Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Colleagues in the field of plant biology. Changes/Problems: The project has centered on events at the transcript and general metabolic levels. It was not possible, because of technical difficulties, to pursue the phospholipids aspect of the original goals. What opportunities for training and professional development has the project provided? Four graduate students have received training on this project, two of whom have graduated already. How have the results been disseminated to communities of interest? Peer-reviewed publications, as listed. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Effects of Drought Stress on Andean Potato Accessions (Mane et al., 2008; Vasquez-Robinet et al, 2008; Watkinson et al., 2008). Drought stress experiments were carried out with three accessions of Solanum tuberosum ssp. andigena that show varying degrees of physiological acclimation (induced after stress exposure) or adaptation (drought resistant from the onset of the experiment) (Watkinson et al., 2006). We correlated changes in expression, and differing constitutive expression levels, of specific pathways and genes with the adaptive and acclimation-related phenotypes exhibited by the three accessions. These include transcripts in anti-oxidant defense, heat shock proteins, metallothioneins, flavonoid biosynthesis, carbon metabolism, chromatin remodeling, and TF genes, including two types of zinc finger and several Myb and bZIP factors, not previously associated with stress responses. In a second series of drought trials, a series of Andean potato genotypes were screened for drought responses in the greenhouse, at the same time as a comparable drought experiment in the field in the Andes. This work was carried out in collaboration with the International Potato Center. Patterns of Gene Expression and Metabolite Changes Accompanying Winter Hardening in Sitka Spruce. (Grene et al., 2012; Collakova et al., 2013a) “Systems biology tools were applied to data from a microarray study of changes in gene expression taking place over the time period of winter hardening in the needles of Sitka spruce (data of Dr. J. Holliday, FREC). Metabolomics data were also available. The MapMan tool and Ontologizer, a Gene Ontology-based tool, revealed enrichment of genes involved in mitochondrial function, antioxidant activity, ethylene metabolism and signaling, secretory pathways, and cell wall function. Changes in metabolite levels over the time course were consistent with the gene expression patterns observed. Our data suggest coordinated metabolic and signaling responses leading to cell wall remodeling. Co-expression of genes encoding proteins associated with biosynthesis of non-structural cell wall carbohydrates was observed, which may be regulated by ethylene signaling components. ) At the same time, numerous genes, whose products are putatively localized to the endomembrane system and involved in both the synthesis and trafficking of cell wall carbohydrates, were up regulated. Taken together, these results suggest a link between ethylene signaling and biosynthesis, antioxidant function, and targeting of cell wall related gene products during the period of winter hardening. Transcriptomic and Metabolomic Transitions Underlying Soybean Seed Development: Seed Filling and Desiccation Tolerance Acquisition (Collakova et al., 2013b). Dehydration stress affects every aspect of plant growth and function, modifying anatomy, morphology, physiology, and biochemistry. During seed development and, especially, seed maturation, desiccation constitutes an active, induced program that can establish extreme drought tolerance potentially lasting millennia.. “Two major developmental and metabolic transitions were captured over the time course of soybean embryo development, enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data from the time course revealed groups of functionally related metabolites and transcripts active in these different developmental programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. Molecular events underlying desiccation acquisition or drought tolerance and dormancy were in the past regarded as being mainly stress related. However, this genetically programmed developmental process proves to be more complex than a simple acquisition of desiccation tolerance. Desiccation acquisition and dormancy induction in orthodox seeds, in fact, involve a specific and active transcriptional and metabolomic program in which 22% of all genes expressed over the course of seed development were activated