Source: TEXAS A&M UNIVERSITY submitted to
SYSTEMS BIOLOGY APPROACHES TO UNDERSTANDING AND IMPROVING PLANT STRESS RESPONSES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0233887
Grant No.
(N/A)
Project No.
TEX09558
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Apr 12, 2013
Project End Date
Apr 11, 2018
Grant Year
(N/A)
Project Director
Rushton, P. J.
Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Dallas-TAMU Agr Res Cntr
Non Technical Summary
Water stress causes major losses to crop plants in rural environments. In urban environments, considerable amounts of often scarce water is used to water turfgrass in lawns and on golf courses. Our research seeks to better understand plants responses to water stress and ultimately both reduce the amount of water used in urban environments and crop losses in rural ones. There is an obvious need for projects that seek to to accomplish both of these goals. We will initially use simple experimental systems where only the amount of water is varied. Later when important genes in water stress responses have been identified, these genes will be further studied in more field-like conditions. In addition to genes, metabolites will also be studied to build a systems-wide knowledge of plant responses to water stress. Outcomes will include the identifcation of key regulators of water stress and genes, metabolites and proteins that are good candidates for plant improvement. If any of our plant species can be improved to require less water, this will be a major benefit and have significant impact.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011820104020%
2012420104010%
2012499104020%
2031820104030%
2032420104020%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean; 2420 - Noncrop plant research; 2499 - Plant research, general;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
My research group will seek to provide both novel insights into the ways that plants respond to water stress and novel strategies to produce plants with improved water usage. Our specific objectives are: SYSTEMS BIOLOGY APPROACHES TO PRODUCING IMPROVED TURFGRASS FOR URBAN ENVIRONMENTS We will start by choosing a turfgrass species that is grown in the Dallas area for our plant system. The possible species include zoysiagrass and bentgrass. As well as growing in the Dallas Metropolitan area, the other criteria for this choice will be 1) The potential for transformation of the plant species and ecotype 2) The ploidy of the species, and 3) The size of the genome. Once chosen, our objectives for the species will be: 1) To sequence and assemble the genome sequence 2) To annotate the genome and produce a good genome-wide set of gene models 3) To use RNA-Seq to determine genes that are up- or down-regulated during water stress 4) To use metabolomics to determine metabolites that are up- or down-regulated during water stress 5) To manipulate the expression of candidate genes to attempt to improve water stress response. WRKY TRANSCRIPTION FACTORS AS TOOLS TO IMPROVE DROUGHT RESPONSES Soybean and tobacco have been our crop species and model species for research for the last five years and we will continue this successful project. Turfgrass is the major new area that we have recently added to our work. Our continuing goal is to identify WRKY genes that regulate drought responses in turfgrass, soybean and tobacco and to develop a tool to identify similar genes in other crop plants that may be less amenable to study. This tool is based on comparative phylogenies, conserved protein domains, expression profiles and promoter analysis. This project has four specific objectives. 1) Which WRKY genes regulate responses to water stress 2) Do related WRKY genes regulate water stress responses 3) Do drought responsive WRKY genes share drought responsive promoter elements 4) How do WRKYs regulate drought responses and can we use them to increase drought tolerance and/or drought resistance PLANT PROMOTERS FOR USE IN BASIC RESEARCH AND PLANT BIOTECHNOLOGY The objectives of this project are to provide well-characterized promoters for use in soybean and turfgrass. These will include 1) pathogen inducible, 2) drought inducible, 3) wound inducible, 4) cold inducible and 5) salt inducible in addition to 6) tissue specific promoters. The final objective is to have over 100 useful promoters with defined expression characteristics that are then available for use.
Project Methods
SYSTEMS BIOLOGY APPROACHES TO PRODUCING IMPROVED TURF GRASS FOR URBAN ENVIRONMENTS We will choose a turfgrass species that grows in Dallas and will sequence the genome using next generation sequencing facilities at College Station. The shotgun sequences will be assembled and a genome browser will be employed to manipulate and annotate the sequences. Gene prediction programs such as FGENESH will be used to produce an initial set of gene models. Turfgrass plants will be subjected to moderate and severe water stress over a time course in a hydroponics system. This will ensure that no other abiotic stresses other than water stress will be encountered by the plants. We will employ RNA-Seq to determine induced or repressed genes and produce a MapMan mapping to visualize these data in a genome-wide manner. Promising gene targets will be manipulated to determine whether they can be used to improve water stress responses. Similar experiments will be performed at the metabolite level. WRKY TRANSCRIPTION FACTORS AS TOOLS TO IMPROVE DROUGHT RESPONSES We have four aims. 1) Which WRKY genes regulate responses to water stress We will use a turf grass species to add to our data sets from soybean and tobacco. 2) Do phylogenetically similar WRKY genes regulate water stress responses We will construct a combined phylogenetic tree of WRKY genes from soybean, rice, tobacco and a turfgrass species.. 3) Do drought responsive WRKY genes share similar drought responsive promoter elements We will test this hypothesis by functionally analyzing drought responsive WRKY promoters for conserved motifs. 4) How do WRKY transcription factors regulate drought responses We will validate candidate genes as drought response regulators using multiple approaches, including knockdown/overexpression coupled with expression profiling, ChIP-seq, biotin streptavidin pulldown assays, yeast two-hybrid assays and mutant analysis. This will reveal target genes of the WRKY transcription factors and provide additional gene targets for improving water stress responses. PLANT PROMOTERS FOR USE IN BASIC RESEARCH AND PLANT BIOTECHNOLOGY Genes with promising expression characteristics have already been chosen from soybean and tobacco and similar genes will be determined in the turf grass species. About a dozen promising promoters have been tested as promoter:reporter constructs in systems of reduced complexity (soybean hairy root and tobacco BY-2 cells) and also transgenic plants. This will expand to include dissection of promoters to discover promoter elements that are both necessary and sufficient for driving the expression characteristics. In addition, a small number of synthetic promoters will be constructed consisting of defined elements. The aim of these synthetic promoters is to produce improved promoters that are better than native promoters at driving transgene expression. This combined in silico and transgenic approach should yield a new toolbox of useful promoters for biotechnological purposes.

