| 8:30 AM
- 8:33 AM |
Overview of Area by Chair, R. Keith Slotkin
(Genetics/Genomics)
LL21C
Chair and Concurrent Symposium Speaker:
|
 |
| 8:33 AM
- 8:53 AM |
Metina: A Transcription factor involved in Iron Deficiency Tolerance in Arabidopsis thaliana
(Genetics/Genomics)
LL21C
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..
Concurrent Symposium Speaker:
|
|
| 8:53 AM
- 9:13 AM |
Identifying regulators and the regulatory network of Kranz anatomy development through Laser-Capture Microdissection
(Genetics/Genomics)
LL21C
C4 leaves are characterized by the Kranz anatomy, in which the vascular bundle is surrounded by one layer of organelle-rich bundle sheath (BS) cells, which is then surrounded by one layer of radially arranged mesophyll (M) cells. Past histological and cell lineage studies in maize revealed that Kranz development starts from three contiguous ground meristem cells, but little is known about the genes and the molecular mechanism involved in Kranz anatomy development. To identify key regulatory genes involved in Kranz development, we compared the tissue specific transcriptomes of different developmental stages of maize embryonic leaf including: 5 stages (ground meristem tissues with only P1, 3, 4, 5, or 6 BS GM cells) of Kranz ground meristem (GM) cells; 4 stages of palisade-like (P1, 3B, 4B, and 5 BS stage) M cells; 2 stages of undifferentiated M ground meristem (1M, 2M) cells by LCM. We obtained high-quality RNAs, and then RNA-seq data. Principal components analysis (PCA) showed that early Kranz and M cells exhibited distinct mRNA populations. These data sets indicate that Kranz and M cells have distinct gene regulatory networks because they arise from distinct genetic origins and that the captured cell types show sufficient diversity at the mRNA level. Differential gene expression and weighted correlation network analysis (WGCNA) identified candidate coexpression modules and gene coexpression networks involved in Kranz development. GO analysis indicated that these modules were enriched for genes involved in anatomical structure, leaf, shoot development, etc. In situ hybridization validated several genes expressed in early Kranz anatomy. Finally, we predicted putative cis-regulatory elements in upstream gene sequences from each gene and validated the predictions by Y1H and protoplast transient assay. Moreover, we constructed a network related to Kranz development. These results provided much insight into the transcriptional regulation of Kranz anatomy development.
Concurrent Symposium Speaker:
|
|
| 9:13 AM
- 9:33 AM |
Functional characterization and identification of distal cis-regulatory elements in plant genomes using chromatin structure
(Genetics/Genomics)
LL21C
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.
Co-author(s):
- Zefu Lu,
- Mary Galli,
- Jordan Rowley,
- Maria Colome-Tatche,
- Nathan Springer,
- Jixian Zhai,
- Maria Mejia-Guerra,
- Victor Corces,
- Alex Marand,
- Frank Johannes,
- William Ricci,
- Lexiang Ji,
- Edward S. Buckler,
- Andrea Gallavotti,
- Jaclyn Noshay,
- Christina Ethridge,
- Xiaoyu Zhang
Concurrent Symposium Speaker:
|
|
| 9:33 AM
- 9:53 AM |
NAD tagSeq for transcriptome-wide identification and characterization of NAD-capped RNAs
(Genetics/Genomics)
LL21C
The 5’ end of a eukaryotic mRNA generally has a methyl guanosine cap (m7G cap) that not only protects the mRNA from decay by 5’-3’ exonucleases, but also plays an essential role in almost all aspects of gene expression. Some RNAs in E. coli, yeast, and mammals were recently found to have NAD+ as a cap. We have developed a new method, termed NAD tagSeq, for transcriptome-wide identification and quantification of NAD+-capped RNAs (NAD-RNAs). The method uses an enzymatic reaction and a click chemistry reaction to label NAD-RNAs with a synthetic RNA tag. The tagged RNA molecules can be enriched and directly sequenced using the Oxford Nanopore sequencing technology. NAD tagSeq not only allows more accurate identification and quantification of NAD-RNAs but can also reveal sequences of whole NAD-RNA transcripts. Using NAD tagSeq, we found that NAD-RNAs in Arabidopsis are mostly produced from a few thousand protein-coding genes. The top 2,000 genes that were found to produce the highest numbers of NAD-RNAs were enriched in the gene ontology terms of responses to stresses, photosynthesis, protein synthesis, and response to cytokinin. For some Arabidopsis genes, over 10% of their transcripts could be NAD-capped. The NAD-RNAs in Arabidopsis have similar overall sequence structures to their canonical m7G-capped mRNAs. NAD tagSeq has been used to identify NAD-RNAs from maize, rice, and other organisms. The identification and quantification of NAD-RNAs and revealing their sequence features provide essential steps toward understanding functions of NAD-RNAs.
Concurrent Symposium Speaker:
|
|
| 9:53 AM
- 10:13 AM |
Initiation of Silencing via expression-dependent de novo RNA-directed DNA Methylation
(Genetics/Genomics)
LL21C
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.
Chair and Concurrent Symposium Speaker:
|
|
| 10:13 AM
- 10:15 AM |
Conditional regulatory logic for the Arabidopsis TCA cycle
(Genetics/Genomics)
LL21C
As a hub of central carbon metabolism, regulation of the tricarboxylic acid (TCA) cycle is crucial to coordinate flux to neighboring metabolic pathways for optimizing growth and development. TCA cycle regulation in plants has largely been studied at the level of the protein or metabolite including post-translational modification, allosteric feedback of enzymes, and metabolite channeling by organizing sequential enzymes into metabolons. However, transcriptional regulation of the TCA cycle in Arabidopsis, and even more broadly, in multicellular organisms, is largely unstudied. Using an enhanced yeast one-hybrid platform, we identified a large number of transcriptional regulators. These predict differential control of TCA targets in the various cellular compartments, potentially enabling flexibility to alter the pathway. Furthermore, no general regulators of the TCA cycle were identified via co-expression analyses, providing an immediate indicator of a novel paradigm for how multicellular organisms regulate this critical metabolic pathway. We selected 17 candidate TFs for conditional transcriptional regulation of the TCA cycle. In total, mutants of all 17 genes were shown to have perturbed TCA cycle function, with a subset having responses that were dependent on specific TCA cycle intermediates. One third of these TF mutants influence growth in a salt stress-dependent manner, and mutations in almost half led to perturbations in the abundance of C, N or C:N ratios. Thus transcription of TCA cycle genes are controlled in the plant to allow fine-tuning of metabolism to meet energetic demands of diverse cell types under various environmental constraints.
|
|
| 8:30 AM
- 8:33 AM |
Overview of Area by Chair, Savithramma Dinesh-Kumar
(Abiotic)
LL20A
Concurrent Symposium Chair:
|
|
| 8:33 AM
- 8:53 AM |
The genetic architecture of osmotic stress tolerance in plants
(Abiotic)
LL20A
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.