during the last three time points of the experiment. “ Effects of Drought Stress on Fertility in Maize, (Kakumanu et al., 2012) “Drought stress affects cereals especially during the reproductive stage. The maize (Zea mays) drought transcriptome was studied using RNA-Seq analysis to compare drought treated and well-watered fertilized ovary and basal leaf meristem tissue, revealing that many more drought responsive genes responded in the ovary compared to the leaf meristem. Gene Ontology (GO) enrichment analysis revealed massive down-regulation of cell division and cell cycle genes in the drought stressed ovary only, in which a lower number of seeds were also observed, compared to well-watered controls. ABA-related processes responded positively, but only in the ovaries. GO enrichment analysis also suggested differential responses to drought between the two tissues in categories such as oxidative stress responses and cell cycle events. Related responses suggested the operation of low glucose sensing in drought stressed ovaries. Our working hypothesis involves signaling events associated with increased ABA levels, together with decreased glucose levels, leading to disruption of ABA/sugar signaling, activation of PCD/senescence through repression of a PLC-mediated signaling pathway, and arrest of the cell cycle in the stressed ovary at 1DAP. Increased invertase levels in the stressed leaf meristem, on the other hand, may have resulted in that tissue maintaining hexose levels at an “unstressed” level, and lower ABA levels, lead to successful resistance to the level of drought stress applied. “ Representation, Visualization, and Modeling of Signaling Pathways in Higher Plants: The Beacon Project. (Utilized in Grene et al., 2012; Kakumanu et al., 2012). Gene expression, protein-protein interaction (PPI), metabolite, and post-transcriptional modification data sets have become the elements of biological discovery. There has been a flood of bioinformatics tools developed to facilitate the task of deriving meaning from such spreadsheet. Common themes are the representation of data using biological networks, the manipulation and computational analysis of those networks, and, lastly, their accumulation for improved inference. One software system for constructing networks comes from the Beacon project, co-lead by Grene and Lenwood Heath (Computer Science), whose goal is to represent, simulate, and infer signal transduction pathways for plant species. The centerpiece of the Beacon system is a network editor for capturing and visualizing signal transduction pathways in the Activity Flow language of the Systems Biology Graphical Notation (SBGN), a standardized topological description of pathways. At a workshop in August, 2012, the Beacon editor was used to capture 11 signal transduction pathways compiled by leading plant biologists. These “founder” pathways are stored in the Beacon database and are publicly accessible online. Other aspects of the Beacon project are under development, see below.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Delasa Aghamirzaie, Mahdi Nabiyouni, Yihui Fang, Curtis Klumas, Lenwood S. Heath, Ruth Grene, and Eva Collakova (2013) Changes in RNA Splicing in Developing Soybean (Glycine max) Embryos. Biology 2:1311-1337.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Collakova E, Klumas C, Suren H, Myers E, Heath LS, Holliday JA, Grene R. (2013) Evidence for extensive heterotrophic metabolism, antioxidant action, and associated regulatory events during winter hardening in Sitka spruce. BMC Plant Biol. 2013 Apr 30;13(1):72
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Eva Collakova, Delasa Aghamirzaie, Yihui Fang, Curtis Klumas, Farzaneh Tabataba, Akshay Kakumanu, Elijah Myers, Lenwood Heath, Ruth Grene (2013). Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) Embryos. Metabolites 3
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
4. Grene R, Klumas C, Suren H, Yang K, Collakova E, Myers E, Heath LS, Holliday JA (2012). Mining and visualization of microarray and metabolomic data reveal extensive cell wall remodeling during winter hardening in Sitka spruce (Picea sitchensis). Front Plant Sci. 2012;3:241. doi: 10.3389/fpls.2012.00241. Epub 2012 Oct 29.
- Type:
Journal Articles
Status:
Published
Year Published:
2012
Citation:
Zhou L, Franck C, Yang K, Pilot G, Heath LS, Grene R (2012). Zhou L, Franck C, Yang K, Pilot G, Heath LS, Grene R (2012). Mining for meaning: visualization approaches to deciphering Arabidopsis stress responses in roots and shoots. OMICS. 2012 16:208-28. Epub 2012 Mar 14.
