ABSTRACT
GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the isoflavonoid biosynthetic pathway, thereby affecting their levels in soybean roots. While GmMYB176 is important for isoflavonoid synthesis, it is not sufficient for the function and requires additional cofactor(s). The aim of this study was to identify the GmMYB176 interactome for the regulation of isoflavonoid biosynthesis in soybean. Here, we demonstrate that a bZIP transcription factor GmbZIP5 co-immunoprecipitates with GmMYB176 and shows protein-protein interaction in planta. RNAi silencing of GmbZIP5 reduced the isoflavonoid level in soybean hairy roots. Furthermore, co-overexpression of GmMYB176 and GmbZIP5 enhanced the level of multiple isoflavonoid phytoallexins including glyceollin, isowighteone and a unique O-methylhydroxy isoflavone in soybean hairy roots. These findings could be utilized to develop biotechnological strategies to manipulate the metabolite levels either to enhance plant defense mechanisms or for human health benefits in soybean or other economically important crops.
Subject(s)
Glycine max/metabolism , Isoflavones/biosynthesis , Soybean Proteins/metabolism , Transcription Factors/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Gene Expression Regulation, Plant , Plant Roots , Protein Binding , Pterocarpans/biosynthesis , Soybean Proteins/genetics , Glycine max/genetics , Transcription Factors/geneticsABSTRACT
Isoflavonoids are a group of plant natural compounds synthesized almost exclusively by legumes, and are abundant in soybean seeds and roots. They play important roles in plant-microbial interactions and the induction of nod gene expression in Rhizobia that form nitrogen-fixing nodules on soybean roots. Isoflavonoids also contribute to the positive health effects associated with soybean consumption by humans and animals. An R1 MYB transcription factor GmMYB176 regulates isoflavonoid biosynthesis by activating chalcone synthase (CHS) 8 gene expression in soybean. Using a combination of transcriptomic and metabolomic analyses of GmMYB176-RNAi silenced (GmMYB176-Si), GmMYB176-overexpressed (GmMYB176-OE), and control soybean hairy roots, we identified a total of 33 differentially expressed genes (DEGs) and 995 differentially produced metabolite features (DPMF) in GmMYB176-Si hairy roots, and 5727 DEGs and 149 DPMFs in GmMYB176-OE hairy roots. By a targeted approach, 25 isoflavonoid biosynthetic genes and 6 metabolites were identified as differentially regulated in GmMYB176-OE and GmMYB176-Si soybean hairy roots. Taken together, our results demonstrate the complexity of isoflavonoid biosynthesis in soybean roots and suggest that a coordinated expression of pathway genes, substrate flux and product threshold level may contribute to the dynamic of the pathway regulation.
ABSTRACT
BACKGROUND: Soybean is a paleopolyploid that has undergone two whole genome duplication events. Gene duplication is a type of genomic change that can lead to novel functions of pre-existing genes. Chalcone synthase (CHS) is the plant-specific type III polyketide synthase that catalyzes the first committed step in (iso)flavonoid biosynthesis in plants. RESULTS: Here we performed a genome-wide search of CHS genes in soybean, and identified 21 GmCHS loci containing 14 unique GmCHS (GmCHS1-GmCHS14) that included 5 newly identified GmCHSs (GmCHS10-GmCHS14). Furthermore, 3 copies of GmCHS3 and 2 copies of GmCHS4 were found in soybean. Analysis of gene structure of GmCHSs revealed the presence of a single intron in protein-coding regions except for GmCHS12 that contained 3 introns. Even though GmCHS genes are located on 8 different chromosomes, a large number of these genes are present on chromosome 8 where they form 3 distinct clusters. Expression analysis of GmCHS genes revealed tissue-specific expression pattern, and that some GmCHS isoforms localize in the cytoplasm and the nucleus while other isoforms are restricted to cytoplasm only. CONCLUSION: Overall, we have identified 21 GmCHS loci with 14 unique GmCHS genes in the soybean genome. Their gene structures and genomic organization together with the spatio-temporal expression and protein localization suggest their importance in the production of downstream metabolites such as (iso)flavonoids and their derived phytoalexins.