Concurrent Symposium Speaker:
|
|
| 8:53 AM
- 9:13 AM |
TOR kinase tunes autophagy and meristem activity for nutrient stress-induced developmental plasticit
(Abiotic)
LL20A
Plants can respond to environmental challenges with comprehensive developmental transitions that allow them to cope with these stresses. It is shown that antagonistic activation of the Target of Rapamycin (TOR) kinase in the root and the shoot is essential for the nutrient deprivation-induced increase in root-to-shoot ratio. We demonstrate that sulfate limitation-induced downregulation of TOR in shoots activates autophagy resulting in carbon allocation to the root. This process is facilitated by specific upregulation of the sucrose-transporters SWEET11/12 in the shoots. SWEET11/12 activation is indispensable to enable sucrose to act as a carbon source for growth and a signal for tuning root apical meristem activity via glucose-TOR signaling. We show that the transcription factor and TOR-substrate, E2Fa, transduces this signal. The sugar-stimulated TOR activity in the root suppresses autophagy and maintains root apical meristem activity to support root growth for mining new sulfate resources in the soil. We expect that our findings not only contribute to the understanding of environment triggered organismal plasticity in plants in general, but enable new modification strategies towards crop plants with enhanced nutrient use efficiency.
Concurrent Symposium Speaker:
|
|
| 9:13 AM
- 9:33 AM |
Time of day regulation of heat stress related growth responses
(Abiotic)
LL20A
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.
Concurrent Symposium Speaker:
|
|
| 9:33 AM
- 9:53 AM |
A novel approach to provide insight on the regulation of Postharvest Chilling Injury in tomato (Solanum lycopersicum L.)
(Abiotic)
LL20A
Most tropical and subtropical produce are so cold-sensitive that refrigeration reduces shelf-life and quality. Tomato fruit experiences Postharvest Chilling Injury (PCI) when stored at 0-12.5°C. Symptoms include surface pitting, and uneven ripening and decay, due to metabolic and physiological dysfunction. Unlike tomato, Arabidopsis thaliana can cold-acclimate partly due to the CBF family of transcription factors (AtCBF1-3). The ectopic and constitutive overexpression of AtCBFs led to higher chilling tolerance but had negative developmental effects in tomato plants, and fruit response to cold stress was not tested. Constitutive overexpression of CBF1 from the cold-tolerant wild tomato relative Solanum habrochaites (ShCBF1) resulted in higher cold-tolerance in Arabidopsis plants. This suggests that increasing the control of transgenic CBF1 expression could be useful to study PCI in tomato fruit without detrimental effects on plant development. We hypothesize that CBF1 overexpression will increase chilling tolerance and ameliorate PCI symptoms during refrigeration. In this study, Micro-Tom tomato plants were independently transformed with three constructs: a dexamethasone system to chemically trigger AtCBF1 expression, and a stress-inducible promoter (RD29A) to induce ShCBF1 or SlCBF1 specifically when fruit are refrigerated. Fruit were stored at 2.5 (PCI-inducing) or 12.5°C (control, non-PCI inducing) for 1- 3 weeks, and transferred to 20°C to promote PCI symptoms. To assess changes in whole-plant cold tolerance, the photosynthetic performance of transgenic lines was measured under cold stress. Results showed that high expression of transgenic CBF1 in fruit as determined by qRT-PCR, was linked to accelerated senescence and an aggravation of PCI symptoms, both quantified by objective color and surface pitting scores. This suggest that PCI may offer an evolutionary advantage in tomato by accelerating fruit breakdown for seed dispersal under extreme stress conditions.
Concurrent Symposium Speaker:
|
|
| 9:53 AM
- 10:13 AM |
The XBAT family of RING-type ubiquitin ligases and plant response to environmental stress
(Abiotic)
LL20A
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.
Concurrent Symposium Speaker:
|
|
| 10:13 AM
- 10:15 AM |
An algorithm to measure root hair response to abiotic stresses in microscopy images
(Abiotic)
LL20A
Improving nutrient and water uptake in crops is one of the major challenges to sustain a fast-growing population that faces increasingly nutrient limited soils. Root hairs, which are specialized epidermal cells, compromise up to 70% of the total root surface area. Therefore, root hairs are important drivers of nutrient and water uptake from the soil. Microscopy provides a mean to record root hairs as digital images. However, quantifying root hairs in microscopy images remains a bottleneck because of their geometry and their complex spatial arrangement. Describing root hairs manually is based on a limited selection of representative root hairs and is only possible in cases, where length and density are sufficiently low to trace individual root hairs. We present a method to automatically quantify phenotypic traits of root hairs in digital microscopy images. Our method uses a machine learning approach that classifies root hair, parent root and the image background. We define local metrics to quantify relatedness between root hair segments that are separated by crossing root hairs or blobs of two or more root hairs. Based on our local metric we can detect individual root hairs by resolving these complexities in a globally optimal way. As a result, we measure the root hair traits, length, number and density. We demonstrate our method on examples of rice, maize and common bean under phosphor, nitrogen and potassium stress. Preliminary results suggest that our measurements of root hair traits strongly correlate with manual measurements (Pearson-correlation up to 0.9 in length). We expect that our method distinguishes subtle differences between genotypes and treatments on the basis of the extracted traits. We believe our study paves a way towards identifying the genetic control of root hair traits and increased agricultural production in future.
|
|
| 8:30 AM
- 8:33 AM |
Overview of Area by Chair, Ravishankar Palanivelu
(Cell/Development/Systems)
LL20D
Chair and Concurrent Symposium Speaker:
|
|
| 8:33 AM
- 8:53 AM |
The Role of CLV2/CRN in Floral Primordium Development in Arabidopsis thaliana
(Cell/Development/Systems)
LL20D
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.