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: Hosted a training workshop on Beacon, an in-house software tool for the depiction of signaling pathways in plants that are responsive to abiotic stress. This is a joint project with a faculty member in the Department of Computer Science. The tool is being expanded to include a database of curated pathways, and the ability for simulation and prediction. In parallel, a phenotypic assay is being developed in the laboratory, using Arabidopsis to test predictions obtained from Beacon concerning the putative functions of individual genes in drought responses and drought tolerance. The assay includes a measurement of the effects of increasing severity of drought stress on seed yield, as well as tests of the extent of drought-mediated expression of key regulatory genes. Preliminary data are encouraging, suggesting a decrease in seed yield of up to 40% under drought stress, and increases in the expression of genes regulating two defense pathways. All aspects of the Beacon project are displayed at a publicly available website, which is frequently updated to reflect progress on the project. PARTICIPANTS: Four graduate students in the VT interdisciplinary Genetics, Bioinformatics, and Computational Biology doctoral program worked on the projects which yielded the publications listed. Expertise in computation was blended with biological knowledge to derive the conclusions reached in each paper. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Drought impacts economic, environmental, and social aspects of life. In fact, drought affects more people than any other natural hazard. According to the National Drought Mitigation Center, loss in agriculture in the United States due to drought was estimated to be approximately $11 billion in 2002. There is increasing demand for methods to ensure food and fiber security and to contribute to sustainable livelihoods in the coming decades. Many crop plants and forest trees are highly susceptible to drought stress. Withholding water reduces leaf number, leaf size, shoot height, and the rate of photosynthesis. Productivity is often limited to areas with adequate rainfall or suitable irrigation and means that the crop has a high and costly water requirement for maximal yield. A better understanding of the effects of drought on plants, especially the integration of drought-responsive events from the molecular and cellular to the whole-plant level, including particular organs such as seeds, is vital to prepare for the genetic engineering of varieties with improved drought tolerance. With the genomics and bioinformatics resources that are currently available for plant species, especially the model plant Arabidopsis thaliana, and, increasingly, crop and forest species, it is now possible to investigate the regulatory mechanisms that underlie relative drought tolerances among closely related genotypes. Winter hardening includes a component of drought stress, hence its inclusion in the report. Although individual components of stress response systems have been studied under water-deficiency, the overall "network logic" of pathways that are operational in drought tolerant plants remains still largely unknown. To further understand drought systems biology, the nature of regulatory control over pathways and networks must be elucidated, and other kinds of data, such as metabolomics and physiological data, and current findings from the literature, incorporated into the system.
Publications
- 1. Mining and visualization of microarray and metabolomic data reveal extensive cell wall remodeling during winter hardening in Sitka spruce (Picea sitchensis). Grene R, Klumas C, Suren H, Yang K, Collakova E, Myers E, Heath LS, Holliday JA. Front Plant Sci. 2012;3:241. doi: 10.3389/fpls.2012.00241. Epub 2012 Oct 29.
- 2. Effects of Drought on Gene Expression in Maize Reproductive and Leaf Meristem Tissue Revealed by RNA-Seq. Kakumanu A, Ambavaram MM, Klumas C, Krishnan A, Batlang U, Myers E, Grene R, Pereira A. Plant Physiol. 2012 Oct;160(2):846-67. doi: 10.1104/pp.112.200444. Epub 2012 Jul 26.
- 3. Mining for meaning: visualization approaches to deciphering Arabidopsis stress responses in roots and shoots. Zhou L, Franck C, Yang K, Pilot G, Heath LS, Grene R. OMICS. 2012 Apr;16(4):208-28. Epub 2012 Mar 14
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Embryo abortion occurs when plants at their reproductive phase experience abiotic stresses, such as drought or temperature extremes. This effect has been reported for wheat, barley, soybean, and chickpea indicating that this is a widespread phenomenon in the plant world . Maize and rice are both affected in this way by drought stress, posing a threat to food security worldwide. Ovaries in tissue subjected to, or with a previous history of, drought stress stop growth within 1-2 days after pollination. Interestingly, embryo abortion is observed even when the drought stress is relieved before fertilization has occurred, suggesting that the vulnerability is caused by an influence of drought on the maternal tissue, which develops extensively both before, and immediately after, fertilization. In fact, tolerance to water stress in female floral parts has been correlated