues. Subsequently, to identify the intracellular localization of cadmium and chelators, the root tissues were analyzed by a confocal laser scanning microscopy using LeadiumTM Green AM that specifically binds cadmium and an NA-specific antibody. We observed colocalization between NA and cadmium and both were into the vacuoles. In this work, we have demonstrated that durum wheat detoxifies roots from cadmium by the compartmentalization into the vacuoles.

Radiocesium, accumulated in the soil by nuclear accidents is a major environmental concern. The transport process of cesium (Cs+) is tightly linked to the indispensable plant nutrient potassium (K+) as they both belong to the group I alkali metal with similar chemical properties. Most of the transporters that had been characterized to date as Cs+ transporters are directly or indirectly linked to K+. Using a combinatorial approach of physiology, genetics, cell biology and direct transport assay, here we identified two ATP-Binding Cassette (ABC) proteins, ABCG37 and ABCG33 as new Cs+ transporters. The gain-of-function mutant of ABCG37 (abcg37-1) showed hypersensitive response to Cs+-induced root growth inhibition, while the double knock out mutant of ABCG33 and ABCG37 (abcg33-1abcg37-2) showed resistance. Single loss-of-function mutant of ABCG33 and ABCG37 did not show any alteration in Cs+ response. Short term uptake experiment with radioactive Cs+ revealed reduced Cs+ uptake in abcg33-1abgc37-2 compared with wild type in presence or absence of K+. Potassium response and content were unaffected in the double mutant background confirming that Cs+ transport by ABCG33 and ABCG37 is independent of K+. Collectively, this work identified two ABC proteins as new Cs+ influx transporters, which act redundantly and independent of K+ transport pathway.

It is estimated that in the U.S. alone, 10 million hectares of military land are contaminated with munitions of which 2,4,6-trinitrotoluene (TNT) is a major component. TNT is highly toxic and recalcitrant to biodegradation and its progressive accumulation in soil, plants and groundwater is a significant concern at military sites. The U.S. DoD estimated that the clean-up of unexploded ordnance, discarded military munitions and munition constituents on its active ranges would cost between $16 billion and $165 billion. Explosives pollution is, however, a global problem with large amounts of land and ground water contaminated, including polluted sites dating back to the First and Second World Wars. A fundamental understanding of the phytotoxicity of TNT, and the enzyme systems plants use to detoxify it, will allow the development of robust plant systems to contain, re-vegetate and remediate explosives pollution effectively in situ. Towards this, we have established that, in Arabidopsis thaliana, monodehydroascorbate reductase 6 (MDHAR6) is responsible for the majority of TNT phytotoxicity. Present in the mitochondria and plastids, MDHAR6 catalyzes the one-electron reduction of TNT to produce a nitro radical, with its spontaneous regeneration back into TNT releasing superoxide. Thus in the presence of only catalytic amounts of TNT, this futile cycle depletes cellular NADH and causes oxidative damage within sensitive organellar environments. To remove TNT from the cellular environment, and the damaging activity of MDHAR6, distinct members of xenobiotic detoxification gene families are expressed, including oxophytodienoate reductases (OPRs), uridine diphosphate (UDP) glycosyltransferases (UGTs), glutathione transferases (GSTs) and cytochrome P450s. Ways in which the knowledge of TNT toxicity and its detoxification can be used to remediate explosives-contaminated environments will be presented.

Nutrient depletion in soils adversely affects crop yield. With soil nutrient deficits being estimated at an average rate of 18.7 N, 5.1 P, and 38.8 K (kg ha -1 yr -1 ) globally, it poses a potential threat to food security. Having no respite from an increasing population, the world needs high performing crops adapted to nutrient deficiencies. Root hairs are responsible for ~40% of the nutrient uptake by a plant. Most studies report changes in length and density after 2-4 weeks of growth under deficiencies. In contrast, we observed distinct phenotypes of root hairs for N, P stress and control already at 3-5 days after germination. We observed them under an inverted light microscope for seedlings germinated in hydroponics with a stress and non- stress nutrient solution respectively. Our lead hypothesis is that nutrient uptake efficiency is already characterized by early root hair phenotypes. To study the functions linked to the newly observed phenotypes, we develop a computational pipeline that characterizes length, density and shape from the microscopy images. Our goal is to enable breeders to select for better stress adapted bean varieties within a few days of germination.

Single-cell RNA sequencing (scRNA-seq) has been used extensively to define and compare gene expression in individual cells from animal tissues, but it has not been widely applied to plants. Here, we present our use of a commercially available droplet-based platform for high-throughput scRNA-seq to obtain more than 10,000 single-cell transcriptomes from Arabidopsis root cell protoplasts (Publication DOI: https://doi.org/10.1104/pp.18.01481)(PMID: 30718350). We find that all major tissues and developmental stages of roots are represented in this single-cell transcriptome population. Further, transcriptomes corresponding to distinct cell sub-populations and rare cell types, including putative quiescent center (QC) cells, were identified. A focused analysis of transcriptomes from the epidermal cells defined individual cells progressing from meristematic through mature stages of root-hair and non-hair epidermal cell differentiation, and pseudotime analysis was used to infer the developmental trajectories for the root-hair and non-hair cell types. In addition, single-cell transcriptomes were obtained from two different root epidermal mutants, enabling a comparative analysis of gene expression at single-cell resolution and providing an unprecedented view of the impact of the mutated genes. Overall, this study demonstrates the feasibility and utility of high-throughput scRNA-seq in plants and provides a first-generation gene expression map of the Arabidopsis root at single-cell resolution.

Single-cell transcriptome profiling of heterogeneous tissues can provide high-resolution windows into developmental dynamics and environmental responses, but its application to plants has been limited. Here, we used the high-throughput Drop-seq approach to profile >12,000 cells from Arabidopsis roots. This identified numerous distinct cell types, covering all major root tissues and developmental stages, and illuminated specific marker genes for these populations. Additionally, we demonstrate the utility of this approach to study the impact of environmental conditions on developmental processes. Analysis of roots grown with or without sucrose supplementation uncovered changes in the relative frequencies of cell types in response to sucrose. Finally, we characterized the transcriptome changes that occur across endodermis development and identified nearly 800 genes with dynamic expression as this tissue matures. Collectively, we demonstrate that single cell RNA-seq can be used to profile developmental processes in plants and show how they can be altered by external stimuli.

Plants synchronize various aspects of developmental and physiological transitions with seasonal environmental changes, including flowering transition that determines reproductive success and productivity of plants. To better understand flowering response to environmental fluctuations in soybean at the regulatory network level, we first elucidated global gene expression patterns under different photoperiod regimes. Transcriptomic signatures of the known maturity loci E1, E2, E3 and E5 in the NILs exhibited unique roles of the E loci in flowering control and identified candidate genes that were controlled by the E loci. To clarify the regulatory gene network controlling soybean photoperiodic flowering, we developed the network inference algorithmic package CausNet. CausNet is implemented in Python 3 and is freely available at https://github.com/Veggente/soybean-network. CausNet captured several regulatory interactions controlling soybean flowering transition that were previously reported, and provided with the predicted soybean circadian clock network and flowering gene networks. While the predicted circadian clock network showed robustness to photoperiods, the flowering gene networks differed drastically under long day and short day, consistently with the photoperiodic nature of soybean flowering control. We demonstrated the predicted regulatory roles of GmCOL1a and GmCOL1b in the flowering gene network using RNA interference. Next, we expanded the above approaches to different combination of photoperiod and temperature conditions. Our preliminary observations show many circadian clock genes exhibit higher amplitude under high temperature conditions, suggesting prominent implications of temperature for the circadian clock. Our results provide novel insights and testable hypotheses in the complex molecular mechanisms of flowering control in soybean and lay a framework for de novo prediction of biological networks controlling important agronomic traits in crops.

Soybean (Glycine Max) is the most produced and consumed oilseed in the world. The majority of storage compounds in the soybean seed accumulate during the maturation phase of seed development. Thus, understanding the initiation and establishment of seed maturation will allow for the development of strategies to improve soybean seed quality. Genome-wide transcriptome analysis allowed us to identify a set of co-expressed genes with a spatial and temporal expression pattern that correlates with the maturation program of the seed. Several transcription factors (TFs) that regulate seed maturation were identified in the cluster, including LEAFY COTYLEDON1 (LEC1), ABA INSENSITIVE3 (ABI3), BASIC LEUCINE ZIPPER67 (bZIP67) and ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3). We performed chromatin immunoprecipitation and differential gene expression analyses to identify potential target genes that are transcriptionally regulated by these TFs. Analysis of target genes showed a complex TF regulatory network in which different combination of TFs are involved in controlling distinct biological programs in soybean embryos, such as storage accumulation, photosynthesis and hormone signaling. Genome-wide analyses of TF binding sites (ChIP-Seq) and accessible chromatin regions (ATAC-Seq) suggest that distinct TF complexes are assembled in cis-regulatory modules to control the expression of target genes. DNA motif analyses suggests that the formation of TFs complexes in cis-regulatory modules are determined by a unique composition of DNA motifs. Transient assays in protoplasts isolated from soybean embryos have been used to validate the functionality of DNA motifs in cis-regulatory modules. We also observed that distinct sets of TF complexes are formed due their ability to physically interact to each other. Our results are providing a framework to understand the complex transcriptional regulatory networks that control distinct biological processes during soybean seed development.

Recent advances in genomic technologies such as DNA Affinity Purification Sequencing (DAP-seq) and Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-seq) have generated large-scale, regulatory genomic data for the multiple plant species. To predict condition specific gene regulatory networks using these data, we developed the Condition Specific Regulatory network inference engine (ConSReg), which combines heterogeneous genomic data using sparse linear model followed by feature selection and stability selection. Using Arabidopsis as a model system, we constructed comprehensive and accurate maps of gene regulation under more than 50 experimental conditions. Our results show that ConSReg accurately predicted gene expressions with an average auROC of 0.84 across these testing datasets. Including ATAC-seq information significantly improves the performance of ConSReg across all tested datasets. We applied ConSReg to Arabidopsis single cell RNA-seq data of two root cell types (endoderims and cortext) and identified five regulators in two root cell types. Three out of the five regulators are supported by existing publications. Finally, we tested our approach in a rice gene expression dataset and were able to identify both known and novel regulatory motifs that control drought response in the rice genome. Our results demonstrated that integrating heterogeneous genomic data can provide novel insights into the regulation of condition-specific and single cell-specific gene expression.

Plant roots are excellent developmental models, as they are transparent, possess reproducible patterns of cell division and are responsive to environmental perturbations. Furthermore, roots are responsible for absorption of water and all non-photosynthetic nutrients. As key tissue in roots, xylem is also an important feature of all vascular plants. Xylem functions in long-distance transport of water and nutrients from roots to shoots in addition to giving plants structural support. Terminal differentiation of xylem is marked by the deposition of secondary cell wall polymers and programmed cell death. In Arabidopsis thaliana, the regulatory network controlling the synthesis of secondary cell wall in xylem cells was previously identified in our lab. However, the mechanisms that control specification of xylem identity in cultivated plants remains poorly understood. Our lab aims to understand the molecular mechanisms of xylem cell identity using Solanum lycopersicum (tomato) roots as a model. To elucidate gene putative regulatory networks that underlie tomato xylem identity, we integrated expression data from ribosome-associated transcripts (TRAP-Seq) and chromatin accessibility information (from ATAC-Seq). Using TRAP-seq, we have defined a set of tomato xylem-specific genes. Using ATAC-seq, we have identified genomic regions that have putative regulatory activity. Within these accessible regions, we have also identified enriched transcription factor motifs that could point to potential upstream regulators of xylem cell fate.

Macroutophagy (hereafter as autophagy) is a conserved metabolic pathways in eukaryotic cells. Autophagy involves a set of Autophagy-related (ATG) genes, but the mechanisms for autophagosome formation are still not fully understood in plant. ATG9 is the only transmembrane protein among the core ATG machinery, and ATG9 vesicles have been long considered as a membrane source for autophagosome formation. Our dynamic and 3D electron tomography analysis demonstrated that, under stress conditions, deficiency of ATG9 leads to a drastic accumulation of autophagosomal tubules with direct connection to the endoplasmic reticulum (ER). Such defect is not detected in other atg mutants, implying that Arabidopsis ATG9 might play a distinct role for autophagosome outgrowth from the ER, in particularly under ER stress. Using a combination of fractionation, cellular and in vitro analysis, here we will present our recent findings on the molecular mechanism of ATG9 vesicle and its trafficking in plant autophagy and autophagosome biogenesis. Supported by grants from the Research Grants Council of Hong Kong (G-CUHK404/18, C4002-17G, R4005-18F, and AoE/M-05/12), and CUHK Research Committee, and the National Natural Science Foundation of China (31670179, and 91854201).

