Source: SOUTH DAKOTA STATE UNIVERSITY submitted to
WRKY TRANSCRIPTION FACTORS AS TOOLS TO IMPROVE DROUGHT RESPONSES.
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0219681
Grant No.
2008-35100-05969
Project No.
SD00G338-09
Proposal No.
2009-05381
Multistate No.
(N/A)
Program Code
56.0B
Project Start Date
Jun 1, 2009
Project End Date
Aug 14, 2013
Grant Year
2009
Project Director
Cheesbrough, T. M.
Recipient Organization
SOUTH DAKOTA STATE UNIVERSITY
PO BOX 2275A
BROOKINGS,SD 57007
Performing Department
Biology & Microbiology
Non Technical Summary
Drought is the major culprit behind crop losses worldwide, with billion dollar losses in the USA. WRKY transcription factors are key regulators of many plant stress responses but until recently, there were only tantalizing pieces of evidence linking WRKYs with drought responses. We now know that over 15% of the rice WRKY gene family (seventeen genes) are highly induced by drought in leaves and/or roots. Similar but less complete evidence comes from other plants. We have identified this as a major gap in our knowledge of drought responses and an opportunity to increase drought tolerance. Our hypothesis is that WRKYs are regulators of water stress responses and that similar genes regulate these responses in different plant species. Our goal is to identify WRKY genes that regulate drought responses in rice and soybean and develop a tool to identify similar genes in other crop plants. The project will not only increase our knowledge of drought responses in crop plants but also lead to new approaches aimed at reducing crop losses in the USA.
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
2031530104030%
2031820104010%
2031999104030%
2032499208030%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1999 - Tobacco, general/other; 2499 - Plant research, general; 1820 - Soybean; 1530 - Rice;

Field Of Science
2080 - Mathematics and computer sciences; 1040 - Molecular biology;
Goals / Objectives
Our goal is to identify WRKY genes that regulate drought responses in rice and soybean and to develop a tool to identify similar genes in other crop plants that may be less amenable to study. This tool will be based on comparative phylogenies, conserved protein domains, expression profiles and promoter analysis. The project has four specific aims. 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
Project Methods
Our hypothesis is that WRKYs are regulators of water stress responses and that similar genes regulate these responses in different plant species. These similarities can be in the primary amino acid sequence of the genes and be clear from phylogenies and/or be similarities in promoter structure and be apparent from expression data and comparative promoter analysis. We aim to investigate both possibilities in this project within our four specific aims. 1) Which WRKY genes regulate responses to water stress We plan to use several different crop plants, both monocot and dicot, to identify drought responsive WRKY genes that are potential regulators of drought responses. There is already Massively Parallel Signature Sequencing (MPSS) data from rice and some data from other plants. We will add to this in a systematic manner using expression profiling with a multiplex oligo array that contains over 1,000 WRKY genes from over 30 plant species. 2) Do phylogenetically similar WRKY genes regulate water stress responses Our initial hypothesis is that related genes will have similar functions in drought responses in different plant species. We will investigate this with a combined phylogenetic tree of WRKY genes. For clades that contain candidate drought response regulators this will be extended to analysis of conserved protein domains outside the WRKY domain using MEME. This translational biology approach will be particularly valuable for crop plants where transformation is difficult and where large mounts of genome sequence and/or EST data is lacking. 3) Do drought responsive WRKY genes share similar drought responsive promoter elements It is also possible, that similarity between drought response regulators lies in the promoters of the genes. We will test this second hypothesis by comparing drought responsive WRKY promoters for conserved motifs. We will perform promoter comparisons (multiple sequence alignments, MotifSampler etc) and some promoter analysis to find conserved cis-acting elements. 4) How do WRKYs regulate drought responses and can we use them to increase drought tolerance We already have candidate WRKY genes in rice and soybean and that number will grow rapidly at the start of the project. The next step is the validation of these genes as drought response regulators. To do this, we have multiple approaches, including knockdown/overexpression coupled with expression profiling, ChIP, biotin streptavidin pulldown assays and mutant analysis. This will reveal target genes of the WRKY TFs.

