GmWRKY49, a Salt-Responsive Nuclear Protein, Improved Root Length and Governed Better Salinity Tolerance in Transgenic Arabidopsis.

Plant WRKY transcription factors (TFs) are active guardians against pathogens' insurgency, key components in developmental processes, contributors in signal transduction pathways, and regulators of diverse biotic and abiotic stress responses. In this research, we isolated, cloned, and functionally characterized a new WRKY TF GmWRKY49 from soybean. GmWRKY49 is a nuclear protein which contains two highly conserved WRKY domains and a C2H2-type zinc-finger structure. The normalized expression (log2 ratio) of GmWRKY49 was 2.75- and 1.90-fold in salt-tolerant and salt-susceptible soybean genotypes, respectively. The transcripts of GmWRKY49 could be detected in roots, stems, leaves, flowers, and almost no expression in pod tissues. The salinity-tolerance response of this gene was studied through overexpression in soybean composite seedlings and transgenic Arabidopsis. The effect of GmWRKY49 overexpression on root length of transgenic Arabidopsis was also investigated. Under salt stress, several parameters including germination rate, survival rate, root length, rosette diameter, relative electrolyte leakage, and proline content were significantly higher in composite seedlings and transgenic Arabidopsis than those in wild-type. Moreover, GmWRKY49 enhanced salinity tolerance in soybean mosaic seedlings and transgenic Arabidopsis. These results suggest that GmWRKY49 is a positive regulator of salinity tolerance in soybean and has high potential utilization for crop improvement.

The nuclear protein GmbZIP110 has transcription activation activity and plays important roles in the response to salinity stress in soybean.

Plant basic-leucine zipper (bZIP) transcription factors play important roles in many biological processes and are involved in the regulation of salt stress tolerance. Previously, our lab generated digital gene expression profiling (DGEP) data to identify differentially expressed genes in a salt-tolerant genotype of Glycine soja (STGoGS) and a salt-sensitive genotype of Glycine max (SSGoGM). This DGEP data revealed that the expression (log2 ratio) of GmbZIP110 was up-regulated 2.76-fold and 3.38-fold in SSGoGM and STGoGS, respectively. In the present study, the salt inducible gene GmbZIP110 was cloned and characterized through phylogenetic analysis, subcellular localization and in silico transcript abundance analysis in different tissues. The functional role of this gene in salt tolerance was studied through transactivation analysis, DNA binding ability, expression in soybean composite seedlings and transgenic Arabidopsis, and the effect of GmbZIP110 on the expression of stress-related genes in transgenic Arabidopsis was investigated. We found that GmbZIP110 could bind to the ACGT motif, impact the expression of many stress-related genes and the accumulation of proline, Na(+) and K(+), and enhanced the salt tolerance of composite seedlings and transgenic Arabidopsis. Integrating all these results, we propose that GmbZIP110 plays a critical role in the response to salinity stress in soybean and has high potential usefulness in crop improvement.

A single-repeat MYB transcription factor, GmMYB176, regulates CHS8 gene expression and affects isoflavonoid biosynthesis in soybean.

Here we demonstrate that GmMYB176 regulates CHS8 expression and affects isoflavonoid synthesis in soybean. We previously established that CHS8 expression determines the isoflavonoid level in soybean seeds by comparing the transcript profiles of cultivars with different isoflavonoid contents. In the present study, a functional genomic approach was used to identify the factor that regulates CHS8 expression and isoflavonoid synthesis. Candidate genes were cloned, and co-transfection assays were performed in Arabidopsis leaf protoplasts. The results showed that GmMYB176 can trans-activate the CHS8 promoter with maximum activity. Transient expression of GmMYB176 in soybean embryo protoplasts increased endogenous CHS8 transcript levels up to 169-fold after 48 h. GmMYB176 encodes an R1 MYB protein, and is expressed in soybean seed during maturation. Furthermore, GmMYB176 recognizes a 23 bp motif containing a TAGT(T/A)(A/T) sequence within the CHS8 promoter. A subcellular localization study confirmed nuclear localization of GmMYB176. A predicted pST binding site for 14-3-3 protein is required for subcellular localization of GmMYB176. RNAi silencing of GmMYB176 in hairy roots resulted in reduced levels of isoflavonoids, showing that GmMYB176 is necessary for isoflavonoid biosynthesis. However, over-expression of GmMYB176 was not sufficient to increase CHS8 transcript and isoflavonoid levels in hairy roots. We conclude that an R1 MYB transcription factor, GmMYB176, regulates CHS8 expression and isoflavonoid synthesis in soybean.

Differential expression of CHS7 and CHS8 genes in soybean.

Chalcone synthase (CHS) catalyzes the first reaction specific for flavonoid and isoflavonoid biosynthesis. The soybean genome consists of nine copies of CHS genes (CHS1-CHS9) and a duplicate copy of CHS1. Even though the soybean CHS gene family members share a high degree of sequence similarity, they play different roles during plant development or in response to environmental stimuli. Our previous work on the comparison of a global gene expression in two soybean cultivars that differ in the level of total isoflavonoid accumulation has denoted the involvement of CHS7 and CHS8 genes in isoflavonoid synthesis. We have extended our effort to understand expression patterns of these two genes in soybean and in transgenic Arabidopsis. Promoter regions of CHS7 and CHS8 genes were isolated and in silico analysis performed to investigate potential transcription factor binding sites (TFBSs). The TFBSs were verified by DNase I footprint analysis. Some unique and several common TFBSs were identified in CHS7 and CHS8 promoters. We cloned beta-glucuronidase (GUS) under CHS7 and CHS8 promoters and monitored the tissue-specific GUS expression in transformed Arabidopsis. Differential GUS activity was observed in young leaves, roots, and mature pod walls of transgenic CHS7 promoter-GUS and CHS8 promoter-GUS plants. The tissue-specific expression patterns of CHS7 and CHS8 genes were determined in soybean by quantitative RT-PCR. Both CHS7 and CHS8 genes were expressed at higher levels in roots; however, overall expression pattern of these genes varied in different tissues. The results suggest that the structural diversity within CHS7 and CHS8 promoters may lead into differential activation of these genes by different inducers as well as developmental stage- and tissue-specific differences in gene expression.