with yield in maize. To advance our understanding of responses to drought stress in early embryos, we conducted a transcriptome analysis using Illumina deep sequencing of RNA populations obtained from well-watered and drought stressed leaf meristem and pollinated ovaries of a fully sequenced maize genotype. The basal leaf meristem regulating vegetative leaf growth was contrasted with ovaries one day after pollination to best capture gene expression events in the ovary that may lead to early embryo abortion and to gain insights into possible causes for this differential sensitivity. PARTICIPANTS: Dr. Andy Pereira, University of Arkansas. A graduate student, Akshay Kakumanu, funded as a GRA, has worked with Grene, since August 2010 on the project. The work of Kakumanu constituted part of his training in the degree program in the life sciences. TARGET AUDIENCES: The analyzed data, and the provisional inferences that we have drawn from them, were presented at the Annual Meeting of the American Society of Plant Biologists, August, 2011. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
An analysis of the RNA Seq data obtained through development of a pipeline and the use of several bioinformatics tools made available through the Discovery Environment hosted by the iPlant Collaborative, a group to which Grene belongs. The data show that many more genes responded to drought stress in the ovary at 1DAP than in the leaf meristem. Gene Ontology (GO) enrichment analysis revealed massive down-regulation of cell division and cell-cycle genes in drought-stressed ovary only. Differences were also apparent among GO categories related to carbohydrate metabolism. Changes in starch and sucrose metabolism- related genes occurred in the ovary, as revealed in MapMan, compatible with a decrease in starch levels, and in sucrose transporter function, with no comparable changes occurring in the leaf meristem. ABA-related processes responded positively, but only in the ovaries. Some evidence for low glucose sensing mediated transcriptional events was obtained. The GO enrichment analysis also suggested differential responses to drought between the two tissues in other categories related to oxidative stress-related events and chromatin remodeling. The data are discussed in the context of the susceptibility of maize kernel to drought stress, and the relative robustness of actively dividing vegetative tissue taken at the same time from the same plant subjected to the same conditions. A hypothesis is formulated, proposing drought-mediated intersecting effects on glucose signaling through hexokinase 1, ABA-dependent and independent signaling, antioxidant responses, PCD, and cell cycle related processes. Our transcriptome analysis also revealed drought-mediated post- transcriptional events (e.g. alternative splicing), which appear to be tissue-specific.
Publications
- No publications reported this period
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: 1 Desiccating and germinating seeds are particularly prone to oxidative stress. Glutathione (GSH) is one of the most abundant and ubiquitous antioxidants in plants. To better understand GSH's role in developing and germinating seeds, wildtype (WT) and a line of seed antisense for glutathione reductase 2 (anGR2), one of two glutathione reductase (GR) genes characterized in the genetic model plant Arabidopsis thaliana, were compared. GSH levels in maturing and germinating seeds were measured by high performance liquid chromatography (HPLC) and GR activity by native Polyacrylamide gel electrophoresis (PAGE). Natural and accelerated aging and germination at high temperature and under water stress were compared for WT and anGR2 seeds. Total GSH pools were two-fold less in anGR2 than in WT after three hours of imbibition. Under high temperature and osmotic stress, germination percentages were lower in anGR2, suggesting a specific relationship between GSH metabolism and stress defense mechanisms early in germination. Germination of anGR2 seeds declined while that of WT seeds increased slightly, during nine months of conventional storage. The relationship between GR2 transcript and activity levels during early imbibition and total GSH pools suggest a key role for GR2 in protection against environmental stress. This study shows that GSH metabolism is one factor that determines the stress tolerance of germinating Arabidopsis seeds. 2 Heat shock proteins (HSPs) are induced not only under heat stress conditions but also under other environmental stresses such as water stress. In plants, HSPs families are larger than those of other eukaryotes. In order to elucidate a possible connection between HSP expression and photosynthetic acclimation or conditioning, we conducted a water stress experiment in loblolly pine (Pinus taeda L.) seedlings involving progressive treatment consisting of one cycle of mild stress, followed by two cycles of severe stress. Net photosynthesis was measured at each stress level. Photosynthetic acclimation occurred in the progressive treatment after the first cycle, but not in the severe treatment, suggesting that a cycle of mild stress conditioned the trees to adapt to a more severe stress. Real time results indicated specific patterns