Progress 06/01/09 to 08/14/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One Postdoctoral researcher (Jun Lin), two graduate students (Roel Rabara & Prateek Tripathi), & four undergraduate students (Tanner J. Langum, Ashley K. Boken, Deena L. Rushton, & Lucas Smidt) were employed at different times on the project in the Rushton laboratory & one graduate student (Angi Liyuan Zhang) in the Shen laboratory. Both Roel Rabara & Prateek Tripathi were graduate students on the project & both have graduated w/ a Ph.D & seven papers to their names (rising to at least ten). Dr. Tripathi now works as a Postdoc at USC & Dr. Rabara at Texas A&M. All four undergraduates appeared on at least one publication & they have obtained valuable wet & dry lab experience. Dr. Jun Lin appeared on three papers before moving to a position in industry. The total number of publications has so far been nine w/ another two in review & one in preparation. Both Prateek Tripathi & Roel Rabara went to several conferences, including two ASPB conferences where work from the project was presented. Prateek Tripathi was an invited speaker at the 2013 ASPB Conference. He was also an invited speaker at the International Conference on Bio-resource & Stress Management (ICBSM-2013)-2013, February 6-9,2013, Kolkata, India. The entire Rushton lab attended the 2011 ASPB Conference in Minneapolis. Both Prateek Tripathi & Roel Rabara attended courses at Cold Spring Harbor. Prateek Tripathi & Roel Rabara also wrote & were awarded a competitive grant “A Novel tobacco BY?2 system for the identification of drought responsive regulatory elements” from The Center of Excellence for Drought Tolerance Research, South Dakota State University. How have the results been disseminated to communities of interest? The results have been disseminated through publications, conference presentations, & databases. What do you plan to do during the next reporting period to accomplish the goals? This is the final report written by Dr. Paul Rushton, PI 2008-2013, who is currently at Texas A&M University. The grant did not move w/ him. This grant provided many new lines of research & many of the aspects of the original project & several of the new discoveries will be the continuing focus of his continuing research in Texas.