Concurrent Symposium Speaker:
|
|
| 8:53 AM
- 9:13 AM |
A Silk-Expressed Pectin Methylesterase Confers Cross-Incompatibility Between Wild and Domesticated Strains of Zea mays
(Cell/Development/Systems)
LL20D
A central problem in speciation is the origin and mechanisms of reproductive barriers that block gene flow between sympatric populations. In sexually reproducing plants, reproductive barriers exist at different stages during reproduction, including pre-pollination, post-pollination and post-fertilization. Post- pollination barriers depend on interaction between the male gametophyte (pollen) and the cells of the female reproductive organs (stigma, style, and ovule). In Zea mays, three haplotypes, Gametophyte factor1-s (Ga1-s), Gametophyte factor2-s (Ga2-s), and Teosinte crossing barrier1-s (Tcb1-s) at three different loci confer Unilateral Cross-Incompatibility by arresting none-self growing pollen tubes. While Ga1-s and Ga2-s are widespread in domesticated maize, Tcb1-s is almost exclusively found in wild teosinte populations. Despite being members of the same species, some strains of wild teosinte maintain themselves as a distinct breeding population by blocking fertilization by pollen from neighboring maize plants. These teosinte strains may be in the process of evolving into a separate species, since formation of reproductive barriers is a critical step in speciation. These teosinte strains typically carry the Tcb1-s haplotype. Tcb1-s contains a female barrier gene that blocks non-self-type pollen and a male function that enables self-type pollen to overcome that block. With genetic and genomic approaches, here we show that the Tcb1-female barrier gene encodes a Pectin Methylesterase38 homolog, implying that pollen cell wall modification is a key cellular mechanism by which these teostine females reject foreign but closely related pollen. Cloning of this female barrier gene in Zea mays represent a major advance in speciation research and opens up exciting working hypotheses to test. Agriculturally, this work may also help to facilitate breeding effort to manage specialty crop populations and enrich crop germplasm by backcrossing to their ancestors.
Concurrent Symposium Speaker:
|
|
| 9:13 AM
- 9:33 AM |
MAS integrates ovular signals and exocytosis to guide pollen tube
(Cell/Development/Systems)
LL20D
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.
Concurrent Symposium Speaker:
|
|
| 9:33 AM
- 9:53 AM |
LORELEI and its most closely related paralog, LLG1, show evidence of regulatory subfunctionalization in Brassicaceae
(Cell/Development/Systems)
LL20D
LORELEI (LRE), and its most closely related paralog LLG1 (LORELEI-LIKE GPI-Anchored Membrane Protein 1) arose from the most recent whole genome duplication (WGD) in Brassicaceae. lre and llg1 mutants in Arabidopsis have no overlapping phenotypes; hence, we hypothesized that LRE and LLG1 were maintained post gene duplication because the two genes split the functions of the ancestral single copy gene (subfunctionalization) found in Cleome violacea (a member of the Cleomaceae, a sister group to Brassicaceae). To test this hypothesis, we performed cross-complementation experiments with LRE and LLG1 and showed that each gene can complement the defects caused by the loss of the other gene. Additionally, we used the single copy gene of LRE/LLG1 in Cleome violacea (CleviLRE//LLG1) to complement Arabidopsis thaliana lre and llg1 mutant phenotypes. Successful cross complementation results led us to propose another explanation for retention of both genes post duplication: the expression domains in the promoters of LRE and LLG1 diverged in a non-overlapping manner, allowing both genes to perform similar functions but in different tissues and cells (regulatory subfunctionalization hypothesis). Using promoter:GUS transcriptional fusions, we found that LRE and LLG1 have distinct expression in Arabidopsis. Additionally, we showed that CleviLRE/LLG1 is expressed in Cleome ovules and vegetative tissues. Additional diversification in the expression of these two genes have been reported, as LRE, but not LLG1, is a maternally-imprinted gene. These results strongly support the regulatory subfunctionalization hypothesis that post gene duplication, LORELEI and LLG1 maintained their molecular functions, but have divergent expression.
Chair and Concurrent Symposium Speaker:
|
|
| 9:53 AM
- 10:13 AM |
Embryo initiation in rice by male-genome expressed BABY BOOM transcription factors
(Cell/Development/Systems)
LL20D
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.
Concurrent Symposium Speaker:
|
|
| 10:13 AM
- 10:15 AM |
Sculpting an imperfect flower: The study of KNUCKLES in primordia regulation
(Cell/Development/Systems)
LL20D
The evolution of sex determination in plants is a central problem in plant evolutionary biology. Currently, there have been limited studies in which the sex determination genes are identified yet we do not know most of the alternative downstream pathways that lead to developmental differences in plants that exhibit sexual dimorphism. Addressing this gap in knowledge is important as it will give insight into the genetic regulation of developmental processes in unisexual flowers and in angiosperm flowers in general. The investigation into the link between the differential expression patters of genetic pathways and the differential expression in floral development involves the differential expression of AG, WUS, and the proposed transcription repressor gene KNUCKLES (KNU) as they relate to the differential formation of floral organ primordia in male and female Spinacia oleracea flowers. Our central hypothesis is that the AG-KNU-WUS pathway regulates the differential morphogenesis of organ primordia between male and female flowers leading to sexual dimorphism in spinach. To test this hypothesis, molecular genetics tools are utilized to quantify KNUCKLES temporal and spatial expression patterns, along with functional testing. Preliminary studies have begun that include characterizing KNU-like gene expression and the phenotypes of KNU-like knockdowns in S. oleracea. Preliminary results show strong phenotypes in the vegetative tissue that are related to the regulation of organ primordia and meristem maintenance.
|
|
| 8:30 AM
- 8:33 AM |
Overview of Area by Chair, Thomas Niehaus
(Biochemistry)
LL21AB
Chair and Concurrent Symposium Speaker:
|
|
| 8:33 AM
- 8:53 AM |
Identification of a pathway for disposal of metabolite damage products formed by the respiratory int
(Biochemistry)
LL21AB
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.