Peroxisomes are organelles present in almost all eukaryotic cells, where they sequester reactions that generate harmful byproducts, such as hydrogen peroxide. In plants, these reactions include the beta-oxidation of fatty acids and hormone processing. Protein import and maintenance for these organelles are sustained by peroxin (PEX) proteins. However, the details of peroxisome homeostasis are not fully elucidated, and the mechanisms that target old, superfluous, or damaged peroxisomes for degradation remain enigmatic, especially in plants. Pexophagy is the selective autophagy of peroxisomes, where a double membrane engulfs a target and then fuses with the plant vacuole for recycling of its constituents. We are investigating pexophagy in Arabidopsis by monitoring peroxisomal protein levels under various autophagy-inducing conditions. Under starvation conditions, many organisms activate autophagy pathways, and in plants, one way to induce starvation is by deprivation of light, and therefore the ability to photosynthesize. We found that when seedlings were subjected to darkness, the levels of several peroxisomal proteins decreased, including peroxisomal malate dehydrogenase, peroxisomal hydroxypyruvate reductase, and PEX5. This decline was not observed in autophagy-defective mutants, and various pex mutations accelerated or impeded the degradation of these proteins, suggesting that perturbing PEX function can impede or accelerate pexophagy in addition to impacting peroxisome biogenesis. Illuminating peroxisomal regulation via pexophagy in Arabidopsis could allow modification of plant metabolism and stress response for agricultural benefit.(This research is supported by the NSF and the Welch Foundation.)

Intra-chloroplast maturation and proteolysis is essential in biogenesis, differentiation and protein homeostasis (proteostasis). However, determinants of chloroplast protein life-time and protease-substrate relationships are poorly understood, even if this is of critical importance for plant life. Protein N-termini are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. To better understand chloroplast protein maturation and stability, and to provide a base line for protein degradation studies, we determined chloroplast protein N-termini using terminal amine isotopic labeling of substrates (TAILS) and mass spectrometry. This showed highly specific N-terminal patterns, suggesting a chloroplast N-end rule for protein stability. The Clp protease system is the most complex and abundant protease in chloroplasts, and consists of a protease core, several chaperones and adaptors. Structural and functional features of the plastid Clp system in Arabidopsis thaliana will be illustrated though reverse genetics analysis combined with biochemical analysis, X-ray crystallography, as well as large scale quantitative proteomics for loss-of-function mutants. Multiple substrates were identified based on their direct interaction with the ClpS1 adaptor (N-recognin), by in vivo trapping on affinity tagged AAA+ CLPC chaperone, and by screening of different loss-of-function protease mutants; we discuss the potential role of Clp in fine-tuning chloroplast metabolism.

Constant oxidative damage is the high cost of photosynthesis and energy production by chloroplasts. As such, a functioning photosynthetic cell must have quality control mechanisms that monitor the turnover and degradation of reactive oxygen species (ROS)-damaged chloroplasts and chloroplast components. We have recently described a conditionally lethal mutation in Arabidopsis that leads to the accumulation of excess protoporphyrin IX (Proto) in the chloroplast and the production of singlet oxygen (1O2). Damaged chloroplasts are subsequently ubiquitinated and selectively degraded. A genetic screen identified the Plant U-Box 4 (PUB4) E3-ligase as being necessary for this process and pub4-6 mutants have defects in stress adaptation and longevity. Together, these results describe a new class of chloroplast signal that leads to the targeted removal of ROS overproducing chloroplasts. To understand the mechanism behind this pathway, we are taking multiple approaches. 1) To identify new genes involved in this pathway, we are mapping several newly isolated mutants including one gain-of-function allele from an activation-tagging genetic screen. 2) We have recently begun identifying metabolite signatures generated during chloroplast 1O2 stress that may act as secondary messengers to initiate cellular degradation. 3) Furthermore, we are aiming to identify the chloroplast protein ubiquitination targets that may regulate selective chloroplast turnover. This has led to the identification of one candidate protein, whose accumulation affects chloroplast development and their ability to withstand severe oxidative stress. With these studies, we hope to understand a fundamental process that ensures productive energy capture and protects photosynthetic cells under dynamic environments. (This work has been generously supported by a Basic Energy Sciences grant from the Department of Energy)

Photosynthesis in many organisms is limited by the slow catalytic rate of the CO2-fixing enzyme Rubisco. Oceanic microalgae make Rubisco run faster by packing it into a phase-separated organelle called the pyrenoid, where the CO2 concentration is much higher. We recently discovered that Rubisco’s clustering in the pyrenoid of the model alga Chlamydomonas is mediated by the intrinsically disordered repeat protein EPYC1; however, the mechanism for this clustering has remained unknown. Here, we demonstrate the structural basis for the clustering of Rubisco by EPYC1. Our discovery of the mechanism of formation of the matrix advances our structural and functional understanding of the pyrenoid, a phase-separated organelle that plays a biogeochemically fundamental role in the global carbon cycle.

Chloroplast biogenesis is initiated by principally the red and far-red photoreceptors, the phytochromes through the light-dependent activation of photosynthesis-associated genes encoded by both the nuclear and plastidial genomes, but how photoreceptors control plastidial gene expression remains enigmatic. Here we show that the photoactivation of phytochromes triggers the expression of photosynthesis-associated plastid-encoded genes (PhAPGs) by stimulating the assembly of the bacterial-type plastidial RNA polymerase (PEP) into a 1000-kDa complex. Using forward genetic approaches, we identified RCB (Regulator of Chloroplast Biogenesis) as a dual-targeted nuclear/plastidial phytochrome signaling component required for PEP assembly. Surprisingly, RCB controls PhAPG expression primarily from the nucleus by interacting with phytochromes and promoting their localization to photobodies for the degradation of the transcriptional regulators PIF1 and PIF3. RCB-dependent PIF degradation in the nucleus triggers the plastids for initiating PEP assembly and PhAPG expression. Thus, our findings reveal the framework of a nucleus-to-plastid phytochrome signaling mechanism linking photobody biogenesis to the regulation of chloroplast transcription.

Soybean meal is an important source of protein for animal feed and the oil is used for human consumption. Improving soybean meal, protein, and oil through enhancing seed composition and increasing yields is of commercial interest and can lead to more sustainable agriculture. We have previously increased soybean oil by engineering the soybean diacylglycerol acyltransferase (DGAT1) protein with 14 amino acid substitutions. CRISPR-edited traits have the potential to decrease regulatory costs, reduce time to market, and may have better public acceptance. To develop a high oil soybean trait via CRISPR editing, we sought enhanced DGAT1 variants with fewer amino acid substitutions. Combinations of one to four amino acid substitutions in DGAT1 were initially screened in yeast and transient tobacco leaf for increased oil accumulation. Methods for screening and quantifying triacylglycerol (TAG) accumulation with increased throughput using a 96-well plate format were also developed. One DGAT1 variant with three amino acid substitutions accumulated TAG to a similar level as the DGAT1 with 14 amino acid substitutions in transient tobacco leaf. Top candidates with one to four amino acid substitutions were then advanced to stable soy over-expression events to confirm the model system results. Based on these results, enhanced soybean DGAT1 variants can be developed via CRISPR-editing to produce high oil soybeans.

Developing technologies to achieve step change increases in crop yield remains a critical unmet need in agriculture and a key challenge for future global food security. Yield10 Bioscience is focused on developing disruptive technologies to achieve step change increases in the inherent yield of crops, a task that will likely require multiple gene modifications to increase photosynthesis and efficiently deliver the increased photosynthate to the target organ, which in most crops is the seed. Genome editing is the favored approach for developing plant lines with enhanced yield since the resulting lines can have non-regulated status under USDA-APHIS rules, significantly reducing the time and expense for commercialization. Yield10 is leveraging our unique expertise in metabolic engineering to identify target genes for genome editing in multiple crops through modeling with subsequent experimental validation. Yield10 has ongoing genome editing programs to increase seed yield in rice, canola, and Camelina sativa, an oilseed that has received attention as a platform crop for the production of omega fatty acid containing oils, biodiesel, jet fuels, oleochemicals, and oils to be used as a replacement for fish oil in aquaculture feeds. For canola and Camelina, additional editing targets designed to increase seed oil content are also being pursued. Results from these editing programs will be discussed with a focus on lines obtained from single and multiplex genome editing of Camelina with gene targets to increase seed yield and/or oil content, some of which have been deemed non-regulated through The USDA-APHIS “Am I Regulated?” process.

Renewable transportation fuels (biodiesel and green diesel) from plant seed oils are considered as environmentally and economically feasible alternatives to petroleum-derived fuels. Camelina sativa, due to its unique seed and oil attributes, has attracted much interest as an emerging crop dedicated for biodiesel and jet fuel production. To increase oil yield, we engineered Camelina by co-expressing the Arabidopsis DGAT1 and yeast GPD1 genes under the control of seed-specific promoters. Transgenic lines exhibited up to 13% higher seed oil content and 52% increase in seed mass compared to wild-type plants. Further, DGAT1- and GDP1 co-expressing lines produced almost double seed and oil yields per plant basis compared to wild-type or plants expressing DGAT1 and GPD1 alone. To identify the bottlenecks for further improving the seed and oil yield in Camelina, we utilized metabolomic and transcriptomic profiling approaches in developing seeds in Camelina overexpressing TAG related genes. Our approach revealed several key genes/gene networks associated with significant changes especially in the TCA cycle and storage/retention of lipids in seeds. Overexpression of candidate genes showed further increase in oil and seed yield. We are now translating this strategy in edible oilseed crops such as Indian mustard and soybean for increasing edible oil contents and seed yields. Further, to enhance plants productivity under the adverse conditions, we constitutively overexpressed a wax synthase gene (WS) for increasing the synthesis of cuticular wax in stem and leaf tissues. WS transgenic plants, when exposed to drought and salinity stresses, exhibited strong tolerance phenotype and had reduced water loss and cuticle permeability due to increased deposition of epicuticular leaf and stem wax loading. Ultimately, our aim is to stack the genes/gene networks responsible for increasing seed and oil yields as well as abiotic stress tolerance to enable these cultivars to growth on margin

Floral color and pigmentation patterning vary greatly among flowering plants, as differences in these traits often alter pollinator behavior and affect floral interactions with the abiotic environment in ways to foster adaptation and speciation. Although much is known how petal hue is specified and varies, far less is known about how pigments are painted into complex patterns during floral development and how these patterns diversify. The ample diversity in floral coloration and preponderant genomic resources for the monkeyflower genus Mimulus make it an ideal group for studying how floral pigmentation patterning develops and evolves. A striking feature of flowers of the common monkeyflower (M.guttatus) is the red anthocyanin spots forming a nectar guide on the ventral lobe of the yellow corolla. By bulked segregant analysis and fine-mapping, we identified independent polymorphisms in the R3-MYB RTO (RED TONGUE) as the genetic bases of blotchy spot variants in multiple wild populations. To validate that these natural loss of function variants cause the variant phenotype, we successfully developed and deployed genome-editing methods for the first time in M. guttatus. Abolishing RTO function with CRISPR/Cas9-introduced frameshifts recapitulated the expanded spot phenotype. Notably, loss of RTO protein function increased RTO expression and expression of the R2R3 MYB transcription factor NEGAN, an activator of anthocyanin production in M.lewisii. RNAi mediated knockdown of NEGAN in M. guttatus confirmed that it promotes petal spot formation and also activates RTO expression. Together, our results from genetic mapping and from newly applying tools for manipulating gene function in M. guttatus indicate that NEGAN and RTO comprise the molecular basis for a reaction-diffusion patterning mechanism. Our data support a model where NEGAN locally activates pigment production and RTO expression, then RTO diffuses to neighboring cells to repress NEGAN and restrict spot expansion.

The luminal binding protein BiP is an endoplasmic reticulum (ER)-located molecular chaperon of the HSP70 stress-related protein family. BiP is essential for proper functioning of the ER, participating in the translocation, folding, assembly, and quality control of newly synthesized proteins targeted to the ER and secretory pathways. The aim of this study was to evaluate the spatiotemporal activity of the upstream region of BiP gene from Douglas-fir, designated as PmBiPPro1 promoter, in different organs and tissues of heterologous host (potato) and determine whether this promoter might be useful to control transgene expression for enhanced stress resistance in crops. Using promoter deletion analysis, the activities of full-length PmBiPPro1 promoter and its two 5′ truncated versions in unstressed leaves and in response to wounding, correlations between promoter activity and promoter length, and the relationship between the number of transgene insertions and protein accumulation were determined. The cis-regulatory functional domains that are important for the spatiotemporal activation of transgene expression under normal plant development and in response to wounding have been identified. Histochemical staining of transgenic plants revealed high activity of truncated PmBiPPro1-3 promoter in mesophyll tissues of young leaves, and in other tissues associated with a high rate of cell division, such as apical and lateral meristems and the procambial regions. The PmBiPPro1 promoters, especially the full-length version, had higher activity in microtubers than in any other potato organ or tissue. The transcriptional activity of the Douglas-fir promoter in potato suggests that the same signal molecules mediate responses in both plant species. The organ-specific activity of the PmBiPPro1 promoters may be useful for targeted expression of heterologous genes in potato tubers.

Plant gene editing begins by delivering gene editing (GE) reagents to somatic plant cells in culture, using either the gene-transferring bacterium, Agrobacterium tumefaciens, or physical means such as particle bombardment. Edited cells are then induced to differentiate into whole plants by exposure to various combinations of plant hormones, namely auxin and cytokinin. Regeneration of plants through tissue culture is not ideal for large-scale, high-throughput production of gene edited plants. The process is often inefficient, requires considerable time, works with limited genotypes, and causes unintended changes to the genome and epigenome. Methods that circumvent these limitations would greatly enhance the ability to create edited plant lines. By editing the stem cells within plant meristems, all tissues derived from the meristem would be expected to contain GE events of interest, leading to vertical transmission. However, direct modification of existing meristematic tissue has proven challenging as it has been historically recalcitrant to genetic modification. Combinations of developmental regulators like WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM), amongst others, have been implicated in the patterning and formation of shoot meristems. Co-opting these types of patterning regulators, a new meristem can be generated from transformed somatic tissues. Using our method of fast treated Agrobacterium co-culture (Fast-TrACC), various combinations of WUS, STM and ISOPENTENYL TRANSFERASE (IPT) were found to facilitate de novo meristem generation in the model species Nicotiana benthamiana. Combining these developmental regulators with GE reagents provides the potential to establish an edited shoot directly from somatic tissue in order to avoid tissue culture. In this regard, de novo meristem induction promises to alleviate the tissue culture bottleneck, allowing for larger collections of genetically engineered germplasm to be assembled to solve agricultural problems.