Impacts
What was accomplished under these goals? Our major hypothesis was that WRKY genes are regulators of water stress responses and we have been able to demonstrate that this hypothesis was indeed true. After the start of the project, several other groups world-wide have also been able to demonstrate this. The project was indeed timely. We have accomplished far more than suggested in the original proposal because we have used systems biology approaches. As a result, we have extensive genome-wide water stress response data at the transcriptome level in both soybean and tobacco. We also have proteomics data for soybean and extensive metabolomics data for both plant species. In addition, we also have some promoter analysis data. Our accomplishments under this project are therefore not limited to the initial four specific aims. Below is a summary of the accomplishments for the four specific aims and then other accomplishments. 1) Which WRKY genes regulate responses to water stress? We have looked at the complete WRKY gene family in soybean and the vast majority of the WRKY family in tobacco. In soybean, we 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). In tobacco, the complete WRKY gene family is not yet available, nevertheless at least 30 genes show inducibilities of at least 7-fold by water stress. These data identify water stress-inducible WRKY genes and validate our initial hypothesis. 2) Do related WRKY genes regulate water stress responses? The answer to this question was determined using a combined phylogenetic analysis of the WRKY families from soybean, Arabidopsis, and tobacco upon which was layered expression data. The results were interesting because although there are some commonalities, there are also family-specific responses. Similarities include the induction by water stress of genes from the I, IIa, IIc and III subfamilies. 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. Only in tobacco are members of the IId subfamily induced by water stress. Arabidopsis group IIb genes, unlike tobacco and soybean, do not appear to contain water stress-inducible members. These analyses suggest that although some WRKY genes form a core component of water stress deficit signaling, that others represent family- or even species-specific responses. 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. To establish whether this is a result of similarities at the promoter level, a data set was constructed containing 1,000 bp of promoter region from the fifty most strongly induced genes at an early time-point (1 h root and 2 h leaf) and a late time-point (5 h in both). These were analyzed by MEME for the presence of conserved sequence motifs. In all four data sets, there was a striking occurrence of the G box-related ABRE sequence motif CACGT/CG. 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. ABRE-like sequences contain potential binding sites for bZIP, NAC, and bHLH TFs and this, together with the transcriptome data suggest the involvement of these types of TFs in regulating water stress. We also studied two water stress-inducible WRKY genes, GmWRKY17 (GLYMA06g06530) and GmWRKY67 (GLYMA13g44730) in more 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. 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. Additional Findings: We have analyzed soybean plants at the system level during moderate and severe water stress. These analyses at the physiological, mRNA, protein, and metabolite levels reveal dynamic changes and identify genes, proteins and metabolites that are key components of water stress responses. MapMan analyses of genome-wide transcriptome data using all Glyma1.0v gene models reveal that changes in regulation play a prominent role in early response. This includes up-regulation of ERF, heat shock, MYB, NAC. bHLH, JAZ/TIFFY and WRKY transcription factors, receptor-like kinases, and calcium signaling components. Genes associated with the phytohormones ABA, JA and ethylene are up-regulated in parallel with increases in the levels of these hormones. Later, downstream target genes are activated including LEA genes, dehydrins, galactinol and raffinose synthases, and osmotins. Metabolite analyses showed that changes in the biochemical profile of root tissues in response to water stress were far more extensive than that observed in leaf tissues. Ammonia detoxification and compatible solute accumulation were major metabolic responses. This included a flow of nitrogen into amino acid metabolism and proteomics studies performed using Shotgun Mass-spectrometry (MuDPIT) revealed this is due to de novo biosynthesis. One major metabolic change was the accumulation of isoflavonoids in root tissue. One of these showed the greatest fold increase (over 100-fold) of any biochemical and may promote reactive oxygen species scavenging. This isoflavanoid is therefore a potential biomarker for water stress in soybean and a promising target for soybean improvement. Similar experiments were performed in tobacco. The earliest responses are the closing of stomata, wilting, changes in gene expression, and an increase in jasmonate-isoleucine in the roots. Gene expression was monitored using a custom oligo array containing 40,000 individual gene space reads with the highest E-value hits to proteins in the database, all TOBFAC transcription factors, and the Version 4.0 DFCI Tobacco Gene Index EST sequences. A MapMan mapping was generated for the oligo array sequences and this showed that several thousand genes are differentially regulated. Metabolomics analyses identified a total of 116 named biochemicals in leaves and 140 in root. The water stress caused considerable alterations in metabolomic profiles in both leaf and root, even at the first time point (20 min). Interestingly, the two tissues seemed to follow different time profiles. Leaf tissue tended to exhibit a biphasic pattern for many compounds, while root tissue had a slow progression of changes during the first hour, followed by much more dramatic differences in the last two time points. There was evidence of drought and oxidative stress responses, as indicated by increased levels of compatible solutes such as sugar alcohols, amino acids, and oligosaccharides, as well as oxidative products of ROS remediation.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Tripathi, P., Rabara, R. C., Reese, R.N., Shen, Q.J., and Rushton, P.J. (2012) Systems wide identification of WRKY transcription factors regulating water stress responses in Soybean (Glycine Max.) 14th Annual Fall Symposia Exploration in Synthetic System Biology, Danforth Plant Science Center, St. Louis, Missouri, USA (PX08).