Chair and Concurrent Symposium Speaker:
|
|
| 8:53 AM
- 9:13 AM |
Regulation of S-Nitrosation of Arabidopsis S-Nitrosoglutathione Reductase (GSNOR) by Thioredoxins an
(Biochemistry)
LL21AB
Nitric oxide (NO) is a short-lived gas that acts as a signaling molecule in all higher organisms, including plants. Despite the clear involvement of NO in multiple plant processes, including germination, root growth and fertility, a basic understanding of the mechanisms by which NO exerts its effects on systems critical for plant growth and development is lacking. Reversible S-nitrosation of critical protein cysteines due to reaction with nitric oxide (NO) and its derivatives is a redox-dependent posttranslational modification that impacts these plant physiological processes. Regulation of NO-levels in planta is predominantly achieved by reaction of reactive nitrogen species (RNS) with glutathione (GSH), thereby forming S-nitrosoglutathione (GSNO), the principal NO reservoir. Mutation of Arabidopsis S-nitrosoglutathione reductase (GSNOR) leads to higher intracellular concentrations of S-nitrosothiols, confirming that the reduction of GSNO by the enzyme is a major route of GSNO catabolism in plants. GSNO-breakdown is believed to help sustain cellular redox poise both by curtailing RNS-bursts and by regenerating GSH. GSNOR contains evolutionary-conserved cysteine residues that are prone to S-nitrosation by different NO donors, leading to a partial loss of enzyme activity that could be recovered by reducing agents in vitro. Protein nitrosation was further confirmed by intact mass spectrometry, for which signals consistent with mono-, di- and tri-nitrosation were observed. In addition, GSNOR denitrosation analysis catalyzed by small oxidoreductases will be addressed. These data implicate a mechanism for RNS signaling by modulating redox-dependent posttranslational modifications of certain proteins. Reduced GSNOR activity is predicted to result in the accumulation of GSNO, itself an agent of protein S-nitrosation. By allowing GSNO to accumulate, inhibition of GSNOR may facilitate more robust NO signaling that regulates plant growth and developmental processes in plants.
Concurrent Symposium Speaker:
|
|
| 9:13 AM
- 9:33 AM |
Structures of Xyloglucan Xylosyltransferases revealed how simple steric rules define patterns of nat
(Biochemistry)
LL21AB
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
Concurrent Symposium Speaker:
|
|
| 9:33 AM
- 9:53 AM |
The entry reaction of the plant shikimate pathway is under highly-complex effector-mediated regulati
(Biochemistry)
LL21AB
The plant shikimate pathway directs bulk carbon flow to support biosynthesis of aromatic amino acids (AAAs) and numerous natural products including phytohormones, cofactors, pigments, phytoalexins, lignin, and more. These aromatic phytochemicals play critical roles in plant physiology and adaptation, and also provide essential nutrients, medicine, and industrial materials to the human society. In microbes, the shikimate pathway is feedback inhibited by AAA effectors at the first enzyme, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DHS). Although the DHS-catalyzed step is also thought to be regulated in plants, effectors that regulate plant DHS have not been identified for decades . Here, we generated recombinant enzymes of all three DHS isoforms of Arabidopsis thaliana (AthDHS1, AthDHS2, and AthDHS3) and conducted their biochemical characterization. Only the AthDHS2 isoform, but not AthDHS1 or AthDHS3, was negatively regulated by tyrosine or tryptophan, whereas phenylalanine had no effects. Chorismate, the final product of the shikimate pathway, strongly inhibited the activity of all three AthDHS enzymes, which was counteracted by a further downstream intermediate, arogenate. Caffeic acid and its derivatives, key intermediates of the phenylpropanoid pathway, were also effective inhibitors of AthDHS enzymes, uncovering a potential regulatory link between the shikimate and phenylpropanoid pathways. DHS activity detected from leaf crude extracts were inhibited by chorismate and caffeic acid, but not by any of AAAs, which appears to be due to the loss of the AthDHS2 AAA-sensitivity in the presence of AthDHS1. These findings reveal unique and highly-complex regulatory mechanisms of the entry reaction of the plant shikimate pathway and provide foundational knowledge to control the production of AAAs and diverse natural products in plants.
Concurrent Symposium Speaker:
|
|
| 9:53 AM
- 10:13 AM |
Polyamines alter primary root growth by modulating biotin synthesis
(Biochemistry)
LL21AB
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.
Concurrent Symposium Speaker:
|
|
| 10:13 AM
- 10:15 AM |
ALA4 and ALA5 are lipid flippases that are critical for vegetative growth in Arabidopsis
(Biochemistry)
LL21AB
Aminophospholipid ATPases (ALAs) are lipid flippases involved in the uptake and translocation of specific lipids across membrane bilayers. Arabidopsis thaliana contains 12 ALAs that sort into five phylogenetic clusters, including five in cluster 2 (ALA8, 9, 10, 11, and 12) and four in cluster 3 (ALA4, 5, 6, and 7). Here we show that double mutants lacking ALA4 and 5 (cluster 3) are severely dwarfed, characterized by reduced growth in rosettes (6.5-fold), roots (4.3-fold), bolts (4.5-fold), and hypocotyls (2-fold). Plant size reductions correlated with reductions in cell size, suggesting that ala4/5 mutants are dwarfed in part due to cellular expansion defects. Dwarfism was also associated with perturbations in the content of both glycerolipids and sphingolipids, most notably a ~2-fold increase in glucosylceramides (GlcCers) which could potentially inhibit growth. Uptake assays in yeast suggested that ALA5 was capable of transporting specific lipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), as well as the sphingolipid sphingomyelin (SM). However, this assay detected no transport for GlcCers, suggesting GlcCer increases in ala4/5 mutants likely arise from indirect pathways. In comparison to other ALAs, the PC > SM > PE > PS transport profile for ALA5 was very similar to that of ALA10 (cluster 2), with the most notable exception being that ALA10 can transport lysophosphatidylcholine. Interestingly, a suppressor mutant screen on mutagenized ala4/5 seedlings was used to identify three dominant suppressor mutants that had near wildtype rosette growth, all of which caused similar disruptions in a putative regulatory domain of a cluster 2 ALA. These results suggest that the biochemical activity of ALA4/5 from cluster 3 is of critical importance for plant and cell growth, and that this distinct activity originates from a putative regulatory domain that differentially controls the activity of cluster 2 and cluster 3 ALAs.
|
|
| 8:30 AM
- 8:33 AM |
Overview of Area by Chair, Rishi Masalia
(Biotic and Applied)
LL21D
Concurrent Symposium Chair:
|
|
| 8:33 AM
- 8:53 AM |
Something Sweet to Eat: Disease Management of Stevia and its Introduction as a New Crop in the southeast United States
(Biotic and Applied)
LL21D
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.