Progress 04/12/13 to 04/11/18

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Project Director is no longer employed with Texas A&M University - nothing to report

Publications


    Progress 10/01/13 to 09/30/14

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Two Postdoctoral researchers (Roel Rabara and Mani Choudhary), one technician (Malini Rao), a visiting Masters student from The University of Gothenberg, Sweden (Naveen Kumar) and an undergraduate from The University of Texas (Marissa Miller) have been trained during this project. Both Postdoctoral researchers have been trained in new techniques and have written manuscripts. The visiting Masters student has performed the research part of his Master’s degree in the laboratory and has been trained in new techniques to accomplish this. The undergraduate student has learned to master several new techniques and has stayed in the laboratory after graduation. All laboratory members are included as authors on at least one manuscript currently in review. Former group member Dr. Prateek Tripathi was awarded a Young Plant Scientist Award by The Society for Applied Biotechnology, Association for the Advancement of Biodiversity Science and Eurasian Academy of Environmental Sciences for the year 2014 as a result of his work on this project. How have the results been disseminated to communities of interest? The results have been disseminated through publications, conference presentations, and databases as follows. Publications: 1) Transcription Factors that regulate defense responses and their uses in increasing disease resistance. Prateek Tripathi, Aravind Galla, Roel C Rabara, Paul J Rushton Biotechnology for Plant Defense Control, Edited by David B Collinge, 07/2014; John Wiley & Sons. Inc. 2) Potential of transcription factor-based genetic engineering in improving crop tolerance to drought Roel C Rabara, Prateek Tripathi, Paul J Rushton Omics A Journal of Integrative Biology 01/2014; Conference Presentations: 1) 2014 World Forum on Biology: What Have We Learned About Synthetic Promoter Construction? Paul J Rushton In Vitro Cellular & Developmental Biology - Animal 05/2014; 50 (Suppl 1:P40). 2) Systems Biology of Water Stress Responses in Soybean (Glycine max.) Reveals Novel Candidates Prateek Tripathi, Roel C Rabara, and Paul J Rushton. Asian Plant Science Conference/International Scientific Conference on Environmental Research: Issues, Challenges and Strategies for Sustainable Development and Livelihood Security, Bhairahawa, Lumbini, Nepal Databases: 1) WRKY.org A WRKY Wide Web (http://www.wrky.org/) Websites: 1) The Lab of Dr. Paul Rushton at Texas A&M AgriLife Research & Extension Center Dallas http://therushtonlab.wordpress.com/ 2) https://www.researchgate.net/profile/Paul_Rushton/?ev=hdr_xprf What do you plan to do during the next reporting period to accomplish the goals? Nuclear- and phosphonuclear-proteomics, shotgun metabolomics, synthetic and native promoter work, additional RNA-seq and yeast 2-hybrid work, identification of key regulatory proteins for further detailed analysis.