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Rabara, R. C., Tripathi, P., Reese, R.N., Shen, Q.J., and Rushton, P.J. (2012) Systems wide identification of WRKY transcription factors regulating water stress responses in Soybean (Glycine Max.) 14th Annual Fall Symposia Exploration in Synthetic System Biology, Danforth Plant Science Center, St. Louis, Missouri, USA (PX09)
  • 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). International Conference on Bio-resource and Stress Management (ICBSM-2013) Kolkata, India.
  • Type: Websites Status: Published Year Published: 2012 Citation: http://therushtonlab.wordpress.com/
  • 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. 2013 Oct 22. [Epub ahead of print] PMID: 24146023.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Tripathi, P., Rabara, R. C., Lin, J., and Rushton, P.J. (2013). GmWRKY53, a water- and salt-inducible soybean gene for rapid dissection of regulatory elements in BY-2 cell culture. Plant Signaling Behavior 8 (5) eLocation ID: e24097.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Tripathi, P., Rabara, R. C., and Rushton, P.J. (2012). Emergence of omics-approaches for crop improvement during abiotic stress. International Journal of Environmental Science and Technology. 1(2):125-128.
  • Type: Journal Articles Status: Published Year Published: 2013 Citation: Rabara, R. C., Tripathi, P., Lin, J., and Rushton, P.J. (2013). Dehydration-Induced WRKY Genes from Tobacco and Soybean Respond to Jasmonic Acid Treatments in By-2 Cell Culture. Biochemical and Biophysical Research Communications. 431(3): 409-14.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Tripathi, P., Rabara, R. C., Langum, T.J., Boken, A.K., Rushton, D.L., Boomsma, D.D., Rinerson, C.I., Rabara, J., Reese, R.N., Chen, X., Rohila, J.S., and Rushton, P.J. (2012). The WRKY transcription factor family from Brachypodium distachyon. BMC Genomics 13: 270.
  • Type: Journal Articles Status: Published Year Published: 2012 Citation: Ling, M. H. T., Rabara, R. C., Tripathi, P., Rushton, P.J. and Ge, X. Extending MapMan Ontology to Tobacco for Visualization of Gene Expression. (2012) Dataset Papers in Biology. 706465. doi: 10.7167/2013/706465.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Rushton, D.L., Tripathi, P., Rabara, R.C., Lin, J., Ringler, P., Boken, A.K., Langum, T.J., Smidt, L., Boomsma, D.D., Emme, N.J., Chen, X., Finer, J.J., Shen, Q.J., and Rushton P.J. (2011) WRKY transcription factors: key components in abscisic acid signalling. The Plant Biotechnology Journal. 10(1): 2-11.
  • Type: Journal Articles Status: Published Year Published: 2011 Citation: Roy, A., Rushton, P.J., and Rohila, J.S. (2011) The Potential of Proteomic Technologies for Crop Improvement under Drought Environments. Critical Reviews in Plant Science. 30:5: 471-490.
  • Type: Journal Articles Status: Published Year Published: 2010 Citation: Rushton, P.J., Somssich, I.E., Ringler, P., and Shen, J. (2010) WRKY transcription factors. Trends in Plant Science 15: 247-58.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Rushton, P.J., Tripathi,P., Boken, A.K., Langum, T.J., Bokoweic, M.T., Sutton, F., Meyerink, B., Geraets, R., Lang, D., Chen, X., Shen, Q.J., Timko, M.P., and Rensing, S.A., The Evolution of WRKY Transcription Factors In the Green Lineage. (W398) Plants and Animal Genome conference JXVIII, San Diego, CA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Tripathi, P., Rabara, R. C., Langum, T.J., Boken, A.K., Rushton, D.L., Smidt, L., and Rushton, P.J. (2011) The molecular and physiological responses of soybean to water stress. Proceedings of the South Dakota Academy of Science, Vol. 90 173.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Rabara, R. C., Lin, J., Tripathi, P., Langum, T.J., Boken, A.K., Rushton, D.L., Smidt, L., and Rushton, P.J. (2011) Drought inducible WRKY genes in tobacco (Nicotiana tabacum L.) Proceedings of the South Dakota Academy of Science, Vol. 90
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Tripathi, P., Rabara, R. C., Boken, A.K., Langum, T.J., Smidt, L., Rushton, D.L., and Rushton, P.J. (2011) The Molecular Responses of Soybean in Water Deificit. Plant Biology 2011, Annual Meeting of The American Society of Plant Biology, Minneapolis, MN, USA (Abstract # P13020): pL68.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Rabara, R. C., Lin, J., Tripathi, P., Boken, A.K., Langum, T.J., Smidt, L., Rushton, D.L., and Rushton, P.J. (2011) Promoter Analysis of drought induced WRKY genes in tobacco (Nicotiana tabacum L.) Plant Biology 2011, Annual Meeting of The American Society of Plant Biology, Minneapolis, MN, USA (Abstract # P13006): pL68.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Rabara, R. C., Tripathi, P., Lin, J., Parett, J., and Rushton, P.J. (2012). A tobacco BY-2 cell system for the identification of drought responsive regulatory elements in plants. American Society of Plant Biology Mid-Westerm Section Meeting, Lincoln, Nebraska, USA. P-39.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Tripathi, P., Rabara, R. C., Reese, R.N., Shen, Q.J., and Rushton, P.J. (2012) Global gene expression in Tobacco in water deficit condition. Plant Biology 2012, Annual meeting of American Society of Plant Biology, Austin, Texas, USA. (Abstract # 14018): pR118.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Rabara, R. C., Tripathi, P., Lin, J., Reese, R.N., Shen, Q.J., and Rushton, P.J. (2012). Global gene expression in Tobacco in water deficit condition. Plant Biology 2012, Annual meeting of American Society of Plant Biology, Austin, Texas, USA. (Abstract # 14020): pR118
  • Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Tripathi, P., Rabara, R. C., Reese, R.N., Shen, Q.J., and Rushton, P.J. (2012) Understanding water stress responses at systems level in Soybean (Glycine Max.) International Conference of Systems Biology 2012, Toronto, ON, Canada (Abstract # 469D): pQ196.