Concurrent Symposium Speaker:
|
|
| 8:53 AM
- 9:13 AM |
Characterization of novel biological processes in fruit crops
(Biotic and Applied)
LL21D
Ripening is a well-characterized process during fruit development. However, fruit crops represent several variations in this ubiquitous process. European pear (Pyrus communis L.), a climacteric fruit, represents one such anomaly where the fruit are harvested at maturity but in an unripe state. Ripening can only be achieved by incubating the fruit in a genetically pre-determined amount of cold during postharvest stages. Further, the use of an ethylene receptor inhibitor, 1-methylcyclopropene (1-MCP) on the unripe fruit results in permanent ‘locking’ of ripening. We identified that the alternate respiratory pathway is activated during pre-climacteric stages, unlike other climacteric fruit, as the fruit undergoes cold conditioning. Using this information, a chemical genomics approach was used to ripen 1-MCP treated fruit. We hypothesize that chemical activation of the alternative respiratory pathway activates the TCA cycle leading to the generation of ethylene. We have utilized this recently patented technology to enable the development of high quality fresh sliced pears. Sweet cherry (Prunus avium L.), taxonomic kin of pear in the Rosaceae family, is characterized as a non-climacteric fruit. However, exogenous application of ethylene induces the formation of an abscission zone at the fruit pedicel junction. We identified an ethylene-inducible variant of an ERF transcription factor in ‘Bing’ cultivar. Incorporation of this allele in breeding strategies or editing could facilitate mechanical harvesting of this important crop.
Concurrent Symposium Speaker:
|
|
| 9:13 AM
- 9:33 AM |
Alteration of seed protein and oil content in soybean by fast neutron induced mutagenesis
(Biotic and Applied)
LL21D
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.
Concurrent Symposium Speaker:
|
|
| 9:33 AM
- 9:53 AM |
Genome-wide association study leads to a future of firmer apples
(Biotic and Applied)
LL21D
Apple (Malus domestica) is one of the world’s most valuable fruit crops and a promising candidate for marker-assisted selection (MAS) due to its lengthy juvenile phase. To discover genotype-phenotype associations, we generated approximately 250,000 SNPs using genotyping-by-sequencing for the Apple Biodiversity Collection (ABC) located in Kentville, Nova Scotia, Canada. In 2017, phenotype data were collected from over 1,300 trees in the ABC, representing over 850 unique accessions. Accessions were also genotyped for several markers previously discovered using linkage mapping and currently used for MAS in apple. Genome-wide association study (GWAS) results confirmed the transcription factor NAC18.1, previously identified in a GWAS of the USDA apple germplasm collection, is a strong functional candidate for fruit firmness and harvest date. In comparison, no significant associations were identified for the firmness markers currently used in apple breeding. NAC18.1 is homologous to the NON-RIPENING (NOR) gene of tomato. While nor mutant fruits fail to ripen, transgenic complementation of nor with the apple NAC18.1 gene rescued its ripening defect, confirming NAC18.1’s role as a conserved regulator of fruit ripening. These results demonstrate that GWAS in a diverse apple collection results in extremely high resolution mapping of putatively causal variants, which holds great potential for continued improvement of apples through MAS.
Concurrent Symposium Speaker:
|
|
| 9:53 AM
- 10:13 AM |
Return of the OG: the original cash crop makes a comeback.
(Biotic and Applied)
LL21D
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.
Chair and Concurrent Symposium Speaker:
|
|
| 10:13 AM
- 10:15 AM |
GOT synthase gene expression is greater under low light levels in Cannabis sativa ‘Sour Diesel’
(Biotic and Applied)
LL21D
Unfertilized female flowers of the dioecious Cannabis sativa plant are a rich source of cannabinoids, including tetrahydrocannabinoid (THC) and cannabidiol (CBD), which may have some medical applications. Research has demonstrated that these cannabinoids are found in higher concentrations levels in apical flowers that are closer to a light source than flowers in under the plant’s canopy. No peer-reviewed literature, however, has shown that gene expression is correlated with varying cannabinoid production levels with respect to light level. Therefore, the objective of this work was to evaluate if the expression of genes encoding enzymes in the cannabinoid biosynthesis pathway changed with respect to three distinct light levels, and if any differences are correlated with changes in cannabinoid end product. We used real-time (quantitative) qPCR to evaluate expression of CBDa synthase, THCa synthase, and geranylpyrophosphate:olivetolate geranyltransferase (GOT) synthase, which produces cannabigerolic acid (CBGa). We employed high-performance liquid chromatography (HPLC) to evaluate the cannabinoid levels in flowers in Cannabis sativa ‘Sour Diesel’. Marijuana plants were grown in a Connecticut Pharmaceutical Solutions LLC’s commercial grow facility. Samples were taken twice during crop development. Expression of GOT synthase was higher at lower light levels than at higher light levels. This was not correlated production of the CBGa as flowers in higher light levels were found to contain significantly greater levels of CBGa than in lower light levels. Higher gene expression may be caused by a shade; however, the photosynthetic rate of flowers receiving less light likely have less carbon to assimilate into cannabinoid end product generation. Expression of other genes in the cannabinoid biosynthetic pathway followed a similar trend; however, significant differences were not found between high and low light levels. Supported by Connecticut Pharmaceutical Solutions LLC.
|
|
| 10:45 AM
- 10:48 AM |
Overview of Area by Chair, Toni M. Kutchan
(Genetics/Genomics)
LL20A
Chair and Concurrent Symposium Speaker:
|
|
| 10:48 AM
- 11:08 AM |
Medicines from plants— the pathway to Veratrum alkaloids
(Genetics/Genomics)
LL20A
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.