    Impacts
    What was accomplished under these goals? SYSTEMS BIOLOGY APPROACHES TO PRODUCING IMPROVED TURF GRASS FOR URBAN ENVIRONMENTS The first RNA-seq data from hydroponically grown and water stressed St Augustine grass have been obtained. The data have been analyzed partly in collaboration with Dr. Rajeev Azad (University of North Texas). The data has been analyzed 1) by de novo assembly and b) by mapping to the sorghum genome. We have identified up-regulation of genes involved in many different processes including targeted protein breakdown, calcium signaling, ethylene biosynthesis, amino acid biosynthesis, heat tolerance, water channel formation, ABA signaling, phenylpropanoid metabolism, compatible solute synthesis, ABA catabolism and ABA level. We have also produced an atlas of water stress induced and repressed transcription factor genes that include genes from the ERF/AP2, homeodomain, WRKY, NAC, MYB, bZIP, PLATZ, EIL, GATA, Zn finger, and bHLH gene families. These represent promising targets for the improvement of turf grass water stress responses. We have also developed methods for the isolation of enriched nuclear fractions and these proteins are being used for both nuclear proteomics and phosphonuclear proteomics. WRKY TRANSCRIPTION FACTORS AS TOOLS TO IMPROVE DROUGHT RESPONSES In addition to water stress induced or repressed WRKY genes from water stressed turf grass, soybean and tobacco, we have also performed yeast two hybrid screens using soybean GmWRKY53 as a bait. GmWRKY53 is induced by water stress at the mRNA level and we have determined that at the protein level, it interacts with three water stress inducible ERF/AP2 transcription factors. One of these ERF transcription factors is the third most strongly early water stress up regulated gene in soybean in root tissue and the second most strongly up-regulated transcription factor. It also interacts with fourteen different VQ proteins that may form components of various signaling networks. Additionally, a strong interaction was found with a soybean ortholog of LHY which encodes a myb-related transcription factor involved in circadian rhythms. These interacting proteins open up new avenues of research aimed at improving drought responses in soybean and other plants. PLANT PROMOTERS FOR USE IN BASIC RESEARCH AND PLANT BIOTECHNOLOGY Dr. Paul Rushton was an invited speaker at the 2014 World Forum on Biology, where he spoke on the construction of synthetic promoters for biotechnological purposes. As a result of this, several new biotechnologically oriented projects have been instigated and external funding has been obtained. THE EVOLUTION OF WRKY TRANSCRIPTION FACTORS AND THEIR FUNCTIONS IN THE PLANT KINGDOM I WRKY transcription factors regulate many important plant processes. The availability of increasing numbers of sequenced genomes made us re-evaluation the evolution of the WRKY transcription factor family that presents a different picture from that previously suggested. We performed searches for WRKY genes, especially divergent forms, in genomes throughout the plant kingdom and in non-plant species. The many diverse species of modern day plants all descended from a single charophyte green alga that colonized the land between 430 and 470 million years ago. The first charophyte genome sequence from Klebsormidium flaccidum therefore filled a gap in the available genome sequences in the plant kingdom between unicellular green algae such as Chlamydomonas reinhardtii that typically have 1-3 WRKY genes and mosses such as Physcomitrella patens that contain 30-40 genes. Based on our phylogenetic analyses and genomic searches we propose that the evolution of WRKY transcription factors includes early lateral gene transfers to some non-plant organisms together with algal WRKY genes that have no counterparts in flowering plants. There are four major WRKY transcription factor lineages in flowering plants, Groups I + IIc, Groups IIa + IIb, Groups IId + IIe, and Group III. We proposed two alternative hypotheses of WRKY gene evolution: The “Group I Hypothesis” sees all WRKY genes evolving from Group I C-terminal WRKY domains. The alternative “IIa+b Separate Hypothesis” sees Groups IIa and IIb with their hallmark VQR intron evolving directly from a single domain ancestral algal WRKY gene separate from the other Group I-derived lineage. Further genome sequences may help determine which of these two alternatives best reflects the evolution of WRKY transcription factors. Both a book chapter and a manuscript on this topic are currently in review.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2014 Citation: Roel C. Rabara, Prateek Tripathi and Paul J. Rushton. The potential of transcription factor-based genetic engineering in improving crop tolerance to drought. OMICS: A Journal of Integrative Biology 2014 doi:10.1089/omi.2013.0177.
    • Type: Book Chapters Status: Accepted Year Published: 2015 Citation: Prateek Tripathi, Aravind Galla,Roel C. Rabara, and Paul J. Rushton.Transcription factors that regulate defence responses and their use in increasing disease resistance. In Biotechnology for Plant Disease Control. Edited by David B. Collinge
    • Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: What Have We Learned About Synthetic Promoter Construction? Paul J Rushton In Vitro Cellular & Developmental Biology - Animal 05/2014; 50(Suppl 1):P40. DOI: 10.1007/s11626-014-9772-z