Progress 06/01/11 to 05/31/12

Outputs
OUTPUTS: The most significant outputs of this project during the reporting period include the following, 1) The Database of Brachypodium distachyon WRKY Transcription factors (http://www.igece.org/WRKY/BrachyWRKY/BrachyWRKYIndex.html), this is a public resource for the plant research community. It was accompanied by a detailed analysis of the WRKY gene family in this model species that was published in BMC Genomics. 2) Our database of WRKY transcription factors that contains the complete WRKY transcription factor family from over 20 plant and algae species has been improved and refined. It is available at http://systemsbiology.usm.edu/BrachyWRKY/WRKY/Species.html and will be updated and improved. 3) Our large systems biology data sets from both soybean and tobacco have revealed many aspects of the responses to water stress and these data have been reported at meetings and in a growing number of publications. We have over 800 potential gene targets for plant improvement in both soybean and tobacco. 4) We have isolated six water stress-responsive promoters from WRKY transcription factor genes (three from tobacco and three from soybean). These promoters direct water stress-inducible expression and are new tools for the analysis and manipulation of water stress responses. 5) At the metabolite level, in both tobacco and soybean, we have identified over 200 metabolites that are changed in level during water stress. In soybean, one phytoalexin increases in level over 200-fold. This compound appears to be a biomarker for water stress and also a potential target to improve water stress responses. 6) We have produced a MapMan mapping to facilitate the use of MapMan software with our tobacco oligo array. This is published and available for the plant community. 7) We have identified the soybean promoter from GmWRKY53, a water- and salt-inducible soybean gene, as an excellent tool for rapid dissection of regulatory elements in BY-2 cell culture. This is also published and freely available. PARTICIPANTS: P. Rushton, PI; Rahuna, Tripathi, Lin - grad students; Reese, Rohila - Collaborators TARGET AUDIENCES: Other plant scientists studying signaling stress responses and metabolomics. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our genome-wide analysis of the WRKY gene family in Brachypodium distachyon has yielded novel insights into tandem gene duplications and the amplification of this gene family in flowering plants. This was published in BMC Genomics. We now have six characterized water stress-inducible WRKY gene promoters and two non WRKY promoters from tobacco. We have successfully developed a soybean hairy root system to characterize these promoters. We have also demonstrated that many of these promoters also direct inducible expression by cold and/or wounding and that several abiotic stress signaling pathways converge at these transcriptional regulators. These promoters promise to have major impacts not only for basic research but also for applied biotechnology products to improve crop plants. Promoters of the most strongly up-regulated genes in our transcrptome analyses were found to be enriched with ABRE-like motifs and two novel motifs. We have produced extensive transcriptome, proteome and metabolome analyses and have identified hundreds of up- or down-regulated genes and metabolites in both soybean and tobacco. Metabolite profiles showed accumulation of proteinogenic amino acids due to de novo biosnthesis as well as compatible solutes such as proline, trehalose and raffinose. Metabolite analyses also revealed a flow of nitrogen into amino acid metabolism. In many cases, expression profiles of our target genes concurred with our metabolome profile. Strikingly, one iso-flavonoid, was found to accumulate over 100-fold in soybean roots during dehydration. This led to a new hypothesis that it might promote dehydration-induced reactive oxygen species scavenging. As a result of these large systems biology data sets, we can say that our hypothesis that WRKY transcription factors are among the major regulatory genes in water stress responses has been validated.