Chair and Concurrent Symposium Speaker:
|
|
| 11:08 AM
- 11:28 AM |
Engineering modular plant-to-plant communication
(Genetics/Genomics)
LL20A
The ability to engineer plants using the tools of synthetic biology is of increasing importance for solving agricultural problems, and for adapting plants to novel uses. A collaboration between five labs has begun to engineer plant-to-plant communication using volatile organic compounds (VOCs). By modifying and enhancing natural hormone pathways in plants we have been able to demonstrate synthetic communication. In particular, we have engineered a modular ethylene sensor, and identified modular promoter structures that are responsive to methyl salicylate and methyl jasmonate. When these are cloned adjacent to reporter genes, gas-dependent production of signals can be observed. In parallel, we have determined that carbon flux through the normal pathway for the production of the volatile ethylene may be limited, and have been able to generate transgenic plants with enhanced ethylene production. When ethylene ‘senders’ are aligned with ethylene ‘receivers,’ plant-to-plant communication can be observed. Along the way, we have developed an extensive new tool kit that allows for the establishment of an Orthogonal Control System (OCS) in plants that operates on top of extant plant regulatory and metabolic systems. We have for the first time created wholly orthogonal transcription factors using dCas9:VP64 as a transcription factor, and shown that these can activate completely artificial promoters. While these demonstrations have so far been shown in model plant species (Nicotiana, Arabidopsis), in parallel we have undertaken an effort to demonstrate that constructs developed can be transported into new species, and to this end have made great progress in ‘taming’ a non-model plant, common dandelion (Taraxacum). Ultimately, by funneling engineered sensor ‘inputs’ through VOC communication channel to appropriate reporter ‘outputs’ it should be possible to allow fields of plants to better serve as self-sentinels against pests and other environmental incursions.
Co-author(s):
- Jeff Tabor,
- Wyatt Rodgers,
- Araceli Cantero-Garcia,
- Hong Qiao,
- Nathaniel Riggan,
- Nestor Rodriguez,
- Jaclyn Tran,
- Andrew Ellington,
- Yogendra Bordiya,
- Inyup Paik,
- Alan Lloyd,
- Sibum Sung,
- Junghyun Kim,
- Jose-angel Torres,
- Shaunak Kar
Concurrent Symposium Speaker:
|
|
| 11:28 AM
- 11:48 AM |
Biosynthesis of complex plant-derived natural products
(Genetics/Genomics)
LL20A
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.
Concurrent Symposium Speaker:
|
|
| 11:48 AM
- 12:08 PM |
Discovery and engineering of colchicine biosynthetic enzymes
(Genetics/Genomics)
LL20A
Historically, plants have played a prominent role in human medicine. The medicinal effects of plants are due to bioactive small molecule natural products, many of which continue to serve as major sources of clinical pharmaceuticals. One such compound is the alkaloid colchicine, which is produced by plants from the Colchicum and Gloriosa genera and is used clinically for treating gout and other inflammatory diseases. Although previous studies have identified specific plant tissues associated with colchicine biosynthesis, along with the precursors from which this molecule is derived, the underlying biosynthetic genes have remained unidentified. To facilitate colchicine biosynthetic pathway discovery, we have performed extensive metabolite profiling in Gloriosa superba and paired this to corresponding RNA-seq expression data, with the hypothesis that relative expression of biosynthetic genes should correlate to accumulation of colchicine alkaloids. By then using a combination of correlative expression analyses and enzymatic logic, we selected a testable number of candidate biosynthetic enzymes for functional screening via heterologous expression in tobacco. Through this methodology, we have discovered and characterized 7 novel enzymes that act to produce a late stage colchicine intermediate from a 1-phenethylisoquinoline substrate that is a known precursor to colchicine. Furthermore, by utilizing enzymes from plant primary metabolism, along with several involved in the biosynthesis of natural products from other plants, we have engineered a 16-enzyme pathway in tobacco that connects the newly discovered biosynthetic steps in colchicine biosynthesis to primary amino acid metabolism from the heterologous tobacco host, thus allowing for metabolic engineering of colchicine alkaloids. This result not only establishes a nearly complete metabolic route to colchicine, but also pushes the boundaries for the rate and magnitude of biosynthetic pathway discovery in plants.
Concurrent Symposium Speaker:
|
|
| 12:08 PM
- 12:28 PM |
Biosynthesis of Pyrethrins in Tanacetum cinerariifolium
(Genetics/Genomics)
LL20A
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.
Concurrent Symposium Speaker:
|
|
| 12:28 PM
- 12:30 PM |
Medicinal Genomics: Exploring the diversity of iridoid compounds in blueberry for human health benef
(Genetics/Genomics)
LL20A
Blueberry (Vaccinium corymbosum) is an economically important fruit crop that is native to North America. Fresh market production of blueberries in the United States was valued at $5.68 billion in 2015 and was planted over 36,349 hectares. In addition to its commercial value, blueberries are prized for their positive health benefits, containing high levels of antioxidants, which has been linked to a decreased risk of cancer and heart disease. Iridoids are another class of known pharmacologically important compounds that have recently been found in blueberries. Iridoids are present in over 15 plant families and are potent natural products with a wide range of biological activities in humans including, anticancer, antibacterial and anti-inflammatory. No work however, has been able to detect monotropein, an iridoid glycoside compound, in any North American blueberry species (V. corybosum, V. angustifolium, V. virgatum), the most commonly used germplasm for cultivated blueberry. To address this research limitation I have collected over 80 berry and leaf samples from multiple species and commercial varieties of blueberry to survey for monotropein production. The glycoside iridoid monotropein was successfully identified in a subset of cultivars in the diversity panel, as well as all wild blueberry species in this panel, indicating iridoid production can be targeted through breeding efforts that incorporate wild germplasm. Currently, both metabolite and transcriptomic data are being leveraged to identify key iridoid biosynthetic pathway genes in blueberry. In addition to providing molecular markers to breed for higher iridoid content, knowing how iridoids are synthesized will enable much improved access to these compounds for future clinical research.
|
|
| 10:45 AM
- 10:48 AM |
Overview of Area by Chair, Thomas Juenger
(Abiotic)
LL20D
Chair and Concurrent Symposium Speaker:
|
|
| 10:48 AM
- 11:08 AM |
Genetic and genomic studies of climate adaptation and genotype-by-environment interaction in switchgrass
(Abiotic)
LL20D
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.
Chair and Concurrent Symposium Speaker:
|
|
| 11:08 AM
- 11:28 AM |
Plant genetics to adapt to and reduce climate change
(Abiotic)
LL20D
Plant populations that currently exist have adapted to past and current climates. Plants are also major controls on the carbon cycle and thus can reduce anthropogenic climate change. I will present findings on the traits and loci that underlie adaptation to climate. I will also introduce more recent work to identify the genetics controlling plant traits that can contribute to carbon sequestration in highly managed agricultural systems.