in needles in the expression of HSP70, HSP90 and sHSP genes for each treatment, both at maximum stress and at recovery. We identified a pine homolog to GRP94 (ER resident HSP90) that was induced after rehydration coincident with acclimation. Further analysis of the promoter region of the pine GRP94 showed putative cis-elements associated with water stress and rehydration, corresponding to the expression pattern observed in our experiment. PARTICIPANTS: Individuals who worked on the project(1) principal investigator(s)/project director(s) (PIs/PDs) Drs. R. Grene and G. Welbaum supervised the project ; and (2) M. Sumugat conducted the work as part of her master's thesis in the Department of Horticulture; Dr. D Cortes, under the supervision of Dr. V. Shulaev, at the Virginia Bioinformatics Institute(VBI), performed analysis of the antioxidant content of the seeds. Collaborators and contacts: Drs. V. Shulaev and D. Cortes, VBI Training of M. Sumugat, Master's student TARGET AUDIENCES: Target Audiences: Seed biologists, seed companies, paper companies, the academic community of plant biologists, horticulturalists, forest biologists, and agronomists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Drought impacts economic, environmental, and social aspects of life. In fact, drought affects more people than any other natural hazard. According to the National Drought Mitigation Center, loss in agriculture in the United States due to drought was estimated to be approximately $11 billion in 2002. There is increasing demand for methods to ensure food and fiber security and to contribute to sustainable livelihoods in the coming decades. Many crop plants and forest trees are highly susceptible to drought stress. Withholding water reduces leaf number, leaf size, shoot height, and the rate of photosynthesis. Productivity is often limited to areas with adequate rainfall or suitable irrigation and means that the crop has a high and costly water requirement for maximal yield. A better understanding of the effects of drought on plants, especially the integration of drought-responsive events from the molecular and cellular to the whole-plant level, including particular organs such as seeds, is vital to prepare for the genetic engineering of varieties with improved drought tolerance. With the genomics and bioinformatics resources that are currently available for plant species, especially the model plant Arabidopsis thaliana, and, increasingly, crop and forest species, it is now possible to investigate the regulatory mechanisms that underlie relative drought tolerances among closely related genotypes. Although individual components of stress response systems have been studied under water-deficiency, the overall "network logic" of pathways that are operational in drought tolerant plants remains still largely unknown. To further understand drought systems biology, the nature of regulatory control over pathways and networks must be elucidated, and other kinds of data, such as metabolomics and physiological data, and current findings from the literature, incorporated into the system.
Publications
- 1 Sumugat MR Donahue JL Cortes DF, Stromberg VK, Grene R Shulaev V Welbaum GE (2010) Seed Development and Germination in an Arabidopsis thaliana Line Antisense to Glutathione Reductase Journal of New Seeds; Vol 11:104-126 2 Vasquez-Robinet C Watkinson JI Sioson AA Ramakrishnan N Heath LS Grene R(2010) Differential expression of heat shock protein genes in preconditioning for photosynthetic acclimation in water-stressed loblolly pine. Plant Physiol Biochem. 48:256-64
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: 1. Taught half of a graduate level course in Plant Stress Physiology, which was centered on molecular and genetic adaptation responses of higher plants to abiotic stresses, including drought. 2. Analyzed gene expression and lipidomic data from a drought stress experiment in Arabidopsis carried out by my former graduate student. 3. Worked with another former student to prepare a manuscript for submission for publication to Plant Physiology and Biochemistry. The paper was accepted, pending revisions, which she and I completed in October, 2009. 4. Worked with Dr. G. Welbaum in Horticulture to analyze data from a drought stress experiment carried out on transgenic Arabidopsis seed. PARTICIPANTS: Dr. G. Welbaum, HORT, VT Dr. V. Shuleav, VBI, VT Dr. Cecilia Vasquez-Robinet, Ludwig-Maximillians Universitat, Munich, Germany TARGET AUDIENCES: Target audience is scientists and students in the research area. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
I have worked throughout the reporting period with The iPlant Collaborative, an NSF-funded project, on devising computational and bioinformatic means for the intelligent compilation of abiotic stress data the currently populate online databases. The goal is to produce tools such that facilitate the generation of novel hypotheses for the interaction of crop plants with their changing environments. This has involved extensive collaboration with researchers from Cornell, UC Berkeley, Cold Spring Harbor, and the University of Texas.
Publications
- No publications reported this period
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