Publications

  • Prateek Tripathi, Roel C. Rabara, Jun Lin and Paul J. Rushton (2013) GmWRKY53, a water- and salt-inducible soybean gene for rapid dissection of regulatory elements in BY-2 cell culture. Plant Signaling & Behavior 8 (5) eLocation ID: e24097
  • Roel C Rabara, Prateek Tripathi, and Paul J Rushton (2012). Emergence of omics-approaches for crop improvement during abiotic stress. International Journal of Environmental Science and Technology. 1(2):125-128.
  • Roel C Rabara, Prateek Tripathi, Jun Lin, and Paul J Rushton (2013. Dehydration-Induced WRKY Genes from Tobacco and Soybean Respond to Jasmonic Acid Treatments in By-2 Cell Culture. Biochemical and Biophysical Research Communications. 01/2013; DOI:doi: 10.1016/j.bbrc.2012.12.156.
  • Prateek Tripathi, Roel C. Rabara, Tanner J. Langum, Ashley K. Boken, Deena L. Rushton, Darius D. Boomsma, Charles I. Rinerson, Jennifer Rabara, R. Neil Reese, Xianfeng Chen, Jai S. Rohila, and Paul J. Rushton (2012). The WRKY transcription factor family from Brachypodium distachyon. BMC Genomics 13:270.
  • Maurice HT Ling, Roel C Rabara, Prateek Tripathi, Paul J Rushton, and Xijin Ge. Extending MapMan Ontology to Tobacco for Visualization of Gene Expression. Dataset Papers in Bioinformatics (Published 30th July 2012. E-pub ahead of print http://www.datasets.com/journals/biology/aip/706465/).


Progress 06/01/10 to 05/31/11

Outputs
OUTPUTS: Over the period from 06/01/2010 to 05/31/2011, we have performed extensive systems biology analyses of water stress responses in both tobacco and soybean. Key outputs were the following: 1) A list of several hundred genes from soybean leaves and roots that are strongly up-regulated by water stress at the mRNA level. 2) A list of several hundred genes from tobacco leaves and roots that are strongly up-regulated by water stress at the mRNA level. 3) A collection of transcription factors that appear to regulate water stress responses in both soybean, tobacco, and rice. 4) A detailed analysis of the WRKY genes that regulate water stress responses and a phylogenetic comparison of similarities in these WRKY genes between soybean and tobacco. 5) Proteomic analyses of the changes in abundant proteins in soybean in the same samples as used for the transcriptome analysis. 6) Promoter:GUS and GFP based validation in transgenic plants of the water stress inducible expression of selected WRKY genes. 7) We have manually curated the complete WRKY gene family from eighteen plant species and constructed two databases: The Database of Brachypodium WRKY transcription factors and The WRKY Wide Web. Development instances are available and production instances will become available in 2012. 8) We have developed a tobacco BY-2 cell system as a system of reduced complexity for the analysis of water stress inducible promoters. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: This project has entered a no cost extension period due to the PI moving from the University of Virginia to South Dakota State University. It is estimated that no more than two years lab work was possible in the first three years of the project due to moving and laboratory start up including the hiring of staff. Nevertheless, the project has been highly successful and the initial hypothesis has been validated.

Impacts
1) We have started detailed anaylses of the promoters of several WRKY genes from both soybean and tobacco. One of these genes from tobacco shows ABA-independent water stress inducible expression in leaves and the expression spreads upwards from the roots initially through the vascular tissue. We believe that this WRKY gene is an important regulator of water stress responses and that the promoter will be a very useful biotechnological tool for expressing genes under water stress in leaves. This is excellent validation of our initial hypothesis that WRKY genes regulate drought responses. 2) We currently are investigating at least a dozen other WRKY genes that are strongly inducible by water stress. These genes are expressed in different tissues and with different kinetics. They appear to occupy different positions in signalling webs (both early and late) and the promoters are currently being tested for possible use in biotechnological projects. 3) Using MapMan to visualize our extensive systems biology results we have gained valuable new insights into the processes in soybean that occur when the plant is subjected to water stress. These results will lead to new projects and continuation of the current project. 4) Our databases will be tools for wet lab research across the globe and we aim to make the WRKY Wide Web the database of choice for research into WRKY transcription factors. These novel findings have identified new gene targets (both WRKY genes and other genes) to improve drought responses in soybean. They validate our initial hypothesis that WRKY genes regulate drought responses, show that there was indeed have a pressing need for the project and suggest that the impact of the project could indeed be high.