Concurrent Symposium Speaker:
|
|
| 11:28 AM
- 11:48 AM |
Isoprene emission affects growth-defense tradeoffs in plants
(Abiotic)
LL20D
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.
Concurrent Symposium Speaker:
|
|
| 11:48 AM
- 12:08 PM |
Physiological and molecular responses of winter wheat exposed to high night-time temperature during
(Abiotic)
LL20D
Night-time temperatures are increasing at a faster pace than day-time temperature, posing a serious threat to wheat production. Different physiological routes through which HDT and HNT induces yield and quality losses have been documented in cereals. A combination of physiological, metabolomic and source-sink starch metabolism related enzymatic responses has helped us to unravel HNT responses in wheat. We imposed a sequential increase in night-time temperatures (from 15o to 27oC) on ten different winter wheat genotypes, to determine the threshold for HNT inducing yield reduction and loss in quality (protein and starch). Based on the identified threshold, source, sink and stem metabolic changes were captured on selected contrasting genotypes exposed to HNT during grain-filling stage. Using leaf and grain samples collected during grain filling, exposed to different night-time temperatures, we have identified key bottlenecks related to sugar translocation and accumulation in wheat grains exposed to HNT. Transmission electron microscopy was employed to ascertain the accumulation of starch, protein and lipids in the endosperm and the germ under different night-time temperatures. Findings generated from controlled chambers and unique field-based heat tents will be presented, with an overall goal to translate findings in to developing HNT tolerant wheat varieties.
Concurrent Symposium Speaker:
|
|
| 12:08 PM
- 12:28 PM |
Developmental scaling of venation architecture in grasses underlies worldwide leaf size distribution
(Abiotic)
LL20D
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.
Co-author(s):
- Yu Zhang,
- Christine Scoffoni,
- Pascal-Antoine Christin,
- Samuel H. Taylor,
- Erika J. Edwards,
- Lawren Sack,
- Christine Vuong,
- Jessica Pasquet-Kok,
- Andrew C Diener,
- Colin P. Osborne,
- Teera Watcharamongkol
Concurrent Symposium Speaker:
|
|
| 12:28 PM
- 12:30 PM |
Developmental changes in leaf physiology and its implications for environmental adaptation
(Abiotic)
LL20D
Plants transition through distinct stages as they develop, the timing of which, significantly impacts large-scale ecological and evolutionary processes. Vegetative phase change (VPC), the developmental transition from juvenile to adult vegetative growth, has been well studied at the molecular level however, little is known about its importance for plant performance and physiological functioning. In this study, variation in physiological and morphological characteristics between juvenile and adult leaves of four diverse species, Zea mays, Passiflora edulis, Populus tremula x alba, and Arabidopsis thaliana, were analyzed. Mutants of miR156, the master regulator of VPC, were used to modulate the timing of VPC in all four species to investigate differences in photosynthetic traits and leaf morphology associated with vegetative development. Further, these mutants were used to determine whether variation in traits between juvenile and adult leaves translate into variation in plant performance under environmental stress. Through this research we found significant variation in photosynthetic properties between juvenile and adult leaves in all species including maximum photosynthetic rates and rates of photosynthetic light induction. Additionally, juvenile and adult leaves showed differences in morphology including specific leaf area, stomatal densities and venation, traits with major implications for foliar function. Lastly, when both mutants and natural genotypes with variation in the timing of VPC were subjected to the environmental stresses of heat, drought and low light, there were significant relationship between plant performance under some of these stresses and the timing of VPC. This research begins to uncover the role of vegetative development in plant physiology, potential mechanisms to be utilized in plant breeding programs and insight into the underpinnings that may have led to the evolutionary conservation of VPC and its master regulator miR156.
|
|
| 10:45 AM
- 10:48 AM |
Overview of Area by Chair, Chunhua Zhang
(Cell/Development/Systems)
LL21C
Chair and Concurrent Symposium Speaker:
|
|
| 10:48 AM
- 11:08 AM |
A heterogeneous nuclear ribonucleoprotein (hnRNP) in Chlamydomonas functions as a cell-cycle repressor upstream of the retinoblastoma tumor suppressor complex
(Cell/Development/Systems)
LL21C
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.
Concurrent Symposium Speaker:
|
|
| 11:08 AM
- 11:28 AM |
Dynamics and Function of the Plant Cell Wall-Plasma Membrane Interface
(Cell/Development/Systems)
LL21C
Living organisms respond and acclimate to a changing environment to survive and thrive. The cell surface is the front line where environmental stimuli are immediately detected and converted to intracellular signaling events. Therefore, a fundamental understanding of the organization, dynamics, and protein components of the cell surface will shed light on the structural and molecular underpinnings of growth and stress responses. In plant cells, it remains largely mysterious as to how the cell wall (CW) and the plasma membrane (PM) are connected, how the CW-PM interface is maintained and remodeled, and how signals are sensed at this interface upon pressures from within or outside the plant cell. We aim to address these questions in the model species Arabidopsis thaliana due to the extensive genetic resources and live-cell imaging marker lines available. To begin to investigate interface dynamics, we fluorescently labeled the outer periclinal CW and the PM, respectively, and tracked their behavior over time during hyperosmotic stress. Compared to the control condition where the CW and the PM are in close proximity all the time, hyperosmotic stress resulted in separation of the PM from the CW. We are currently analyzing the images to determine the existence (and if so, distribution) of CW-PM attachment sites during hyperosmotic stress. To identify the molecular components that serve as “pins” to hold together the CW and the PM, we surveyed a collection of mutants deficient in each major class of wall components or glycosylphosphatidylinositol-anchored proteins. We found that mutants associated with cellulose biosynthesis and organization are hypersensitive to osmotic stress and have defects in growth recovery. Contrastingly, mutants related to rhamnogalacturonan-I exhibit hyposensitivity to osmotic stress. We are currently investigating the cellular and subcellular details, such as cytosolic Ca2+ profiles, apoplast pH signatures, among other processes in these mutants.
