A combinatorial action of GmMYB176 and GmbZIP5 controls isoflavonoid biosynthesis in soybean (Glycine max)

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.

Fig. 1. Co-immunoprecipitation of GmMYB176 interacting proteins from soybean hairy roots.

a Subcellular localization of GmMYB176-YFP and YFP-GmMYB176 in hairy roots. Both GmMYB176-YFP and YFP-GmMYB176 fusion proteins were localized in the nucleus and the cytoplasm of soybean hairy root cells as observed by confocal microscopy. Scale bar = 50 μm. b Crude protein extracts were subjected for Co-IP assay using anti-GFP microbeads and µMAC epitope tag protein isolation system. Samples from each step were separated on an SDS-PAGE and visualized by silver staining (top gel). The bottom image shows Western blot analysis using anti-GFP monoclonal antibody. The arrow indicates the estimated size of GmMYB176-YFP protein in the eluate. Crude: crude protein extract from soybean hairy roots; Flow through: crude extract incubated with anti-GFP microbeads and applied to μcolumn, with the flow through collected; Wash: sequential wash steps with lysis buffer; Eluate: elution of bound proteins from the column; –ve control: crude extract from control hairy roots. c Venn diagram showing the overlap of GmMYB176-YFP, YFP-GmMYB176, and YFP-only interacting candidate proteins in soybean hairy roots identified by LC–MS/MS analysis. The YFP interacting protein candidates were obtained from our previous study. d ‘GO’ annotations of the 716 candidate GmMYB176-interacting proteins. List of soybean genes encoding the candidate proteins was used in PhytoMine to generate annotations regarding the biological process, cellular component, and the molecular function of the candidates.

Fig. 2. GmMYB176-interacting transcription factors and their DNA binding activity.

a GmMYB176- transcription factors obtained by co-IP assay GO annotation “Biological process” was retrieved and compared with the list of transcription factors obtained from in silico analysis of 30 bp GmCHS8 promoter fragment (Supplementary Table 2) using a Venn diagram. b Protein–protein interactions of GmMYB176 and GmMYB176S29A in planta with candidate transcription factors obtained by Co-IP. The interaction between the proteins was assayed by co-expression of translational fusions of candidate proteins with N-terminal (YN) and C-terminal (YC) halves of YFP. The proximity of the two fragments results in a functional fluorophore. The fluorescence indicates the presence and location of the interaction between GmMYB176 or GmMYB176S29A with the candidate transcription factors. Fluorescent intensity parameters were kept constant in all images. Scale bars = 50 μm. c GmCHS8 promoter (30 bp fragment) binding activity of GmMYB176 interacting candidates. Yeast cells carrying 30 bp GmCHS8 tandem repeats (30 bpTR) as a bait, were transformed with prey constructs fused to a GAL4 activation domain. Growth on SD lacking leucine and in the presence of Aureobasidin A (SD/-Leu/AbA) shows the activation of the reporter and indicates DNA binding activity. As a negative control, pGADT7 vector only was used. d Tissue-specific expression pattern of GmMYB176, GmbZIP4, and GmbZIP5 in soybean. RNA-seq data across different tissues were extracted from soybean whole genome database in Phytozome (https://phytozome.jgi.doe.gov/pz/portal.html#!info?alias=Org_Gmax) and a heatmap was constructed. Numbers to the right indicate the maximum value of fragments per kilobase of million mapped reads (FPKM). The color scale indicates expression values, green indicating low transcript abundance, and red indicating high levels of transcript abundance.

Fig. 3. Effect of GmbZIP silencing and overexpression of GmMYB176-GmbZIP fusion complex on isoflavonoid level in soybean hairy roots.

a Accumulation of GmbZIP4 and GmbZIP5 transcripts in multiple independent pools of transgenic or control hairy root samples determined by quantitative (q) RT-PCR. Values were normalized against the CONS4 reference gene. b Effect of RNAi silencing of GmbZIP4 (GmbZIP4-Si) or GmbZIP5 (GmbZIP5-Si) on isoflavonoid content using hairy roots. Control indicates untransformed hairy roots. Data correspond to mean isoflavonoid levels in ten biological replicates. The asterisk (*) denotes statistically significant expression (one-tail t test, p < 0.0001). c Schematic diagram showing GmMYB176-GmbZIP4 and GmMYB176-GmbZIP5 gene fusion for overexpression in soybean hairy roots. Expression levels of the gene fusion were determined by qRT-PCR in multiple independent pools of control or transgenic roots normalized against the reference gene CONS4. Control indicates untransformed hairy roots. d Effect of overexpression of GmMYB176-GmbZIP4 or GmMYB176-GmbZIP5 on isoflavonoid levels. Data correspond to mean isoflavonoid levels in ten biological replicates. The asterisk (*) denotes statistically significant expression (one-tail t test, p < 0.01).

Fig. 4. Overexpression of GmMYB176–GmbZIP5 upregulates isoflavonoid biosynthesis.

Multiple arrows indicate multiple steps in the pathway and dotted arrows indicate speculated steps. PAL phenylalanine ammonia-lyase, C4H cinnamate-4-hydroxylate, 4CL 4-coumarate-CoA-ligase, CHS chalcone synthase, CHR chalcone reductase, CHI chalcone isomerase, IFS 2-hydroxyisoflavanone synthase, 2HID 2-hydroxyisoflavanone dehydratase, IOMT isoflavone O-methyltransferase, UGT uridine diphosphate glycosyltransferase, MT malonyltransferase. This pathway is adopted and modified. The blue highlighted metabolites are accumulated at higher level in GmMYB176–GmbZIP5 overexpression lines compared to control.