This symposium will cover a range of topics and a range of scales related to sustainably feeding a growing human population as the planet’s climate continues to warm. Beginning with a broad brush focus on food system sustainability and resilience, the symposium will then delve more deeply into the challenges and opportunities afforded by modern approaches to plant breeding and the rapidly growing area of plant-based meats. The symposium will conclude with a talk on the ways in which big data can inform and improve agricultural practices.

One way or another, agriculture and the broader food system represent the footprints of humankind on the planet; our use of and impacts on land, water and labor are primary examples. There is an urgent need to pursue sustainable solutions that accommodate not only our growing demand for food, but also our changing dietary preferences and the changing climate. Inherent in this focus on sustainability is the recognition that tradeoffs and synergies exist among sustainability domains, including environmental, social and economic domains, all of which will need to be considered. It is also essential to explore technological and ecological approaches simultaneously, rather than viewing them as mutually exclusive, and we must recognize that all sectors – public, private, non-profit, and academic – have roles to play in developing and implementing solutions. In this presentation, I will identify barriers, opportunities and approaches to get from here to there.

The use of data for seed research and development at commercial seed companies has evolved from pencil and paper note taking to highly automated, high volume data collection and decision-making systems that enable continued increases in genetic gain. Collection and use of DNA information illustrates the dramatic shift in data and technology supporting genetic improvement. Early methods relying on manual lab processes, data scoring and endpoint selection/remediation decisions have been replaced by lab robotics with continual process monitoring, data collection and decision making via highly automated systems. This evolution in workflows, along with the opportunity to scale the density of genomic information and population size through inexpensive lab analyses, has caused vast increases in data volume with associated increases genetic gain potential and challenges for data management. The availability of deep pedigree information and automated phenotypic and environmental data collection provide additional layers of complexity and opportunity to identify and advance superior genetics.

For thousands of years and in cultures around the globe, humans have had the desire to eat meat – and the demand is only rising. Unfortunately, due to dramatically high greenhouse gas emissions, water consumption, and land utilization, animal agriculture is now the most environmentally destructive industry on the planet. A solution is needed to motivate consumers to reduce their consumption of animal-based products. To create the sensory experience that meat eaters demand, we studied meat at the molecular level to identify the biochemical principles underlying the unique flavor and textural attributes. We then searched the plant kingdom for materials that can recreate those specific properties. One of these discoveries was that heme – the iron-containing co-factor in myoglobin and hemoglobin – is responsible for the flavor of meat. The bloody metallic flavor of beef in its raw state is from heme. Also, when cooked, heme generates beefy flavors and aromas that are critical for the sensory experience of beef. Heme is ubiquitous in nature, and we found heme proteins in plants are also capable of the same flavor characteristics. One example is the leghemoglobin (LegH) found in the root nodules of soybeans. We then combined heme-protein LegH with simple nutrients, plant proteins and fats to assemble a plant-based beef burger that has all the sensory and cooking experiences of beef. Our newly identified plant based flavor system enables the creation of meat products that carnivores crave with a fraction of the environmental impact. This allows us to feed our growing human population while expanding forested land, increasing freshwater, and decreasing greenhouse gases. With millions of Impossible burgers being served in thousands of restaurants across the US and Asia, the food revolution is underway.

Societies around the world are facing unprecedented challenges in the endeavor to provide food security to all people. Two billion people—nearly a third of global population—are currently food insecure, a number that will increase by 30% in the next four decades. Climatic variability is projected to decrease the yields of California’s crops by approximately 15% over the same time period. The potential for prolonged drought adds further uncertainty. At the same time, sustainable crop production and humane food-animal production are areas of growing concern for many consumers. Imagine a world with food production systems capable of feeding and nourishing 10 billion people and eliminating food insecurity while sustaining our planet. A farm where smart machines use sensors to rapidly learn the needs of each plant and animal and provide them with the individualized care they need to thrive. A place where a new generation of biologists and environmental scientists will transform plant and animal breeding and welfare to rise to the challenges of low resource utilization, climate change, and environmental sustainability and accelerate the creation of superior plants and intimate animal care to thrive in the farm of the future. Imagine, too, a future when farmers and ranchers worldwide employ breakthrough technologies for a more caring, resilient, efficient, productive and sustainable food production system in harmony with our natural environment. The Smart Farm Initiative at UC Davis is a vision for the farm of the future where multidisciplinary teams of engineers, computer and life scientists will provide new innovative solutions, education, and training to lead the way forward into this new era of agriculture.

All attendees are invited to attend the Town Hall meeting to both learn about ASPB's activities over the last year and to offer input to guide future trajectories for the society. Members of the ASPB leadership will be present to share updates and respond to feedback from members.

Join the Primarily Undergraduate Institution (PUI) section for a business meeting and share your ideas about activities for this newly formed section. Workshop requires preregistration.

Graduate training of plant biologists occurs primarily in universities, and students have little exposure to plant biology careers apart from the academic path. This workshop will provide discussion of a diversity of career options available to those with graduate degrees in plant biology. Panelists will be selected to represent a variety of careers including in those in large and small private companies, government, etc. The workshop format will include introduction of career options by the panelists followed by small group discussions. Workshop requires preregistration.

More and more data are available in plant science that have fueled ground breaking discoveries. Beyond the original intents of the experiments, these data can be used to discover even more. This is where machine learning come in - using computers to learn from data and generate models that can predict a biological phenomenon of interest - e.g. will this gene be lethal when it is knocked-out, or which genetic variants can meaningfully predict a phenotype of interests. Specifically, this workshop will touch on the following topics: What is machine learning and why is it useful? How does machine learning work? What are some example machine learning applications in plant science? How can we feed data into machine learning tools to make discoveries? What are the best practices when doing machine learning? Where to go to learn more? The workshop will include presentations, discussions, and a short hand-on section using online machine learning resources. Workshop has a fee to attend and requires preregistration. WORKSHOP FULL To be added to the wait list please email PBregistration@aspb.org.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Have you seen the evidence about the effectiveness of using active learning methods in undergraduate STEM courses? Want to hear about an NSF-funded program that will provide you with a teaching mentor for up to a year to learn how to implement active learning in your own courses? Or, would you be interested in being a mentor for someone wanting to learn how to do this? This Conversation Circle will discuss the Promoting Active Learning and Mentoring (PALM) Network, the community of practice that it supports, and how to apply to be a Fellow or mentor in the program. ASPB is a founding member of the PALM Network, and the PALM PI and a former PALM Fellow, both members of ASPB, will be on hand to answer your questions.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Interested in using social media to increase the visibility of your research? Let's chat! We will go over ways ASPB uses social media, including Pinterest and Instagram; we'll talk about WeChat, Plantae, and how to optimize your social media interactions.

Interested in finding effective classroom activities to use in your undergraduate plant biology or other biology courses? Interested in documenting your scholarly teaching efforts, accomplishments, and innovations? Come learn about the peer-reviewed, open access online journal, CourseSource, and how to publish your classroom activities in it. A key feature of CourseSource is the alignment of articles with learning goals developed by scientific professional societies, including the American Society for Plant Biologists. This Conversation Circle will introduce you to features of the journal, show how prospective CourseSource authors can write up their work for publication, and include information on how to present classroom activities so that journal readers can replicate them.

Ever wondered what happens to your manuscript after you push the "submit" button? ASPB Publications staff will be here to explain the process and answer all your burning questions!

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Learn about opportunities available for summer internships through ASPB's Summer Undergraduate Research Fellowship (SURF) program.

Are you considering going to grad school? Not sure what lies beyond? Join your peers to discuss pros and cons on earning a graduate degree in plant biology.

Are you a Community Network Leader on Plantae, or interested in starting a new network? Come chat with others about the process and your ideas!

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

In February 2019, members of the ASPB community attended the Plant Summit 2019 meeting, organized by the Plant science Research Network. The Summit convened nearly 50 plant scientists with backgrounds in ecology, molecular biology, data sciences, crop sciences, and other areas to discuss plant science research agenda and strategic priorities for the next 10 years (2020-2030). Summit participants will share outcomes from the Summit and gather feedback from ASPB members on the major goals outlined in the report. Join the PSRN online at plantae.org/PSRN to learn more and get involved.

Are you a 2018 or 2019 author in Plant Physiology, The Plant Cell or Plant Direct? Join your fellow authors for a meet & greet.

Want to lead a 20 minute conversation on a topic of your choice? Sign up for a time slot at the conversation circle at Registration Area Conversation Circle!

Since debuting during Plant Biology 2018 in Montreal, #ASPBForward has come to describe ways in which ASPB might embrace changing cultures and climates, including increased transparency, improved diversity, greater inclusion, and an enriched set of engagement opportunities for members. The society has made its commitment. Now we need talk about what can be done and how to implement it! Join us for a conversation of vision, ideas, and a glimpse into the future of ASPB.

Iron (Fe) is vital micronutrient for living organisms. Plants are the principal source of dietary Fe. Fe deficiency leads to developmental defects and excess can cause toxicity. Plants tightly control the Fe homeostasis for optimal Fe absorption. In order to identify new key players in maintaining Fe homeostasis, the molecular components involved from Fe acquisition from root and transportation to sink are required to be studied comprehensively. To identify key players in Fe homeostasis, IRT1 (Iron Regulated Transporter 1) promoter-driven luciferase (PIRT1:LUC) system was used for genetic screening. This reporter system is activated under Fe-deficient conditions and repressed under Fe-sufficient conditions. EMS (Ethyl Methane Sulfonate) mutagenesis approach was used to screen novel candidates. The idt1 (Iron Deficiency Tolerant 1) mutant was identified with constitutive IRT1/IRT1 expression. The Fe specific mutant idt1 is Metal Tolerance and Iron Accumulator (Metina) i.e. resistant to Fe deficiency, excess zinc (Zn), cadmium (Cd), copper (Cu), cobalt (Co), nickel (Ni) and lead (Pb) and accumulates more Fe. Quantitative analysis for Fe accumulation in idt1 shows that in excess Cd, Zn and other heavy metals the Fe content is higher. Transcriptomic analysis reveals that Fe deficiency signaling pathway including IRT1, FRO2 (Ferric Reduction Oxidase 2), FIT (Fer Like Iron-Deficiency Induced Transcription Factor) and bHLH100/101/38/39 is constitutively expressed in idt1. Optimal overexpression of IDTA320V in WT leads to “Metina” phenotype which results in enhanced protein localization in nucleus. Current data indicates that optimal expression of IDT can improve Fe bio-fortification in crops and counterbalancing the heavy metal toxicity which manifests its importance in phytoremediation..

C4 leaves are characterized by the Kranz anatomy, in which the vascular bundle is surrounded by one layer of organelle-rich bundle sheath (BS) cells, which is then surrounded by one layer of radially arranged mesophyll (M) cells. Past histological and cell lineage studies in maize revealed that Kranz development starts from three contiguous ground meristem cells, but little is known about the genes and the molecular mechanism involved in Kranz anatomy development. To identify key regulatory genes involved in Kranz development, we compared the tissue specific transcriptomes of different developmental stages of maize embryonic leaf including: 5 stages (ground meristem tissues with only P1, 3, 4, 5, or 6 BS GM cells) of Kranz ground meristem (GM) cells; 4 stages of palisade-like (P1, 3B, 4B, and 5 BS stage) M cells; 2 stages of undifferentiated M ground meristem (1M, 2M) cells by LCM. We obtained high-quality RNAs, and then RNA-seq data. Principal components analysis (PCA) showed that early Kranz and M cells exhibited distinct mRNA populations. These data sets indicate that Kranz and M cells have distinct gene regulatory networks because they arise from distinct genetic origins and that the captured cell types show sufficient diversity at the mRNA level. Differential gene expression and weighted correlation network analysis (WGCNA) identified candidate coexpression modules and gene coexpression networks involved in Kranz development. GO analysis indicated that these modules were enriched for genes involved in anatomical structure, leaf, shoot development, etc. In situ hybridization validated several genes expressed in early Kranz anatomy. Finally, we predicted putative cis-regulatory elements in upstream gene sequences from each gene and validated the predictions by Y1H and protoplast transient assay. Moreover, we constructed a network related to Kranz development. These results provided much insight into the transcriptional regulation of Kranz anatomy development.

Significant progress has been made in recent years in plant genome assembly and gene annotation. However, the systematic identification of plant cis-regulatory DNA elements remains a challenge, as methods that are highly effective in animals do not translate to plants. A comprehensive and well-curated data set of plant cis-regulatory DNA elements is instrumental to understanding transcriptional regulation during development and/or in response to external stimuli. In addition, cis-regulatory DNA elements are also hotspots for genetic variations underlying key agronomical traits. We have discovered a plant-specific chromatin signature that is indicative of cis-regulatory DNA elements. We are using this newly identified signature in combination with high-throughput validation assays to systematically identify, analyze and functionally validate cis-regulatory elements and their evolution in important crop species.

