Represent your science through art. Using your imagination, draw anything that represents what you do, how you do it and/or why you do it. No artisitic skills needed. Located near registration.

Mark on the map where your traveled from to attend Plant Biology 2019. Let's see who came the furthest. Located near registration.

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.

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.

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.

The variation among individuals of a species is critical for adaptation and for crop improvement. This variation begins with genetic and epigenetic variation that can influence the nature and levels of gene products which in turn influence phenotypic outcomes. We have been studying the natural variation of maize to better understand the sources of variation in crop plants. There is significant variation in the transcriptome of different maize inbreds. This variability is often manifest through changes in cis-regulatory elements that influence tissue-specific changes in expression. In order to understand the potential cis-regulatory variation among different haplotypes the genome structure and content of maize lines has been compared. Maize genomes exhibit significant structural variation. Much of this is due to changes in transposable element content. Transposable element polymorphisms can alter the regulation of genes through genetic and epigenetic mechanisms and as well as changes in gene content. Together, these analyses paint a picture of a dynamic genome and points to the underlying mechanisms that create variable phenotypes.

Why is it rewarding for plant scientists to engage the public? If one is interested, how does one begin? Do public engagement activities disrupt a research career? How can we as plant scientists cultivate a community of researchers dedicated to making scientific research accessible, relevant, and interesting to everyone? How can we provide the next generation of scientists with the training, support, and tools they need to become effective communicators? How can we infuse scientifically sound information into the public discourse? Ronald will address these questions in light of her career as a research scientist and science communicator.

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

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.

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.

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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!

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 initiatives, and T-training opportunities.

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.

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

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".

Are you interested in learning more about commercialization in plant science, as well as meeting entrepreneurs and like-minded individuals? Come to the Commercialization in Plant Science workshop and networking event, where attendees will get the opportunity to learn from, and ask questions to a panel of scientists, media specialists, investors, entrepreneurs and strategic partners. A networking event with take place at the end of the workshop to facilitate introductions, cultivate relationships, and foster discussion on all things entrepreneurial, IP, investment and partnership. This networking event is sponsored by the wonderful folks at Indigo Ag. Questions? Please reach out to Phil Taylor (phil.taylor@bayer.com) or Rishi Masalia (rishimasalia@gmail.com).

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

Cellulose, xylan and lignin are the principal macromolecules of lignocellulosic biomass in bioenergy crops such as grasses and fast-growing trees. Lignin abundance is considered to be the major recalcitrance factor to the enzymatic digestion of cellulose from biomass into fermentable sugar. However, biomass recalcitrance is multi-facetted, multi-scale and conversion-specific, and not confined to inhibitory interactions of lignin on carbohydrate conversion. Disassembly of lignin in woody particles by Ni/C-catalyzed delignification (CDL) improved yields of glucose by enzymatic digestion or catalytic conversion to biofuel substrates. Although lignin was completely removed by CDL, recalcitrance to enzymatic digestion of cellulose from the high-Syringyl lignin lines was reduced compared to other lignin variants. However, cellulose still exhibited considerable recalcitrance to complete enzymatic digestion or catalytic conversion after complete delignification. Low-temperature swelling of the CDL-treated wood particles in trifluoroacetic acid resulted in nearly complete enzymatic hydrolysis, regardless of original lignin composition. Genetic modification of lignin composition can enhance the portfolio of aromatic products obtained from lignocellulosic biomass while promoting disassembly into biofuel and bioproduct substrates. Our results inform a ‘no carbon left behind’ strategy to convert total woody biomass into lignin, cellulose, and hemicellulose value streams for the future biorefinery. Supported by the Department of Energy, Office of Science, Basic Energy Sciences Center for the direct Catalytic Conversion of Biomass to Biofuels (C3Bio). Matheus Benatti1, Rucha Karve1, Chien-Yuan Lin2, Baoyuan Liu3, Hao Luo3, Jeong Im Kim1, Anna Olek1, Phillip Rushton1, Hui Wei2, Haibing Yang1, Ximing Zhang1, Mahdi Abu-Omar3, Clint Chapple1, Peter Ciesielski2, Mike Crowley2, Bryon Donohoe2, Mike Himmel2, Lee Makowski4, Maureen McCann1, Rick Meilan1, Nate Mosier1, Cynthia Stauffacher1, Mel Tucker2, and Nick Carpita1 1Purdue University; 2National Renewable Energy Lab, 3University of California Santa Barbara; 4Northeastern University

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.

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.

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.

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.

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You've read the headlines. You've seen the nonstop partisan fights on cable news. But what are policy makers in Washington DC really doing, and what does it mean for you as a plant scientist? Join us for a discusson of the state of play of the FY 2020 appropriations process and other science policy-related actvities.

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.

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.

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.

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.

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.

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.

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

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.

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.

Cellular functions are organized and regulated through specific protein-protein interactions. Identifying these interactions are challenging, particularly for the transient and dynamic interactions such as those between protein-modifying enzymes and their substrates. Proximity labeling has immerged as a powerful method for studying subcellular proteomes and protein-protein interactions. Here we apply a proximity dependent biotin identification (BioID) system using a mutated biotin ligase (BirA) that display enhanced enzymatic activity, which is named TurboID. Using a pair of known interacting proteins of the brassinosteroid (BR) signaling pathway, the GSK3-like kinase BIN2 and its substrate BZR1, we showed that BIN2-TurboID enriches BZR1 about 10x more effectively than the traditional co-immunoprecipitation method. Using the homologs of the BIN2 family and PP2A family that function in the BR pathway, we showed specific interaction of BZR1 with the protein family members that are involved in BR signaling but not those that do not play an important role in BR signaling, demonstrating good specificity of TurboID. We have used TurboID to identify interacting proteins of known kinases and phosphatases, and this has led to an expanded protein networks that regulate plant growth responses to hormonal and environmental signals.

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.

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.

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.

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.

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.

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!

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

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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.

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.

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.

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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!

Discussion led by Tim Griffin

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 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.

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: Tanya Berardini, Arabidopsis Group; Marcela K. Tello-Ruiz, CSHL •TreeGenes, Citrus, GDR, Hardwood Genomics : Dorrie Main, Washington State University •SGN/BrAPI/Nicotiana Database : Lukas Mueller/Hartmut Foerster, Boyce Thompson Institute •Gramene : Marcela K. Tello-Ruiz - 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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).

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.

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.

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.

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

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.

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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..

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.

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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.