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.
Highlights
GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the isoflavonoid biosynthetic pathway, thereby affecting their levels in soybean roots
We performed in silico analysis of 30 bp GmCHS8 promoter region (23 bp with 7 bp flanking sequence) for regulatory elements binding sites as this region is critical for GmMYB176-mediated gene expression[19]
Since relatively stronger protein–protein interaction was observed between GmMYB176S29A and GmbZIP4/GmbZIP5 compared to GmMYB176 and GmbZIP4/GmbZIP5, we examined the DNA binding ability of GmMYB176S29A and discovered that the DNA binding activity of GmMYB176 does not depend on its phosphorylation state
Summary
GmMYB176 is an R1 MYB transcription factor that regulates multiple genes in the isoflavonoid biosynthetic pathway, thereby affecting their levels in soybean roots. 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. Genes involved in flavonoid biosynthesis are well conserved in higher plants and are regulated by a combinatorial action of transcriptional regulatory factors expressed in temporal and spatially controlled fashion[14,15]. The expression of early biosynthetic genes involved in flavonoid biosynthesis, such as Phenylalanine ammonia-lyase, CHS, Chalcone isomerase (CHI), Flavonol 3′-hydroxylase, Flavonol synthase (FLS) is regulated by MYB transcription factors in a coordinated manner[16] while the late biosynthetic genes are regulated by an MBW ternary complex consisting of a R2R3 MYB transcription factor, a basic helix-loophelix (bHLH) transcription factor and a WD repeat protein[17,18]
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