    Progress 04/12/13 to 09/30/13

    Outputs
    Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One Postdoctoral researcher (Roel Rabara) and one technician (Malini Rao) have been employed on the project. An additional Postdoc (Mani Choudhary) and a visiting Masters student (Naveen Kumar) will join the project before the end of 2013. Dr. Prateek Tripathi was also involved with the project before moving to USC and continues to be a collaborator on the project. Prateek Tripathi was an invited speaker at the 2013 ASPB Conference. He was also an invited speaker at the International Conference on Bio-resource and Stress Management (ICBSM-2013)-2013, February 6-9,2013, Kolkata, India. One publications has so far been published in the short period that the grant has been running. How have the results been disseminated to communities of interest? The results have been disseminated through publications, conference presentations, and databases as follows. Publications: 1) Tripathi, P., Rabara, R. C., and Rushton, P.J. (2013) A systems biology perspective on the role of WRKY transcription factors in drought responses in crops. Planta. Oct 22. [Epub ahead of print] PMID: 24146023. Conference Presentations: Mini symposia 1) Tripathi, P., Rabara, R. C., Reese, R. N., Shen, Q. J. and Rushton, P.J. (2013) Comprehensive Systems based analyses of water stress responses in soybean (Glycine max.). Electronic conference proceedings (abstract), Plant Biology 2013, Providence Rhode Island, July 2013. 2) Tripathi, P., Rabara, R. C., Reese, R.N., Shen, Q.J., and Rushton, P.J. (2013) Systems Biology of Drought Tolerance in Soybean (Glycine Max). Electronic conference proceedings (abstract), International Conference on Bio-resource and Stress Management (ICBSM-2013) Kolkata, India. Databases: 1) WRKY.org A WRKY Wide Web (http://www.wrky.org/) Websites: 1) The Lab of Dr. Paul Rushton at Texas A&M AgriLife Research & Extension Center Dallas http://therushtonlab.wordpress.com/ What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