The 5’ end of a eukaryotic mRNA generally has a methyl guanosine cap (m7G cap) that not only protects the mRNA from decay by 5’-3’ exonucleases, but also plays an essential role in almost all aspects of gene expression. Some RNAs in E. coli, yeast, and mammals were recently found to have NAD+ as a cap. We have developed a new method, termed NAD tagSeq, for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses an enzymatic reaction and a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq not only allows more accurate identification and quantification of NAD-RNAs but can also reveal sequences of whole NAD-RNA transcripts. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis are mostly produced from a few thousand protein-coding genes. The top 2,000 genes that were found to produce the highest numbers of NAD-RNAs were enriched in the gene ontology terms of responses to stresses, photosynthesis, protein synthesis, and response to cytokinin. For some Arabidopsis genes, over 10% of their transcripts could be NAD-capped. The NAD-RNAs in Arabidopsis have similar overall sequence structures to their canonical m7G-capped mRNAs. NAD tagSeq has been used to identify NAD-RNAs from maize, rice, and other organisms. The identification and quantification of NAD-RNAs and revealing their sequence features provide essential steps toward understanding functions of NAD-RNAs.

Eukaryotic cells have the ability to reliably ascertain which regions of their genome should be expressed (such as genes) and which regions should be transcriptionally repressed as constitutive heterochromatin. This affords them the ability to protect their genomes from parasitic forms of DNA, such as transposable elements, while this same defense mechanism is also triggered during transgenesis when newly introduced genes are unintentionally targeted for silencing. This transcriptional silencing is epigenetic in nature, as once DNA methylation, histone modification and nucleosome compaction set in, they can be maintained over subsequent cell divisions. The field of epigenetic silencing is replete with labs studying how transcriptional silencing is epigenetically maintained, or in some cases re-targeted, across cell divisions and generations. On the other hand, the initiation of that silencing in the first place, especially for DNA that is “new” to the genome, is not well understood. Furthermore, the de novo initiation of transgene silencing is of high importance to plant biology, as understanding how transgenes are targeted for silencing has significant implications for genome engineering and crop production. Our data in the powerful model plant Arabidopsis demonstrates that de novo initiation of transgene silencing is expression-dependent and utilizes a host of small RNA classes that function specifically in the initiation of silencing to guide the first round of DNA methylation. We find that a non-transcribing transgene can avoid silencing altogether, demonstrating that the transcript or RNA Pol II transcription itself is the key trigger for the initiation of transgene silencing, with small RNA biogenesis being secondary. I plan to present our ongoing work on the molecular mechanisms of silencing initiation, focusing on the key difference between small RNA factors required to maintain the silenced state from those needed to initiate de novo silencing.

As a hub of central carbon metabolism, regulation of the tricarboxylic acid (TCA) cycle is crucial to coordinate flux to neighboring metabolic pathways for optimizing growth and development. TCA cycle regulation in plants has largely been studied at the level of the protein or metabolite including post-translational modification, allosteric feedback of enzymes, and metabolite channeling by organizing sequential enzymes into metabolons. However, transcriptional regulation of the TCA cycle in Arabidopsis, and even more broadly, in multicellular organisms, is largely unstudied. Using an enhanced yeast one-hybrid platform, we identified a large number of transcriptional regulators. These predict differential control of TCA targets in the various cellular compartments, potentially enabling flexibility to alter the pathway. Furthermore, no general regulators of the TCA cycle were identified via co-expression analyses, providing an immediate indicator of a novel paradigm for how multicellular organisms regulate this critical metabolic pathway. We selected 17 candidate TFs for conditional transcriptional regulation of the TCA cycle. In total, mutants of all 17 genes were shown to have perturbed TCA cycle function, with a subset having responses that were dependent on specific TCA cycle intermediates. One third of these TF mutants influence growth in a salt stress-dependent manner, and mutations in almost half led to perturbations in the abundance of C, N or C:N ratios. Thus transcription of TCA cycle genes are controlled in the plant to allow fine-tuning of metabolism to meet energetic demands of diverse cell types under various environmental constraints.

Single cell models such as yeast have aided the identification of core osmo-sensory pathways in non-plant cells. To facilitate defining such pathways in plants, we have utilized high-throughput genetic screening methods in the alga, Chlamydomonas. We have recently found that mutants in putative osmosensory pathways in Arabidopsis are necessary for survival of Chlamydomonas after hyperosmotic shock indicating conservation across the plant lineage (Vilarrasa-Blasi et al., in preparation). Initial genome-scale mutant screens have identified hundreds of loci that are necessary for growth under hyperosmotic conditions including putative signal transduction components not previously associated with the osmotic stress response. Transcriptomic and proteomic profiling of Chlamydomonas under these conditions has enabled a systems level understanding of osmoregulation without the complications of a multicellular context. Characterization of these genes in Arabidopsis has revealed broad conservations of the newly uncovered pathways.

Cultivated chickpea, Cicer arietinum, has ~ 100-fold less genetic variation relative to its immediate wild progenitor, Cicer reticulatum, possibly harboring drought tolerance traits. We used a composite source site aridity index to prioritize two C. reticulatum accessions (Savur_63 and Kalkan_64) and compared their performance to two cultivars contrasting in water use (ICC14778 and ICC8058). These plants were grown under unstressed, mild or severe soil water deficit. Using a network analysis approach, we associated network-derived gene co-expression modules with the severity of drought treatment; physiological and metabolic responses; and host genotype. Leaf area and photosynthetic traits responded to increasing drought stress similarly in Savur_63, Kalkan_64 and ICC14778 (drought tolerant), compared to ICC8058 (drought sensitive). Network analysis identified nine distinct co-expression modules in shoots, with frequent species-specific components. Of these, three and five modules were strongly associated with drought severity in C. arietinum and C. reticulatum respectively. Pathways responding to drought stress tended to be conserved across genotypes and species, including genes implicated in metabolic pathways, plant hormone signaling and photosynthesis. Among species-specific responses, terpenoid biosynthesis was positively correlated with increasing water deficit in C. reticulatum, while sphingolipid metabolism was downregulated only in cultivars. Ureide transport was specifically downregulated in C. reticulatum. Interestingly, the hub genes were often unique in individual genotypes. Comparative analysis of C. reticulatum and C. arietinum provides a broader view of Cicer’s core drought response patterns associated with tolerance and susceptibility. We suggest that incorporation wild specific modules into cultivated backgrounds will expand drought response strategies and serve to prioritize candidate genes for breeding for drought tolerance.

The circadian clock enables organisms to synchronize their metabolism, physiology, and development with changes in the environment that ultimately optimizes fitness in plants. Temperatures extremes such as heat stress can affect normal clock function and also growth and productivity. Together, temperature and the clock control many aspects of plant growth and fitness through extensive regulation of gene expression. Mechanistically, the clock is able to regulate the expression of these stress responsive genes by controlling the magnitude or occurrence of the transcriptional response based on time of day. We performed a transcriptomic analysis to gain a global understanding and determine to what extent time of day and the circadian clock contribute to differential transcriptional responses under heat stress during the day period when plants are exposed to maximum heat stress and likely primed for high temperature. From the thousands of genes that were differentially expressed, we identified genes where the occurrence or the magnitude of the transcriptional response was specific to the time of day the stress was applied. A subset of these responses is dependent on the proper expression of specific clock genes. Characterization of selected genes suggests that the observed transcriptional changes in response to temperature stress directly influences flowering and plant thermotolerance in a time of day dependent context. Insights from our studies can help to guide similar research in crop species aimed at optimizing growth, yield, and resilience.

Most tropical and subtropical produce are so cold-sensitive that refrigeration reduces shelf-life and quality. Tomato fruit experiences Postharvest Chilling Injury (PCI) when stored at 0-12.5°C. Symptoms include surface pitting, and uneven ripening and decay, due to metabolic and physiological dysfunction. Unlike tomato, Arabidopsis thaliana can cold-acclimate partly due to the CBF family of transcription factors (AtCBF1-3). The ectopic and constitutive overexpression of AtCBFs led to higher chilling tolerance but had negative developmental effects in tomato plants, and fruit response to cold stress was not tested. Constitutive overexpression of CBF1 from the cold-tolerant wild tomato relative Solanum habrochaites (ShCBF1) resulted in higher cold-tolerance in Arabidopsis plants. This suggests that increasing the control of transgenic CBF1 expression could be useful to study PCI in tomato fruit without detrimental effects on plant development. We hypothesize that CBF1 overexpression will increase chilling tolerance and ameliorate PCI symptoms during refrigeration. In this study, Micro-Tom tomato plants were independently transformed with three constructs: a dexamethasone system to chemically trigger AtCBF1 expression, and a stress-inducible promoter (RD29A) to induce ShCBF1 or SlCBF1 specifically when fruit are refrigerated. Fruit were stored at 2.5 (PCI-inducing) or 12.5°C (control, non-PCI inducing) for 1- 3 weeks, and transferred to 20°C to promote PCI symptoms. To assess changes in whole-plant cold tolerance, the photosynthetic performance of transgenic lines was measured under cold stress. Results showed that high expression of transgenic CBF1 in fruit as determined by qRT-PCR, was linked to accelerated senescence and an aggravation of PCI symptoms, both quantified by objective color and surface pitting scores. This suggest that PCI may offer an evolutionary advantage in tomato by accelerating fruit breakdown for seed dispersal under extreme stress conditions.

The ubiquitination pathway involves the attachment of ubiquitin, a small, highly conserved protein to select substrates. The attachment of a chain of ubiquitin molecules targets the modified protein to the multi-proteolytic 26S proteasome complex for degradation. At the center of the pathway is a large and diverse family of substrate recruiting ubiquitin ligases (E3s). The Arabidopsis genome is encodes for ~500 RING-type E3s, many of which are known to regulate abiotic stress signalling. Of interest are the seven XBAT (XB3 ortholog in Arabidopsis thaliana) E3s, each of which has a distinct role including regulating ethylene biosynthesis, abscisic acid (ABA) signalling, cell death and pathogen defense. I will discuss our recent findings for two members, XBAT31 and XBAT35, both of which are alternatively spliced to produce two isoforms. XBAT31.1, but not XBAT31.2, is involved in regulating iron deficiency response, increasing root iron uptake when availability is low. Overexpression of XBAT35.2, but not XBAT35.1, is known to induce cell death and reduce susceptibility to bacterial pathogens. The regulatory role of XBAT35.2 is linked to its ability to promote the proteasome-dependent degradation of Accelerated Cell Death 11 (ACD11) in the presence of pathogen. Also, XBAT35.2 promotes its own turnover and pathogen infection leads to stabilization of the E3. Interestingly, we have recently uncovered a role for XBAT35.1 and XBAT35.2 in abiotic stress tolerance. Expression of both isoforms increase in response to ABA and high salinity stress. However, the xbat35 mutant is more tolerant of salt stress, suggesting that, in contrast to its role in pathogen defence, the E3 is a negative regulator of abiotic stress response. We are continuing to examine the dual, but conflicting, roles of XBAT35, and the function of XBAT31 in stress tolerance by identifying substrates and determining how these enzymes are regulated to affect growth under suboptimal conditions.

Improving nutrient and water uptake in crops is one of the major challenges to sustain a fast-growing population that faces increasingly nutrient limited soils. Root hairs, which are specialized epidermal cells, compromise up to 70% of the total root surface area. Therefore, root hairs are important drivers of nutrient and water uptake from the soil. Microscopy provides a mean to record root hairs as digital images. However, quantifying root hairs in microscopy images remains a bottleneck because of their geometry and their complex spatial arrangement. Describing root hairs manually is based on a limited selection of representative root hairs and is only possible in cases, where length and density are sufficiently low to trace individual root hairs. We present a method to automatically quantify phenotypic traits of root hairs in digital microscopy images. Our method uses a machine learning approach that classifies root hair, parent root and the image background. We define local metrics to quantify relatedness between root hair segments that are separated by crossing root hairs or blobs of two or more root hairs. Based on our local metric we can detect individual root hairs by resolving these complexities in a globally optimal way. As a result, we measure the root hair traits, length, number and density. We demonstrate our method on examples of rice, maize and common bean under phosphor, nitrogen and potassium stress. Preliminary results suggest that our measurements of root hair traits strongly correlate with manual measurements (Pearson-correlation up to 0.9 in length). We expect that our method distinguishes subtle differences between genotypes and treatments on the basis of the extracted traits. We believe our study paves a way towards identifying the genetic control of root hair traits and increased agricultural production in future.

Understanding the pathways that control plant development is critical in building a more complete account of how plants grow, especially with changing environmental conditions. This process still lacks mechanistic knowledge at the level of receptors and ligands. In the shoot, the CLE peptide CLAVATA3 (CLV3) limits stem cell production by signaling through receptor-like kinases such as CLAVATA1 (CLAVATA1) and the receptor complex of CLAVATA2 (CLV2) and CORYNE (CRN). Mutations in any of these genes cause an over-proliferation of stem cells and thus an excess of floral organs. We find that crn and clv2 mutants exhibit an additional phenotype that involves a pause in development along with a period of floral primordia termination. Interestingly, floral primordia termination is both temperature- and light-dependent which is often indicative of an imbalance of auxin, which we find to be disrupted in crn and clv2 backgrounds. Furthermore, we find that this pathway is CLV1-independent, which points to a novel pathway involving CLV2/CRN that is specific to floral primordia development and that likely relies on additional non-CLV3 CLE ligands. We show a unique function of the CLV2/CRN receptor complex in adapting to various environmental conditions for proper floral development. In this way, we provide a new mechanistic look at a pathway that can integrate external environmental signals and translate it into intercellular hormone signals to allow for proper floral development.