Publications

  • Rushton, D.L., Tripathi, P., Rabara, R.C., Lin, J., Ringler, P., Boken, A.K., Langum, T.J., Smidt, L., Boomsma, D.D., Emme, N.J., Chen, X., Finer, J.J., Shen, Q.J., and Rushton P.J. (2011) WRKY transcription factors: key components in abscisic acid signalling. The Plant Biotechnology Journal. Jun 22. doi: 10.1111/j.1467-7652.2011.00634.x.
  • THE MOLECULAR AND PHYSIOLOGICAL RESPONSES OF SOYBEAN TO WATER DEFICIT Prateek Tripathi, Roel C. Rabara, Tanner J. Langum, Ashley K. Boken, Deena L. Rushton, Lucas Smidt and Paul J. Rushton* Proceedings of the South Dakota Academy of Science, Vol. 90 (2011) 177
  • DROUGHT-INDUCIBLE WRKY GENES IN TOBACCO (NICOTIANA TABACUM L.) Roel C. Rabara, Jun Lin, Prateek Tripathi, Tanner J. Langum, Ashley K. Boken, Deena L. Rushton, Lucas Smidt and Paul J. Rushton* Proceedings of the South Dakota Academy of Science, Vol. 90 (2011) 178


Progress 06/01/09 to 05/31/10

Outputs
OUTPUTS: Activities: 1) Which WRKY genes regulate responses to water stress We performed water stress experiments using either soybean or tobacco grown under well-defined hydroponic conditions. Oligo array analysis was performed in the two plant species in parallel. In tobacco leaves, thirteen WRKY genes were induced at least 8-fold by water stress and an additional ten were induced at least 5-fold. In the parallel soybean experiments, 21 WRKY genes were induced at least 8-fold by water stress. This represents over 10% of the soybean WRKY gene family. 2) Do phylogenetically similar WRKY genes regulate water stress responses We performed a phylogenetic analysis of the WRKY gene family in the green lineage and compared the water stress-regulated WRKY genes from tobacco and soybean. WRKY genes from all subfamilies are up-regulated by water stress, but we found clear "hotspots" in the phylogeny in Groups I, IIa and III. This forms the first part of our predictive phylogenetic tree. 3) Do drought responsive WRKY genes share similar drought responsive promoter elements Five promoters from tobacco WRKY genes that are strongly up-regulated by water stress are currently being evaluated for the direction of water-stress inducible gene expression in several systems including transgenic plants. Twelve soybean WRKY promoters are being analyzed in transgenic soybean. 4) How do WRKY genes regulate drought responses and can we use them to increase drought tolerance We have identified at least 800 potential gene targets for the improvement of drought responses. These may be regulated by WRKY transcription factors and we are focusing on the identification of downstream target genes. In the rice portion of the project, OsWRKYb and OsWRKYf are likely to be the key regulators of drought responses. Genotyping and phenotyping of OsWRKYf knockout mutants is being performed. Overexpression, RNAi, and promoter-GUS constructs are also being tested, together with three transposon insertional knockout mutants for OsWRKYf. Phenotyping of these OsWRKYf mutants under drought and ABA treatments are ongoing. We are also pursuing a characterization of the OsWRKYb and OsWRKYf proteins and results indicate that OsWRKYb, but not OsWRKYf, can bind to W boxes. It is likely that both proteins are positive regulators of drought response in rice, but that they regulate different subsets of genes. Events: Paul Rushton established his laboratory at South Dakota State University and was an invited speaker at the Plant and Animal Genome Conference in San Diego in January 2010. Services: In 2010, Paul Rushton served as an ad hoc reviewer for the NSF. Together with Dr Imre Somssich (Cologne, Germany) the PI and Co-PI, wrote a review on WRKY transcription factors for Trends in Plant Science. Dissemination of results: Some project data were presented at the Plant and Animal Genome Conference in San Diego in January 2010. PARTICIPANTS: PARTICIPANTS: During the last year, the personnel on the project has grown to include one postdoc (Jun Lin), two graduate students (Prateek Tripathi and Roel Rabara) and four undergraduate student (Deena Rinerson, Ashley Boken, Tanner Langum and Lucas Smidt). TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Change in knowledge: This year's work has provided evidence that 10-20% of WRKY genes appear to be involved in drought responses in soybean, rice and tobacco. In addition, there appear to be "hotspots" in the phylogenetic tree of the WRKY gene family (Groups I, IIa and III) where orthologous WRKY genes may regulate water stress responses in multiple species and these genes may represent a commonality in water stress responses in plants. We now have many promising candidate WRKY genes and their promoters to form the focus of continuing work. The transcriptome work has also identified at least 800 potential gene targets for the improvement of drought responses in crop plants.

Publications

  • Rushton, P.J., Somssich, I.E., Ringler, P., and Shen, J. (2010) WRKY transcription factors. Trends in Plant Science Volume: 15 Issue: 5 Pages: 247-258