Concurrent Symposium Speaker:
|
|
| 11:28 AM
- 11:48 AM |
Motors take a pause: A new role for myosin XI in secretory vesicle tethering
(Cell/Development/Systems)
LL21C
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.
Concurrent Symposium Speaker:
|
|
| 11:48 AM
- 12:08 PM |
Enzymatic activities of protein complexes could affect their cellular trafficking in plants
(Cell/Development/Systems)
LL21C
Like in other eukaryotic cells, plant membrane trafficking pathways transport proteins among organelles and play essential roles in growth and development. The cargo proteins for membrane trafficking have diverse functions such as cell wall biosynthesis, signaling, and nutrient uptake. How plant cells accurately control protein transport in a spatiotemporal manner has not been well characterized. Cellulose synthase complexes (CSCs) are large membrane-associated protein complexes that catalyze the synthesis of cellulose at the plasma membrane. CSCs are delivered to the plasma membrane through membrane trafficking pathways for their proper functions. Using chemical genetic approach, we identified a small molecule that targets the catalytic domain of plant cellulose synthases. Combining small molecule treatment, live cell imaging and quantitative image analysis, we found that the catalytic activity of CSCs affected efficient exocytic transport of these large protein complexes. Inhibition of CSCs catalytic activity reduced the transport of CSCs at early steps of exocytic trafficking, although these protein complexes might have been assembled properly. Our results add to current understanding of how plant membrane trafficking machineries regulate spatiotemporal delivery of proteins for plant growth and environment adaptation.
Chair and Concurrent Symposium Speaker:
|
|
| 12:08 PM
- 12:28 PM |
The auxin transport inhibitor targets villin-generated actin bundles to regulate polar auxin transport in Arabidopsis
(Cell/Development/Systems)
LL21C
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.
Concurrent Symposium Speaker:
|
|
| 12:28 PM
- 12:30 PM |
TOR coordinates plant growth by dynamically regulating cell-cell transport
(Cell/Development/Systems)
LL21C
The coordinated redistribution of sugars from mature “source” leaves to support the growth of developing “sink” leaves requires tight regulation of sugar transport between cells via plasmodesmata (PD). Although fundamental to plant physiology, the mechanisms that control PD transport and thereby support development of new leaves have remained elusive. From a forward genetic screen for altered PD transport, we discovered that PD transport is regulated by the conserved eukaryotic glucose-TOR (TARGET OF RAPAMYCIN) signaling hub. TOR is significantly more active in mature leaves photosynthesizing excess sugars than in young, growing leaves, and this shift in activity impacts rates of PD transport. Genetic, chemical, and physiological treatments promoting or disrupting TOR activity support the model that glucose-activated TOR controls PD transport in leaves. An established TOR effector in plants, PP2A, contributes to the control of PD transport during shoot development. We conclude that plant cells regulate PD trafficking in response to changing carbohydrate availability monitored by the TOR pathway.
|
|
| 10:45 AM
- 10:48 AM |
Overview of Area by Chair, Alexander Jones
(Biochemistry)
LL21AB
Chair and Concurrent Symposium Speaker:
|
|
| 10:48 AM
- 11:08 AM |
Dynamic regulation of cellular gibberellin and abscisic acid distributions influencing plant growth patterning
(Biochemistry)
LL21AB
Regulated distribution of plant hormones across tissues and over time is fundamental to plant growth and development and optical biosensors for plant hormones are beginning to shed light on hormone distributions in planta. We have engineered improved versions of the previously published optogenetic biosensors for the hormones gibberellin (GPS1) and abscisic acid (ABACUS1). These next-generation biosensors detect nanomolar levels of hormone at the cellular level with reduced effects on endogenous hormone signaling in Arabidopsis compared with first generation GPS1 and ABACUS1. Gibberellin gradients detected with GPS1 biosensors correlated with gradients of cell length in rapidly elongating roots and dark-grown hypocotyls, but it remained unclear how these gradients arise from the ensemble activities of gibberellin enzymes and transporters. We now present evidence in support of patterned gibberellin biosynthesis and permeability driving gibberellin patterns in roots. The effect of gibberellin enzyme and transport mutants on gibberellin patterning in roots will be discussed in the context of understanding how gibberellin gradients are determined and how they, in turn, influence patterning of plant cell growth. ABA patterns detected with next generation ABACUS biosensors will be discussed in the context of ABA accumulation and elimination rates and their effects on plant development.
Chair and Concurrent Symposium Speaker:
|
|
| 11:08 AM
- 11:28 AM |
Regulation of CDC48/p97 Dependent Plant Growth by the Phytohormone Gibberellin
(Biochemistry)
LL21AB
The phytohormone Gibberellin (GA) regulates various aspects of plant growth and development, including seed germination, stem elongation, flower induction and fruit set through the destabilization of the growth repressor, DELLA. CDC48/p97 is a highly-conserved homohexameric AAA-ATPase molecular chaperone that uses the energy of ATP hydrolysis to unfold and/or extract client proteins from membranes, protein complexes and other cellular structure. The Plant ubiquitin regulatory X (UBX)-containing protein, PUX1, negatively regulates CDC48/p97 by promoting the disassembly of the active homohexameric complex into its inactive monomers. Preliminary data indicate that the Gibberellin receptor, GID1 (GA-INSENSITIVE DWARF1), interacts with two forms of PUX1, a 38kD full-length and a 34 kD putative truncated form suggesting a previously undefined molecular mechanism by which GA controls plant growth and development. In vitro characterization of the interaction have shown that the binding of PUX1 to GID1 is GA-independent and mediated through a region of PUX1 containing the UBX domain. Additionally, PUX1/GID1/CDC48 was showed to form a ternary complex that was also GA-independent. Furthermore, in vivo studies of fluorescently tag PUX1 in plants have shown that PUX1 is also localize in the nucleus. Whereas GA biosynthesis mutant suffer from dwarfism, lines with elevated levels of GA show increased growth due increase cell division and elongation. Similar to lines that have enhance GA-signaling, pux1 loss-of-function mutants exhibit accelerated growth compared to wild type and behave like GA-hypersensitive mutants when treated with Paclobutrazol, a GA biosynthesis inhibitor. Together our findings suggests a new mechanism by which GA-signaling promotes plant growth and development in part by inhibiting PUX1 function, thereby maintaining levels of active CDC48/p97 necessary for proper cell division and expansion.
Co-author(s):
|