A central problem in speciation is the origin and mechanisms of reproductive barriers that block gene flow between sympatric populations. In sexually reproducing plants, reproductive barriers exist at different stages during reproduction, including pre-pollination, post-pollination and post-fertilization. Post- pollination barriers depend on interaction between the male gametophyte (pollen) and the cells of the female reproductive organs (stigma, style, and ovule). In Zea mays, three haplotypes, Gametophyte factor1-s (Ga1-s), Gametophyte factor2-s (Ga2-s), and Teosinte crossing barrier1-s (Tcb1-s) at three different loci confer Unilateral Cross-Incompatibility by arresting none-self growing pollen tubes. While Ga1-s and Ga2-s are widespread in domesticated maize, Tcb1-s is almost exclusively found in wild teosinte populations. Despite being members of the same species, some strains of wild teosinte maintain themselves as a distinct breeding population by blocking fertilization by pollen from neighboring maize plants. These teosinte strains may be in the process of evolving into a separate species, since formation of reproductive barriers is a critical step in speciation. These teosinte strains typically carry the Tcb1-s haplotype. Tcb1-s contains a female barrier gene that blocks non-self-type pollen and a male function that enables self-type pollen to overcome that block. With genetic and genomic approaches, here we show that the Tcb1-female barrier gene encodes a Pectin Methylesterase38 homolog, implying that pollen cell wall modification is a key cellular mechanism by which these teostine females reject foreign but closely related pollen. Cloning of this female barrier gene in Zea mays represent a major advance in speciation research and opens up exciting working hypotheses to test. Agriculturally, this work may also help to facilitate breeding effort to manage specialty crop populations and enrich crop germplasm by backcrossing to their ancestors.

Plants use Ca2+ signaling to trigger universal cellular signaling pathways in development and response to the environment. But how the extracellular signals are translated to trigger the Ca2+ flux is poorly understood. The pollen tube as an invasive growing cell is beaconed by diverse female signals and transduces these signals into the intracellular growth machineries, such as the Ca2+ signaling, for the navigation into the embryo sac. How the pollen tube realizes this molecular integration from outside to the inside Ca2+ dynamics for the guided growth is unknown and important to understand the reproduction and adaption strategies of plant cells. Here we report a mechanism for the directional exocytosis of cargos in pollen tube response to the female signals. Mutants of MALE SENSOR (MAS), which encodes a plasma membrane protein, show abnormal pollen tube response to the secreted ovular cues in Arabidopsis thaliana. Protein affinity-based mass spectrometry showed that MAS forms a physical complex with a cysteine-rich peptide and a receptor-like kinase that regulate pollen tube guidance. Molecular and biochemical studies reveal that MAS selectively tether Ca2+-related cargos to the plasma membrane where the extracellular signals are perceived through the SNARE proteins in a trans mode. These results reveal a new mechanism of molecular integration of extracellular cues and selective exocytosis, and will shed light on the general regulation of cell response to the environment.

LORELEI (LRE), and its most closely related paralog LLG1 (LORELEI-LIKE GPI-Anchored Membrane Protein 1) arose from the most recent whole genome duplication (WGD) in Brassicaceae. lre and llg1 mutants in Arabidopsis have no overlapping phenotypes; hence, we hypothesized that LRE and LLG1 were maintained post gene duplication because the two genes split the functions of the ancestral single copy gene (subfunctionalization) found in Cleome violacea (a member of the Cleomaceae, a sister group to Brassicaceae). To test this hypothesis, we performed cross-complementation experiments with LRE and LLG1 and showed that each gene can complement the defects caused by the loss of the other gene. Additionally, we used the single copy gene of LRE/LLG1 in Cleome violacea (CleviLRE//LLG1) to complement Arabidopsis thaliana lre and llg1 mutant phenotypes. Successful cross complementation results led us to propose another explanation for retention of both genes post duplication: the expression domains in the promoters of LRE and LLG1 diverged in a non-overlapping manner, allowing both genes to perform similar functions but in different tissues and cells (regulatory subfunctionalization hypothesis). Using promoter:GUS transcriptional fusions, we found that LRE and LLG1 have distinct expression in Arabidopsis. Additionally, we showed that CleviLRE/LLG1 is expressed in Cleome ovules and vegetative tissues. Additional diversification in the expression of these two genes have been reported, as LRE, but not LLG1, is a maternally-imprinted gene. These results strongly support the regulatory subfunctionalization hypothesis that post gene duplication, LORELEI and LLG1 maintained their molecular functions, but have divergent expression.

In flowering plants, the fertilization of an egg cell by a sperm cell results in embryo development. The molecular pathways that control embryo initiation and prevent its occurrence without fertilization, are not well understood. Our previous transcriptome analysis of rice gametes and zygotes identified transcription factors that are specifically induced in zygotes after fertilization, including AP2-family transcription factors from the PLETHORA/ BABY BOOM clade (Anderson et al. 2017 Developmental Cell 43: 349–358). One of these factors, BABY BOOM1 (BBM1), is expressed in sperm cells and only from the male allele in zygotes immediately after fertilization. However, the expression becomes biparental before the first zygotic division, likely arising from the capability of BBM1 auto-activation, which we observed in leaf cells by using an inducible BBM1-GR protein. Ectopic expression of BBM1 in the egg cell resulted in parthenogenesis and the production of haploid progeny (Khanday et al. 2018 Nature 565: 91–95). Thus, expression of this single male-genome expressed transcription factor in the egg cell is sufficient to overcome the fertilization block and initiate embryogenesis. Putative targets of BBM1 include auxin biosynthetic genes, implying that embryo initiation after fertilization involves auxin signaling. Triple knockouts of BBM1 and two other BBM-like genes (BBM2 and BBM3) result in embryo arrest. Embryo arrest was observed even if a wild-type copy of BBM1 is inherited from the female parent, but is fully rescued by wild-type BBM1 from the male parent. A parent-of-origin dependent embryogenesis phenotype was also observed for BBM2. Thus, expression of BBM-family transcription factors from the paternal genome plays a key role in early embryogenesis in rice. More generally, these findings suggest that at least in rice and related cereals, the fertilization requirement for embryogenesis might act in part through male-transmitted pluripotency factors.

The evolution of sex determination in plants is a central problem in plant evolutionary biology. Currently, there have been limited studies in which the sex determination genes are identified yet we do not know most of the alternative downstream pathways that lead to developmental differences in plants that exhibit sexual dimorphism. Addressing this gap in knowledge is important as it will give insight into the genetic regulation of developmental processes in unisexual flowers and in angiosperm flowers in general. The investigation into the link between the differential expression patters of genetic pathways and the differential expression in floral development involves the differential expression of AG, WUS, and the proposed transcription repressor gene KNUCKLES (KNU) as they relate to the differential formation of floral organ primordia in male and female Spinacia oleracea flowers. Our central hypothesis is that the AG-KNU-WUS pathway regulates the differential morphogenesis of organ primordia between male and female flowers leading to sexual dimorphism in spinach. To test this hypothesis, molecular genetics tools are utilized to quantify KNUCKLES temporal and spatial expression patterns, along with functional testing. Preliminary studies have begun that include characterizing KNU-like gene expression and the phenotypes of KNU-like knockdowns in S. oleracea. Preliminary results show strong phenotypes in the vegetative tissue that are related to the regulation of organ primordia and meristem maintenance.

Cellular thiols such as cysteine and glutathione spontaneously and readily react with the respiratory intermediate fumarate, resulting in the formation of stable S-(2-succino)-adducts. Fumarate-mediated succination of thiols increases in certain tumors and in response to glucotoxicity associated with diabetes. Therefore, S-(2-succino)-adducts such as S-(2-succino)cysteine (2SC) are considered oncometabolites and biomarkers for human disease. 2SC has not been detected in plants suggesting that they have a mechanism to prevent its accumulation. Recently, a pathway for 2SC disposal was discovered in Bacillus subtilis, but this pathway is only present in firmicute bacteria. A comparative genomics analysis identified two putative alternate pathways for 2SC disposal in prokaryotes; the enzymes of one of these pathways have close homologs in plants. The predicted plant-pathway is initiated by an acetyltransferase (At2g39000 or At4g28030) that acetylates 2SC. A glutathione-S-transferase-like enzyme (At5g44990 or At5g44000) then cleaves acetylated-2SC into succinate and N-acetylcysteine. Biochemical and genetic characterization of this pathway is ongoing. This pathway, if confirmed, represents a metabolite damage control system that could have applications in improving stress tolerance and metabolic engineering in plants. This work also nicely exemplifies the use of cross-kingdom comparative genomics to predict the function of unknown genes in plants.

Nitric oxide (NO) is a short-lived gas that acts as a signaling molecule in all higher organisms, including plants. Despite the clear involvement of NO in multiple plant processes, including germination, root growth and fertility, a basic understanding of the mechanisms by which NO exerts its effects on systems critical for plant growth and development is lacking. Reversible S-nitrosation of critical protein cysteines due to reaction with nitric oxide (NO) and its derivatives is a redox-dependent posttranslational modification that impacts these plant physiological processes. Regulation of NO-levels in planta is predominantly achieved by reaction of reactive nitrogen species (RNS) with glutathione (GSH), thereby forming S-nitrosoglutathione (GSNO), the principal NO reservoir. Mutation of Arabidopsis S-nitrosoglutathione reductase (GSNOR) leads to higher intracellular concentrations of S-nitrosothiols, confirming that the reduction of GSNO by the enzyme is a major route of GSNO catabolism in plants. GSNO-breakdown is believed to help sustain cellular redox poise both by curtailing RNS-bursts and by regenerating GSH. GSNOR contains evolutionary-conserved cysteine residues that are prone to S-nitrosation by different NO donors, leading to a partial loss of enzyme activity that could be recovered by reducing agents in vitro. Protein nitrosation was further confirmed by intact mass spectrometry, for which signals consistent with mono-, di- and tri-nitrosation were observed. In addition, GSNOR denitrosation analysis catalyzed by small oxidoreductases will be addressed. These data implicate a mechanism for RNS signaling by modulating redox-dependent posttranslational modifications of certain proteins. Reduced GSNOR activity is predicted to result in the accumulation of GSNO, itself an agent of protein S-nitrosation. By allowing GSNO to accumulate, inhibition of GSNOR may facilitate more robust NO signaling that regulates plant growth and developmental processes in plants.

We have obtained the crystal structures of Arabidopsis xyloglucan xylosyltransferase 1 (XXT1) without ligands and in complexes with the substrates, UDP and cellohexaose. XXT1 initiates side-chain extensions from a linear glucan polymer by transferring the xylosyl group from UDP-xylose during xyloglucan biosynthesis. XXT1, a homodimer and member of the GT-A fold family of glycosyltransferases, binds UDP analogously to other GT-A fold enzymes. Structures and the properties of mutant XXT1s are consistent with a SNi-like catalytic mechanism. Distinct from other systems is the recognition of cellohexaose by way of an extended cleft. Steric conflicts in the acceptor binding cleft disallow XXT1 alone to produce the complete xylosylation patterns observed for native xyloglucans. Homology modeling of XXT2 and XXT5, the other two xylosyltransferases involved in xyloglucan biosynthesis, reveals the presence of an empty pocket in XXT5 that is large enough to encompass the xylose of a partially xylosylated glucan chain. The structural organization of three XXTs, unraveled in our study, support the existence of an organized multi-enzyme complex involved in the xyloglucan synthesis and explain how the particular XXXG pattern is synthesized. Results from computational docking suggest subunit interfaces of the homodimer XXT1 and the heterodimer XXT2-XXT5 are similar; however, different surfaces of the XXT1 homodimer and the XXT2 subunit in the XXT2-XXT5 heterodimer can interact to form a linear trimer of dimers in which the XXT1 homodimer occupies the central position, thus confirming our experimental observations. We propose a model of a multi-enzyme complex organization to produce the specific xylosylation patterns of the native xyloglucan in which the high substrate specificity of each of the XXT is mediated by steric constraints within their acceptor substrate active site cleft. This model significantly extends our limited understanding of polysaccharide biosynthesis in Golgi

The plant shikimate pathway directs bulk carbon flow to support biosynthesis of aromatic amino acids (AAAs) and numerous natural products including phytohormones, cofactors, pigments, phytoalexins, lignin, and more. These aromatic phytochemicals play critical roles in plant physiology and adaptation, and also provide essential nutrients, medicine, and industrial materials to the human society. In microbes, the shikimate pathway is feedback inhibited by AAA effectors at the first enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DHS). Although the DHS-catalyzed step is also thought to be regulated in plants, effectors that regulate plant DHS have not been identified for decades . Here, we generated recombinant enzymes of all three DHS isoforms of Arabidopsis thaliana (AthDHS1, AthDHS2, and AthDHS3) and conducted their biochemical characterization. Only the AthDHS2 isoform, but not AthDHS1 or AthDHS3, was negatively regulated by tyrosine or tryptophan, whereas phenylalanine had no effects. Chorismate, the final product of the shikimate pathway, strongly inhibited the activity of all three AthDHS enzymes, which was counteracted by a further downstream intermediate, arogenate. Caffeic acid and its derivatives, key intermediates of the phenylpropanoid pathway, were also effective inhibitors of AthDHS enzymes, uncovering a potential regulatory link between the shikimate and phenylpropanoid pathways. DHS activity detected from leaf crude extracts were inhibited by chorismate and caffeic acid, but not by any of AAAs, which appears to be due to the loss of the AthDHS2 AAA-sensitivity in the presence of AthDHS1. These findings reveal unique and highly-complex regulatory mechanisms of the entry reaction of the plant shikimate pathway and provide foundational knowledge to control the production of AAAs and diverse natural products in plants.

