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Document Type

Dissertation - University Access Only

Award Date


Degree Name

Doctor of Philosophy (PhD)


Biology and Microbiology

First Advisor

Paul J. Johnson


We have used systems level approaches to investigate the WRKY transcription factors regulating water stress responses. The number of genes in the WRKY transcription family was determined to generate a comprehensive data set from soybean for further analysis and similar data was generated from Medicago truncatula and Brachypodium for comparative and computational analysis. Soybean plants were grown under hydroponic conditions, removed and subjected to dehydration. Plants underwent dehydration stress as confirmed by measuring total water content, osmotic potential, stomatal conductance and phytohormone level. We found that drought intensity ranged from severe to extreme, as confirmed by rehydration experiments. Transcriptome analyses using a custom made NimbleGen oligoarray containing all gene models from the Glyma1.0v of the soybean genome showed that over 3000 genes and 25% of soybean WRKY genes are up regulated at least 8-fold in either root or leaf tissue. Promoters of up-regulated genes were found to be enriched with ABRE-like motifs and two novel motifs. The Group IIa genes of WRKY transcription factors and Group IX genes of ERF transcription factors are among the major groups up regulated during dehydration. Metabolite analyses revealed a flow of nitrogen into amino acid metabolism and an elevation of amino acids due to de novo biosynthesis. This was confirmed by proteomics studies performed using Shotgun Mass-spectrometry (MuDPIT) and it constitutes a potential dehydration tolerance strategy. Strikingly, coumestrol an isoflavonoid was a prominent metabolite found to accumulate over 100-fold in roots during dehydration. This led to our hypothesis that increases in coumestrol level might promote dehydration-induced reactive oxygen species scavenging. Promoter activity of GmWRKY17 and GmWRKY67 in soybean hairy roots and GmWRKY53 in tobacco BY-2 cells suggests that WRKY gene activation during water stress is largely ABA-dependent. Promoter activity of GmWRKY53 and GmWRKY112 clearly shows that use of ABA rather than PEG is better at simulating water stress in BY-2 cells. These results provide many novel insights into the roles of WRKY transcription factors during water stress in soybean and provide valuable inputs towards a systems-wide understanding of water-stress signaling.

Library of Congress Subject Headings

Soybean -- Genetics
Soybean -- Effect of stress on
Soybean -- Effect of drought on


Includes bibliographical references (page 264)



Number of Pages



South Dakota State University


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