    Impacts
    What was accomplished under these goals? SYSTEMS BIOLOGY APPROACHES TO PRODUCING IMPROVED TURF GRASS FOR URBAN ENVIRONMENTS We have chosen two turf grass species to sequence their genomes. The first species was chosen because of the diploid nature of the grass and its economic importance in Texas. The second species was chosen because of extensive breeding populations and expertise. BAC library construction and construction of maps have been started. We have also established a hydroponics system for gene, protein, and metabolite discovery. The first samples for RNAseq have been prepared. WRKY TRANSCRIPTION FACTORS AS TOOLS TO IMPROVE DROUGHT RESPONSES This is a continuing focus of the laboratory and we continuously produce new data. 1) Which WRKY genes regulate responses to water stress? In soybean, we recently established that there are 178 WRKY genes and 37 potential pseudogenes. Of these, 48 genes show inducibilities by water stress of at least 7-fold in either root or leaf tissue. Notably, two WRKY genes (Glyma06g13090 and Glyma04g41700) were among the top 30 most highly water stress induced genes in leaf tissue (out of over 50,000 expressed genes). 2) Do related WRKY genes regulate water stress responses? This was determined using a combined phylogenetic analysis of the WRKY families from soybean, Arabidopsis, and tobacco upon which was layered expression data. The results show there are some commonalities, but there are also family-specific responses. This includes notable hotspots of co-expressed genes in the group IIc and group IIa subfamilies. There are also striking differences between the plant species. For example, only in tobacco are members of the IId subfamily induced by water stress. 3) Do drought responsive WRKY genes share drought responsive promoter elements? Our transcriptome analysis identified about 3,000 genes that are strongly up regulated by water stress. We determined that there was a striking occurrence of the G box-related ABRE sequence motif CACGT/CG in up-regulated genes. These ABRE-like sequences were not only over-represented in the promoters, but they were also clustered in the 200 bp upstream of the start of transcription. 4) How do WRKYs regulate drought responses and can we use them to increase drought tolerance? Our discovery phase has identified over 75 WRKY genes that respond to water stress and that therefore may be regulating drought responses. The most promising are GmWRKY137, GmWRKY161, GmWRKY19, GmWRKY25, and GmWRKY53 that show over 20-fold increases in mRNA level and GmWRKY155 that shows a 14-fold reduction in mRNA level. These genes are currently being used to construct overexpression and knockdown lines and these lines are being assessed for drought tolerance. PLANT PROMOTERS FOR USE IN BASIC RESEARCH AND PLANT BIOTECHNOLOGY We have studied two water stress-inducible WRKY genes, GmWRKY17 (GLYMA06g06530) and GmWRKY67 (GLYMA13g44730) in detail. Promoter:GFP constructs were transformed into soybean roots via hairy-root transformation and transgenic roots were subjected to dehydration stress. GFP quantification confirmed water stress inducibility and revealed that the promoter of GmWRKY17 directed 11.1-fold and 12.7-fold inducible expression after three hours and five hours respectively. The GmWRKY67 promoter directed 4.8-fold and 4.6-fold inducible expression. Additionally, both promoters directed ABA-inducible expression, demonstrating that they are part of the ABA-inducible water stress signaling network. In tobacco, NtWRKY69 promoter:GFP constructs directed water stress and ABA independent gene expression in leaves. Expression followed the vascular tissue through the stem, into the leaf and finally into all leaf tissue. This is potentially a very useful promoter for plant biotechnology. THE EVOLUTION OF WRKY TRANSCRIPTION FACTORS AND THEIR FUNCTIONS IN THE PLANT KINGDOM In addition to phylogenetic analyses, expression studies, and the analysis of conserved protein domains, functional studies are also being employed. WRKY genes from several lower plants including Physcomitrella patens and Selaginella moellendorfii will be used for expression analysis, targeted gene knockdown, overexpression, TAP tagging and ChIP-SEQ.

    Publications

    • Type: Journal Articles Status: Published Year Published: 2013 Citation: Tripathi, P., Rabara, R. C., and Rushton, P.J. (2013) A systems biology perspective on the role of WRKY transcription factors in drought responses in crops. Planta. Oct 22. [Epub ahead of print] PMID: 24146023.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Tripathi, P., Rabara, R. C., Reese, R. N., Shen, Q. J. and Rushton, P.J. (2013) Comprehensive Systems based analyses of water stress responses in soybean (Glycine max.). Electronic conference proceedings (abstract), Plant Biology 2013, Providence Rhode Island, July 2013.
    • Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Tripathi, P., Rabara, R. C., Reese, R.N., Shen, Q.J., and Rushton, P.J. (2013) Systems Biology of Drought Tolerance in Soybean (Glycine Max). Electronic conference proceedings (abstract), International Conference on Bio-resource and Stress Management (ICBSM-2013) Kolkata, India.