Cadaverine, a polyamine produced by plants and microbes, modulates root architecture by decreasing primary root growth, promoting lateral root development, and altering root skewing and waving. To identify genes involved in cadaverine response, a forward genetic screen was carried out in Arabidopsis thaliana. One of the identified hypersensitive mutations was mapped to a nonsynonomous polymorphism in the biotin-synthesis BIO3-BIO1 gene, affecting the catalytic pocket of DAPA synthase. Treatment with exogenous biotin suppressed the inhibitory effect of cadaverine on primary root growth in wild-type seedlings, and it alleviated cadaverine hypersensitivity of bio3-bio1 mutant, suggesting the biotin synthesis pathway is a target for cadaverine action. Arabidopsis BIO3-BIO1 enzyme was expressed in E. coli, affinity-purified and tested in in vitro enzymatic reactions leading to DTB synthesis, using KAPA as a substrate. Both putrescine and cadaverine were found to function as efficient amino donors. However, cadaverine appeared to somewhat inhibit the reaction when added together with putrescine. These preliminary data suggest that both putrescine and cadaverine can function as amino donors, but cadaverine is either catabolized less efficiently, or more strongly retained in the binding site of the enzyme, relative to putrescine thereby inhibiting the reaction. Biotin is an important molecule used as a co-factor in a number of carboxylation and decarboxylation reactions involved in central metabolism. Quantification of biotinylated proteins in cadaverine-treated seedlings showed reduced amounts of BCCP1, a subunit of Acetyl-CoA carboxylase. A lipidomic analysis revealed significant changes in the lipid profiles of cadaverine-treated seedlings relative to the control. We propose that cadaverine controls root growth by inhibiting biotin synthesis, thereby affecting central metabolic pathways. This works is supported by a UW-Madison HATCH grant.

Aminophospholipid ATPases (ALAs) are lipid flippases involved in the uptake and translocation of specific lipids across membrane bilayers. Arabidopsis thaliana contains 12 ALAs that sort into five phylogenetic clusters, including five in cluster 2 (ALA8, 9, 10, 11, and 12) and four in cluster 3 (ALA4, 5, 6, and 7). Here we show that double mutants lacking ALA4 and 5 (cluster 3) are severely dwarfed, characterized by reduced growth in rosettes (6.5-fold), roots (4.3-fold), bolts (4.5-fold), and hypocotyls (2-fold). Plant size reductions correlated with reductions in cell size, suggesting that ala4/5 mutants are dwarfed in part due to cellular expansion defects. Dwarfism was also associated with perturbations in the content of both glycerolipids and sphingolipids, most notably a ~2-fold increase in glucosylceramides (GlcCers) which could potentially inhibit growth. Uptake assays in yeast suggested that ALA5 was capable of transporting specific lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), as well as the sphingolipid sphingomyelin (SM). However, this assay detected no transport for GlcCers, suggesting GlcCer increases in ala4/5 mutants likely arise from indirect pathways. In comparison to other ALAs, the PC > SM > PE > PS transport profile for ALA5 was very similar to that of ALA10 (cluster 2), with the most notable exception being that ALA10 can transport lysophosphatidylcholine. Interestingly, a suppressor mutant screen on mutagenized ala4/5 seedlings was used to identify three dominant suppressor mutants that had near wildtype rosette growth, all of which caused similar disruptions in a putative regulatory domain of a cluster 2 ALA. These results suggest that the biochemical activity of ALA4/5 from cluster 3 is of critical importance for plant and cell growth, and that this distinct activity originates from a putative regulatory domain that differentially controls the activity of cluster 2 and cluster 3 ALAs.

The leaves of stevia (Stevia rebaudiana) contain numerous glycosides extracted for use as nonnutritive sweeteners, which are utilized in a rapidly expanding portfolio of food and beverage products. Increased global demand prompted the investigation of stevia as a new crop in the southeast United States beginning in 2011. Since the first commercial planting in North Carolina, numerous diseases including southern blight (Athelia rolfsii syn. Sclerotium rolfsii) and Septoria leaf spot (Septoria steviae) emerged as economically relevant due to their potential for dramatic yield loss. Leaf lesions caused by a Septoria sp. were present throughout the season in 2015, spreading rapidly up the plant and causing significant defoliation during favorable environmental conditions prior to harvest. Type culture isolates of Septoria steviae, collected in Japan in 1982, were obtained for comparison to NC isolates. All isolates were sequenced for seven loci: actin, β-tubulin, calmodulin, internal transcribed spacer, nuc rDNA 28S subunit, RNA polymerase II second largest subunit, and translation elongation factor-1alpha. The isolates from NC formed a well-supported monophyletic group with the ex-type culture of S. steviae confirming it as a unique species. Management strategies for Septoria leaf spot as well as other diseases affecting overwintering survival of this perennial plant are needed. Fungicide trials were conducted from 2014-2018 to evaluate product efficacy and potential for US fungicide labels. In spring 2016 and 2017, plants that received an application of strobilurin (QoI) fungicide had higher overwintering emergence and reduced isolation of Macrophomina phaseolina, an important soilborne pathogen that may limit perennial production without visible foliar symptoms. Continued understanding of pathogen biology and disease management will be critical to the expansion of acreage and long-term establishment of stevia as a viable crop in the US.

Ripening is a well-characterized process during fruit development. However, fruit crops represent several variations in this ubiquitous process. European pear (Pyrus communis L.), a climacteric fruit, represents one such anomaly where the fruit are harvested at maturity but in an unripe state. Ripening can only be achieved by incubating the fruit in a genetically pre-determined amount of cold during postharvest stages. Further, the use of an ethylene receptor inhibitor, 1-methylcyclopropene (1-MCP) on the unripe fruit results in permanent ‘locking’ of ripening. We identified that the alternate respiratory pathway is activated during pre-climacteric stages, unlike other climacteric fruit, as the fruit undergoes cold conditioning. Using this information, a chemical genomics approach was used to ripen 1-MCP treated fruit. We hypothesize that chemical activation of the alternative respiratory pathway activates the TCA cycle leading to the generation of ethylene. We have utilized this recently patented technology to enable the development of high quality fresh sliced pears. Sweet cherry (Prunus avium L.), taxonomic kin of pear in the Rosaceae family, is characterized as a non-climacteric fruit. However, exogenous application of ethylene induces the formation of an abscission zone at the fruit pedicel junction. We identified an ethylene-inducible variant of an ERF transcription factor in ‘Bing’ cultivar. Incorporation of this allele in breeding strategies or editing could facilitate mechanical harvesting of this important crop.

Soybean has been subjected to genetic manipulation by various approaches such as breeding, mutation, and transgenesis to produce value added quality traits. Among those approaches, mutagenesis through fast neutrons radiation is intriguing because it yields a variety of mutations, including single/multiple gene deletions and/or duplications. Characterizing the seed composition of the fast neutron mutants and its relationship with gene mutation is useful towards understanding oil and protein traits in soybean. From a large population of fast neutrons mutagenic plants, we selected ten mutants based on a screening of total oil and protein content using near infra-red spectroscopy. The mutant 2R29C14Cladecr233cMN15 (nicknamed as L10) showed the highest protein and lower oil content compared to wild type, followed by three other lines (L03, L05, and L06). We have physically mapped the position of the deletion or duplications of genes in each mutant using comparative genomic hybridization (CGH). All ten lines had one or more deletions and/or duplications. We selected the L03 mutant for detailed proteomic analysis because it exhibited 55% protein while only showing a homozygous deletion encompassing few genes. A proteomic profiling of the wild type and L03 revealed 3,502 proteins, of which 206 proteins exhibited increased abundance and 214 decreased abundance. Among the abundant proteins, basic 7S globulin increased four-fold, followed by vacuolar-sorting receptor and protein transporters. The differentially expressed proteins were mapped to the global metabolic pathways. A higher enrichment in ribosomal, endoplasmic reticulum, protein export and purine metabolic pathways were observed. A shift of carbon metabolism towards amino acid formation was also observed. The deletion of the sequence-specific DNA binding transcription factor along with 22 other genes may have caused a cascade effect on protein synthesis, resulting in an increased amount of 7S globulin.

Apple (Malus domestica) is one of the world’s most valuable fruit crops and a promising candidate for marker-assisted selection (MAS) due to its lengthy juvenile phase. To discover genotype-phenotype associations, we generated approximately 250,000 SNPs using genotyping-by-sequencing for the Apple Biodiversity Collection (ABC) located in Kentville, Nova Scotia, Canada. In 2017, phenotype data were collected from over 1,300 trees in the ABC, representing over 850 unique accessions. Accessions were also genotyped for several markers previously discovered using linkage mapping and currently used for MAS in apple. Genome-wide association study (GWAS) results confirmed the transcription factor NAC18.1, previously identified in a GWAS of the USDA apple germplasm collection, is a strong functional candidate for fruit firmness and harvest date. In comparison, no significant associations were identified for the firmness markers currently used in apple breeding. NAC18.1 is homologous to the NON-RIPENING (NOR) gene of tomato. While nor mutant fruits fail to ripen, transgenic complementation of nor with the apple NAC18.1 gene rescued its ripening defect, confirming NAC18.1’s role as a conserved regulator of fruit ripening. These results demonstrate that GWAS in a diverse apple collection results in extremely high resolution mapping of putatively causal variants, which holds great potential for continued improvement of apples through MAS.

Cannabis fever is taking over the United States. Used medicinally, recreationally and functionally (i.e., fiber, construction, etc.) for millennia, Cannabis has a global distribution and deep ties to human culture. In recent history, the Cannabis field in the US has been heavily influenced by its Schedule I classification. Despite this prohibition, Cannabis cultivation still persists underground and in isolation. With public opinion quickly changing, more states are legalizing Cannabis, enabling the scientific community to investigate this plant, allowing us to pose basic biological questions about genetic diversity and ancestry. To explore the current breadth of genetic diversity in Cannabis and how that relates to naming conventions, we present analyses of genome-wide SNP data from a large collection of Cannabis accessions. Our results indicate discrepensives in Cannabis labeling and genetic clustering based on classic Cannabis ideotypes (i.e., Indica and Sativa). As prohibition of Cannabis wanes, understanding genetic diversity and its relationship with cultivar identification is imperative for advancing the Cannabis field.

Unfertilized female flowers of the dioecious Cannabis sativa plant are a rich source of cannabinoids, including tetrahydrocannabinoid (THC) and cannabidiol (CBD), which may have some medical applications. In fact, the FDA has recently approved a highly purified form of CBD as an efficacious pharmaceutical drug called Epidiolex® for reducing seizures in some epileptic conditions. No research has documented which cannabinoid biosynthesis pathway enzyme(s) contribute to rate-limitation of carbon flow to end-products (THC and CBD). The objective here was to monitor expression of genes encoding these enzymes in developing flowers of female hemp (< 0.3% THC) cannabis and examine if changes in expression of any of these genes is associated with increasing CBD. We used real-time (quantitative) qPCR to monitor expression of CBDa synthase, geranylpyrophosphate (GPP):olivetolate geranyltransferase (GOT) synthase, olivetolic acid synthase, GPP synthase, and THCa synthase. High-performance liquid chromatography (HPLC) was used to monitor cannabinoid levels in developing flowers in three commercial strains of hemp. These strains include: ‘Wife’, ‘Abacus’, and (Otto 2 x BaOx) x (Colorado Cherry x BaOx). Hemp plants were grown in a greenhouse, and weekly samples were taken to assess gene expression and cannabinoid content. As the flowers matured, there was a strong upregulation in CBDa synthase expression. This was accompanied by a significant rise in CBD content. Expression of the gene (GOT synthase) encoding the enzyme that generates the first cannabinoid in the pathway cannabigerol (CBG) did not change during flower development. In addition, the high-level CBD producing strain, (Otto 2 x BaOx) x (Colorado Cherry x BaOx), had higher levels of CBDa synthase expression compared to the two other varieties that produced lower levels of CBD. Supported by Dr. David Levine research support fund and Connecticut Pharmaceutical Solutions LLC.

The chemical diversity of plant natural products has provided humans with a variety of intriguing structures and biological activities. The biological function of most plant natural products remains unstudied, but in general their presence is believed to increase organismal fitness. Commonly, many natural products are thought to play a role in communication of the plant with its environment, as these compounds possess an array of biological activities. Due to these biological activities, 25% of medicines today are either derived directly from plants or are structural modifications of plant natural products. An understanding of how these molecules are formed would serve a dual role to enable a study of the in planta function, as well as development of a synthetic biology production platform. Natural products typically do not accumulate to high levels in the plant. If the source plant for a novel drug is not amenable to cultivation, drug development can be precluded. Engineering of a natural product biosynthetic pathway into an easily cultivated host plant can result in a sustainable supply of a drug. The first obstacle to this approach, however, is knowledge of the underlying biosynthetic genes. Biochemical pathway elucidation in non-model systems has often taken decades to complete. A prominent example is the well-known plant natural product morphine produced by the opium poppy Papaver somniferum. Though discovered in the early 1800’s, the biosynthetic pathway to morphine was not completely elucidated until 2014. Next-gen sequencing technology enables revolutionary new approaches to biochemical pathway discovery in the non-model system. A combination of bioinformatics and next-gen sequencing has the potential to shorten natural product pathway discovery in non-model systems from several decades to several years. Presented herein are results obtained to date elucidating and refactoring a pathway to steroid alkaloids.

The ability to engineer plants using the tools of synthetic biology is of increasing importance for solving agricultural problems, and for adapting plants to novel uses. A collaboration between five labs has begun to engineer plant-to-plant communication using volatile organic compounds (VOCs). By modifying and enhancing natural hormone pathways in plants we have been able to demonstrate synthetic communication. In particular, we have engineered a modular ethylene sensor, and identified modular promoter structures that are responsive to methyl salicylate and methyl jasmonate. When these are cloned adjacent to reporter genes, gas-dependent production of signals can be observed. In parallel, we have determined that carbon flux through the normal pathway for the production of the volatile ethylene may be limited, and have been able to generate transgenic plants with enhanced ethylene production. When ethylene ‘senders’ are aligned with ethylene ‘receivers,’ plant-to-plant communication can be observed. Along the way, we have developed an extensive new tool kit that allows for the establishment of an Orthogonal Control System (OCS) in plants that operates on top of extant plant regulatory and metabolic systems. We have for the first time created wholly orthogonal transcription factors using dCas9:VP64 as a transcription factor, and shown that these can activate completely artificial promoters. While these demonstrations have so far been shown in model plant species (Nicotiana, Arabidopsis), in parallel we have undertaken an effort to demonstrate that constructs developed can be transported into new species, and to this end have made great progress in ‘taming’ a non-model plant, common dandelion (Taraxacum). Ultimately, by funneling engineered sensor ‘inputs’ through VOC communication channel to appropriate reporter ‘outputs’ it should be possible to allow fields of plants to better serve as self-sentinels against pests and other environmental incursions.

Plants are a rich source of unique molecules, including 25% of natural-product-derived drugs. However, the discovery, synthesis, and overall material supply chains for sourcing plant-based medicines remain ad hoc, biased, and tedious. While microbial biosynthesis presents compelling alternatives to traditional approaches based on extraction from natural plant hosts, many challenges exist in the reconstruction of plant specialized metabolic pathways in microbial hosts. We have developed approaches to address the challenges that arise in the reconstruction of complex plant biosynthetic pathways in microbial hosts. We have utilized these strategies to develop yeast production platforms for an important class of plant alkaloids, which include the medicinal opioids and noscapinoids. The intersection of synthetic biology, genomics, and informatics will lead to transformative advances in how we make and discover essential medicines.

Historically, plants have played a prominent role in human medicine. The medicinal effects of plants are due to bioactive small molecule natural products, many of which continue to serve as major sources of clinical pharmaceuticals. One such compound is the alkaloid colchicine, which is produced by plants from the Colchicum and Gloriosa genera and is used clinically for treating gout and other inflammatory diseases. Although previous studies have identified specific plant tissues associated with colchicine biosynthesis, along with the precursors from which this molecule is derived, the underlying biosynthetic genes have remained unidentified. To facilitate colchicine biosynthetic pathway discovery, we have performed extensive metabolite profiling in Gloriosa superba and paired this to corresponding RNA-seq expression data, with the hypothesis that relative expression of biosynthetic genes should correlate to accumulation of colchicine alkaloids. By then using a combination of correlative expression analyses and enzymatic logic, we selected a testable number of candidate biosynthetic enzymes for functional screening via heterologous expression in tobacco. Through this methodology, we have discovered and characterized 7 novel enzymes that act to produce a late stage colchicine intermediate from a 1-phenethylisoquinoline substrate that is a known precursor to colchicine. Furthermore, by utilizing enzymes from plant primary metabolism, along with several involved in the biosynthesis of natural products from other plants, we have engineered a 16-enzyme pathway in tobacco that connects the newly discovered biosynthetic steps in colchicine biosynthesis to primary amino acid metabolism from the heterologous tobacco host, thus allowing for metabolic engineering of colchicine alkaloids. This result not only establishes a nearly complete metabolic route to colchicine, but also pushes the boundaries for the rate and magnitude of biosynthetic pathway discovery in plants.

Pyrethrum (Tanacetum cinerariifolium) plants have been known since antiquity for the presence of a group of natural pesticides, called pyrethrins, in its flowers. The six types of pyrethrins produced in pyrethrum are all esters of a monoterpenoid acid (chrysanthemic acid or pyrethric acid) and a jasmonic acid-derived alcohol (jasmolone, pyrethrolone or cinerolone). Recently, we have begun to identify the enzyme responsible for the synthesis of the alcohols. By comparing the structure of these alcohols with that of JA, we hypothesized that jasmolone may be generated by hydroxylation of jasmone, a catabolite of JA, and that pyrethrolone could be derived from jasmolone by additional oxidation step. Through correlation analysis of transcriptomic data and metabolomics data of different pyrethrum tissues, eleven P450 candidate genes were selected. The candidate genes were transiently expressed in Nicotiana benthamiana leaves which were fed with jasmone, and the tobacco tissues examined for the production of new alcohols. Using this approach, one introduced P450 gene was shown to encode an enzyme capable of hydroxylating jasmone to give jasmolone. This gene was accordingly named Jasmone Hydroxylase (TcJMH). Furthermore, by coexpressing TcJMH with the rest of the P450 candidates in the tobacco system, a second gene was found to encode an enzyme that converts jasmolone to pyrethrolone directly, and this gene was named Pyrethrolone Synthesis (TcPYS). The enzymatic activities of TcJMH and TcPYS were further verified in in vitro assays. Coexpressed of TcJMH and TcPYS with TcGLIP, the enzyme that forms the pyrethrin esters, in tobacco leaf fed with jasmone and chrysanthemic acid, led to the production the pyrethrin molecules jasmolin I (the ester of chrysanthemic acid with jasmolone) and pyrethrin I (the ester of chrysanthemic acid with pyretrolone), indicating the possibility of engineering the production of these human-safe natural pesticide in other plant species.

Blueberry (Vaccinium corymbosum) is an economically important fruit crop that is native to North America. Fresh market production of blueberries in the United States was valued at $5.68 billion in 2015 and was planted over 36,349 hectares. In addition to its commercial value, blueberries are prized for their positive health benefits, containing high levels of antioxidants, which has been linked to a decreased risk of cancer and heart disease. Iridoids are another class of known pharmacologically important compounds that have recently been found in blueberries. Iridoids are present in over 15 plant families and are potent natural products with a wide range of biological activities in humans including, anticancer, antibacterial and anti-inflammatory. No work however, has been able to detect monotropein, an iridoid glycoside compound, in any North American blueberry species (V. corybosum, V. angustifolium, V. virgatum), the most commonly used germplasm for cultivated blueberry. To address this research limitation I have collected over 80 berry and leaf samples from multiple species and commercial varieties of blueberry to survey for monotropein production. The glycoside iridoid monotropein was successfully identified in a subset of cultivars in the diversity panel, as well as all wild blueberry species in this panel, indicating iridoid production can be targeted through breeding efforts that incorporate wild germplasm. Currently, both metabolite and transcriptomic data are being leveraged to identify key iridoid biosynthetic pathway genes in blueberry. In addition to providing molecular markers to breed for higher iridoid content, knowing how iridoids are synthesized will enable much improved access to these compounds for future clinical research.

Plants live in an ever-changing and unpredictable environment. As sessile organisms, they must cope with perturbations to their particular microhabitat in space and time. Which environments matter most? What physiological or metabolic mechanisms buffer responses to the environment and climatic change? How are these responses encoded in genomes and how do they evolve? Genome-enabled research has characterized the myriad expression and metabolite responses of many species to common stresses including drought, temperature extremes, light stress and salinity. The challenge now is to disentangle evolved and adaptive responses of plants to stress from the deleterious results of stress. A promising avenue is the use of locally adapted natural variation to winnow the beneficial responses from the maladaptive consequences of stress. Switchgrass (Panicum virgatum) is a polyploid C4 perennial grass that is native to North America and has been championed as a promising biofuel feedstock. It is a common member of most native prairie communities and exhibits extensive phenotypic variability and adaptation across its range, especially related to latitude and precipitation gradients. Much of this variability is associated with evolved lowland and upland ecotypes. Here, I report on the development of genetic and genomic resources for switchgrass, as well as present results from field experiments aimed at understanding upland/lowland ecotype divergence and local adaptation. In particular, I present preliminary results from QTL studies aimed at detecting gene-by-environment interactions for a variety of traits utilizing collaborative common garden experiments across the species latitudinal/climatic range.

Plant populations that currently exist have adapted to past and current climates. Plants are also major controls on the carbon cycle and thus can reduce anthropogenic climate change. I will present findings on the traits and loci that underlie adaptation to climate. I will also introduce more recent work to identify the genetics controlling plant traits that can contribute to carbon sequestration in highly managed agricultural systems.

Some, but not all, plants make isoprene, which is lost from the plants and causes substantial effects on atmospheric chemistry. Plants making isoprene, or exposed to isoprene, are better able to tolerate some stresses, especially high temperature, but tolerance of other stresses is not affected. Tolerance of chilling stress appears to be reduced by isoprene and isoprene emission is reduced at low temperature. Gene expression changes are consistent with a role of isoprene in preparing plants to tolerate stress. Genes involved in synthesis of jasmonic acid and abscisic acid are expressed at higher levels when isoprene is present although salicylic acid related genes are not affected. Isoprene also affects plant growth. In some cases, growth is enhanced and in some cases it is suppressed. Expression of transcription factors likely to affect DELLA and PIF proteins, which are related to growth, is altered by isoprene when growth is stimulated. Development is also stimulated and expression of genes related to cytokinins is found to be altered by isoprene. It appears that isoprene can cause widespread changes in gene expression that alters growth/defense tradeoffs and results in plants better prepared for warm season stresses. This is consistent with the observations of very large temperature effects on isoprene emission capacity.

Night-time temperatures are increasing at a faster pace than day-time temperature, posing a serious threat to wheat production. Different physiological routes through which HDT and HNT induces yield and quality losses have been documented in cereals. A combination of physiological, metabolomic and source-sink starch metabolism related enzymatic responses has helped us to unravel HNT responses in wheat. We imposed a sequential increase in night-time temperatures (from 15o to 27oC) on ten different winter wheat genotypes, to determine the threshold for HNT inducing yield reduction and loss in quality (protein and starch). Based on the identified threshold, source, sink and stem metabolic changes were captured on selected contrasting genotypes exposed to HNT during grain-filling stage. Using leaf and grain samples collected during grain filling, exposed to different night-time temperatures, we have identified key bottlenecks related to sugar translocation and accumulation in wheat grains exposed to HNT. Transmission electron microscopy was employed to ascertain the accumulation of starch, protein and lipids in the endosperm and the germ under different night-time temperatures. Findings generated from controlled chambers and unique field-based heat tents will be presented, with an overall goal to translate findings in to developing HNT tolerant wheat varieties.

The tendency for dicotyledonous species of drier climates to have smaller leaves is one of the most famous global biogeographic trends. One explanation has focused on their shared leaf developmental program that leads to larger leaves having lower vein length per leaf area, such that smaller leaves gain in drought tolerance. These patterns have not been previously tested in grasses (family Poaceae), a lineage that radiated across climates and ecosystems, dominates ≈40% of the Earth’s land surface, and accounts for 33% of terrestrial primary productivity. We show that across grasses worldwide, species adapted to drier climates had narrower leaves with higher major vein density, associated with narrower leaves, a trend confirmed for 1753 globally distributed grass species. We present a synthetic model for leaf development for grasses, analogous to that of dicot leaves, based on published histological data, which predicts general scaling relationships for venation traits with leaf dimensions. Tests on 26 C3 and C4 grass species grown in a common garden showed that venation architecture showed the predicted developmental scaling, such that species with wider leaves had lower major vein densities, and species with longer leaves had greater major vein diameters, whereas the venation architecture of minor veins was independent of leaf dimensions. These trends can explain the distribution of shorter, narrower leaves in drier climates, and provide a strong example of how the differential elaboration of a conserved developmental process can determine worldwide macroecological patterns.

Plants transition through distinct stages as they develop, the timing of which, significantly impacts large-scale ecological and evolutionary processes. Vegetative phase change (VPC), the developmental transition from juvenile to adult vegetative growth, has been well studied at the molecular level however, little is known about its importance for plant performance and physiological functioning. In this study, variation in physiological and morphological characteristics between juvenile and adult leaves of four diverse species, Zea mays, Passiflora edulis, Populus tremula x alba, and Arabidopsis thaliana, were analyzed. Mutants of miR156, the master regulator of VPC, were used to modulate the timing of VPC in all four species to investigate differences in photosynthetic traits and leaf morphology associated with vegetative development. Further, these mutants were used to determine whether variation in traits between juvenile and adult leaves translate into variation in plant performance under environmental stress. Through this research we found significant variation in photosynthetic properties between juvenile and adult leaves in all species including maximum photosynthetic rates and rates of photosynthetic light induction. Additionally, juvenile and adult leaves showed differences in morphology including specific leaf area, stomatal densities and venation, traits with major implications for foliar function. Lastly, when both mutants and natural genotypes with variation in the timing of VPC were subjected to the environmental stresses of heat, drought and low light, there were significant relationship between plant performance under some of these stresses and the timing of VPC. This research begins to uncover the role of vegetative development in plant physiology, potential mechanisms to be utilized in plant breeding programs and insight into the underpinnings that may have led to the evolutionary conservation of VPC and its master regulator miR156.

Coordination of growth and division in eukaryotic cells is thought to be mediated by size checkpoints, but the mechanisms for size homeostasis are largely unknown. The green alga Chlamydomonas divides by a multiple fission cell cycle, where the Commitment checkpoint ensures enough growth for completion of at least one division, and the DNA synthesis/mitosis checkpoint ensures mother cells undergo the correct number of divisions to produce uniform-sized daughters. tny1-1 was identified in a insertional mutagenesis screen and exhibits a recessive small phenotype due to defects at both checkpoints. TNY1 encodes a predicted hnRNP A-related RNA binding protein with two N-terminal RNA recognition motifs and a low complexity glycine-rich C-terminus—a structure shared by many eukaryotic hnRNPs. Microscopy showed that TNY1 is cytosolic throughout the cell cycle. Immunoblotting revealed that daughter cells are born with a fixed amount of TNY1, whose absolute abundance remains constant on a per-cell basis during G1 phase, but whose overall cellular concentration decreases as cells grow. TNY1 mRNA and protein levels peak during cell division and are reset to the highest concentration in newly-formed daughters. Altering the dosage of TNY1 in diploids impacted daughter cell size, indicating that TNY1 is limiting for size control. Epistasis experiments placed TNY1 upstream of cyclin dependent kinase CDKG1, one of whose substrates is MAT3/RB (retinoblastoma tumor suppressor homolog). In wild-type cells CDKG1 is produced before division and eliminated upon mitotic exit, but in post-mitotic tny1-1 mutants CDKG1 remains detectable, suggesting TNY1 inhibits CDKG1 accumulation. North-Western assays showed that TNY1 binds to the unusually long and uridine-rich 3’ UTR of CDKG1 mRNA but not to its CDS or 5’ UTR. Taken together, our data suggest a model where TNY1 influences size homeostasis through dosage-dependent repression of CDKG1, possibly through direct binding to the CDKG1 3’UTR.

Living organisms respond and acclimate to a changing environment to survive and thrive. The cell surface is the front line where environmental stimuli are immediately detected and converted to intracellular signaling events. Therefore, a fundamental understanding of the organization, dynamics, and protein components of the cell surface will shed light on the structural and molecular underpinnings of growth and stress responses. In plant cells, it remains largely mysterious as to how the cell wall (CW) and the plasma membrane (PM) are connected, how the CW-PM interface is maintained and remodeled, and how signals are sensed at this interface upon pressures from within or outside the plant cell. We aim to address these questions in the model species Arabidopsis thaliana due to the extensive genetic resources and live-cell imaging marker lines available. To begin to investigate interface dynamics, we fluorescently labeled the outer periclinal CW and the PM, respectively, and tracked their behavior over time during hyperosmotic stress. Compared to the control condition where the CW and the PM are in close proximity all the time, hyperosmotic stress resulted in separation of the PM from the CW. We are currently analyzing the images to determine the existence (and if so, distribution) of CW-PM attachment sites during hyperosmotic stress. To identify the molecular components that serve as “pins” to hold together the CW and the PM, we surveyed a collection of mutants deficient in each major class of wall components or glycosylphosphatidylinositol-anchored proteins. We found that mutants associated with cellulose biosynthesis and organization are hypersensitive to osmotic stress and have defects in growth recovery. Contrastingly, mutants related to rhamnogalacturonan-I exhibit hyposensitivity to osmotic stress. We are currently investigating the cellular and subcellular details, such as cytosolic Ca2+ profiles, apoplast pH signatures, among other processes in these mutants.

Cellulose microfibrils, the major tensile components of the plant cell wall, play essential roles in plant growth and development. In flowering plants, cellulose is synthesized at the cell surface by plasma membrane (PM)-localized cellulose synthase (CESA) complexes (CSCs). Cellulose production is influenced by the abundance of CSCs at the PM which is thought to be coordinated by intracellular trafficking events and the cytoskeleton. The cortical actin cytoskeleton has been implicated in trafficking of CSCs to the PM, but the exact mechanism remains unclear. Here, we demonstrate that myosin XI and the actin cytoskeleton mediate CSC delivery to the PM by coordinating the exocytosis of CESA-containing compartments. Measurement of cellulose content indicated that cellulose biosynthesis was significantly reduced in a myosin xik xi1 xi2 triple knockout (xi3KO) mutant. By combining genetic and pharmacological disruption of myosin activity with quantitative live-cell imaging of functional YFP-CESA6, we observed decreased abundance of PM-localized CSCs and reduced delivery rate of CSCs in myosin-deficient cells. These phenotypes correlated with a significant increase in failed vesicle secretion events at the PM as well as an abnormal accumulation of CESA-containing compartments at the cell cortex. Through high-resolution spatiotemporal assays of cortical vesicle behavior, we identified defects in CSC vesicle tethering and fusion at the PM. Furthermore, colocalization studies showed transient association of MYOSIN XIK with CSCs during vesicle tethering. These findings reveal a previously undescribed role for myosin in vesicle secretion and cellulose production at the cytoskeleton–PM–cell wall nexus.

Like in other eukaryotic cells, plant membrane trafficking pathways transport proteins among organelles and play essential roles in growth and development. The cargo proteins for membrane trafficking have diverse functions such as cell wall biosynthesis, signaling, and nutrient uptake. How plant cells accurately control protein transport in a spatiotemporal manner has not been well characterized. Cellulose synthase complexes (CSCs) are large membrane-associated protein complexes that catalyze the synthesis of cellulose at the plasma membrane. CSCs are delivered to the plasma membrane through membrane trafficking pathways for their proper functions. Using chemical genetic approach, we identified a small molecule that targets the catalytic domain of plant cellulose synthases. Combining small molecule treatment, live cell imaging and quantitative image analysis, we found that the catalytic activity of CSCs affected efficient exocytic transport of these large protein complexes. Inhibition of CSCs catalytic activity reduced the transport of CSCs at early steps of exocytic trafficking, although these protein complexes might have been assembled properly. Our results add to current understanding of how plant membrane trafficking machineries regulate spatiotemporal delivery of proteins for plant growth and environment adaptation.

Plant development and tropisms are largely dependent on the polar transport of the phytohormone auxin. Actin cytoskeleton regulates auxin transport by controlling polar localization of auxin transporters such as PIN2. Inhibitors of polar auxin transport have been essential tools in understanding auxin-dependent plant development. One mode of their inhibitory effect is to affect actin dynamics, however, the underlying mechanisms remain unclear. In this study, we demonstrate that auxin transport inhibitor such as 2,3,5-triiodobenzoic acid (TIBA) target villin-mediated actin bundles in Arabidopsis to inhibit auxin transport. Multiple villins isovariants are targeted by TIBA, among which, villin4 (VLN4) has the highest affinity to this inhibitor. Mutants of VLN4 have significantly reduced TIBA sensitivity. Loss of VLN4 results in low abundance of actin bundles. Furthermore, VLN4-dependent actin bundling controls the plasma membrane presence of auxin exporter and subsequent auxin transport, which is critical for the inhibitory effect of TIBA. Biochemical approaches and docking simulation demonstrate that TIBA directly interacts with the C-terminal headpiece domain of Arabidopsis villins. The VHP-TIBA interaction promotes villin to oligomerize, which facilitate cross-linking of actin filament. Villin C-terminal headpiece confers in-vivo TIBA sensitivity. VLN4 mutant lacking VHP domain fails to mediate the action of TIBA on both actin cytoskeleton and auxin transport in plant. Collectively, our study provides evidence that villins mediates the action of TIBA on actin dynamics in Arabidopsis; Villin-generated actin bundles determine downstream location of auxin efflux transporters and regulate polar auxin transport.

The coordinated redistribution of sugars from mature “source” leaves to support the growth of developing “sink” leaves requires tight regulation of sugar transport between cells via plasmodesmata (PD). Although fundamental to plant physiology, the mechanisms that control PD transport and thereby support development of new leaves have remained elusive. From a forward genetic screen for altered PD transport, we discovered that PD transport is regulated by the conserved eukaryotic glucose-TOR (TARGET OF RAPAMYCIN) signaling hub. TOR is significantly more active in mature leaves photosynthesizing excess sugars than in young, growing leaves, and this shift in activity impacts rates of PD transport. Genetic, chemical, and physiological treatments promoting or disrupting TOR activity support the model that glucose-activated TOR controls PD transport in leaves. An established TOR effector in plants, PP2A, contributes to the control of PD transport during shoot development. We conclude that plant cells regulate PD trafficking in response to changing carbohydrate availability monitored by the TOR pathway.

Regulated distribution of plant hormones across tissues and over time is fundamental to plant growth and development and optical biosensors for plant hormones are beginning to shed light on hormone distributions in planta. We have engineered improved versions of the previously published optogenetic biosensors for the hormones gibberellin (GPS1) and abscisic acid (ABACUS1). These next-generation biosensors detect nanomolar levels of hormone at the cellular level with reduced effects on endogenous hormone signaling in Arabidopsis compared with first generation GPS1 and ABACUS1. Gibberellin gradients detected with GPS1 biosensors correlated with gradients of cell length in rapidly elongating roots and dark-grown hypocotyls, but it remained unclear how these gradients arise from the ensemble activities of gibberellin enzymes and transporters. We now present evidence in support of patterned gibberellin biosynthesis and permeability driving gibberellin patterns in roots. The effect of gibberellin enzyme and transport mutants on gibberellin patterning in roots will be discussed in the context of understanding how gibberellin gradients are determined and how they, in turn, influence patterning of plant cell growth. ABA patterns detected with next generation ABACUS biosensors will be discussed in the context of ABA accumulation and elimination rates and their effects on plant development.

The phytohormone Gibberellin (GA) regulates various aspects of plant growth and development, including seed germination, stem elongation, flower induction and fruit set through the destabilization of the growth repressor, DELLA. CDC48/p97 is a highly-conserved homohexameric AAA-ATPase molecular chaperone that uses the energy of ATP hydrolysis to unfold and/or extract client proteins from membranes, protein complexes and other cellular structure. The Plant ubiquitin regulatory X (UBX)-containing protein, PUX1, negatively regulates CDC48/p97 by promoting the disassembly of the active homohexameric complex into its inactive monomers. Preliminary data indicate that the Gibberellin receptor, GID1 (GA-INSENSITIVE DWARF1), interacts with two forms of PUX1, a 38kD full-length and a 34 kD putative truncated form suggesting a previously undefined molecular mechanism by which GA controls plant growth and development. In vitro characterization of the interaction have shown that the binding of PUX1 to GID1 is GA-independent and mediated through a region of PUX1 containing the UBX domain. Additionally, PUX1/GID1/CDC48 was showed to form a ternary complex that was also GA-independent. Furthermore, in vivo studies of fluorescently tag PUX1 in plants have shown that PUX1 is also localize in the nucleus. Whereas GA biosynthesis mutant suffer from dwarfism, lines with elevated levels of GA show increased growth due increase cell division and elongation. Similar to lines that have enhance GA-signaling, pux1 loss-of-function mutants exhibit accelerated growth compared to wild type and behave like GA-hypersensitive mutants when treated with Paclobutrazol, a GA biosynthesis inhibitor. Together our findings suggests a new mechanism by which GA-signaling promotes plant growth and development in part by inhibiting PUX1 function, thereby maintaining levels of active CDC48/p97 necessary for proper cell division and expansion.

Plant roots are usually the first organ to perceive water-deficit stress and soil-borne diseases, but how the root system adapts to the unfavorable environment is still unclear. In rice, an intrinsically disordered protein with about 40% proline, REPETITIVE PROLINE-RICH PROTEIN (RePRP), is induced by water-deficit stress, abscisic acid (ABA) and jasmonic acid (JA) specifically in the root elongation zone. Ectopic expression of RePRP confers a ‘short but heavy’ root phenotype, resembling the effect of water-deficit stress or ABA treatment; and this phenotype is reduced in RePRP RNA-interference knockdown transgenic rice, which suggests that RePRP is sufficient and necessary for water-deficit stress- or ABA-repressed root development. RePRP interacts with the highly ordered cytoskeleton components, actin and tubulin, both in vitro and in vivo. The binding of RePRP reduces the abundance of actin filaments and impairs non-cellulosic polysaccharide transport to the cell wall. RePRP also reorients the microtubule network, which leads to disordered cellulose microfibril organization in the cell wall. These cell wall modifications inhibit the elongation of root cells and promotes biomass accumulation in the ‘heavy’ root, which facilitates plant survival under adverse conditions. We demonstrate a novel role of intrinsically disordered proteins controlling cell expansion via an ‘order-by-disorder” mechanism for development of ‘short-but-heavy’ roots as an adaptive response to water deficit in rice. In addition, RePRP mediates several ABA- and JA-regulated defense mechanisms leading to the enhanced synthesis of lignin, phytoalexins and chitinases. Consequently, the rice plants are more resistant to the root pathogenic fungus, Pythium. As an intrinsically disordered protein, RePRP may have many conformations capable of interacting with multiple partners, thus it appears to function as an integrator for responses to diverse stress conditions.