GmMYB176 Regulates Multiple Steps in Isoflavonoid Biosynthesis in Soybean

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

Soybean AROGENATE DEHYDRATASES (GmADTs): involvement in the cytosolic isoflavonoid metabolon or trans-organelle continuity?

Soybean (Glycine max) produces a class of phenylalanine (Phe) derived specialized metabolites, isoflavonoids. Isoflavonoids are unique to legumes and are involved in defense responses in planta, and they are also necessary for nodule formation with nitrogen-fixing bacteria. Since Phe is a precursor of isoflavonoids, it stands to reason that the synthesis of Phe is coordinated with isoflavonoid production. Two putative AROGENATE DEHYDRATASE (ADT) isoforms were previously co-purified with the soybean isoflavonoid metabolon anchor ISOFLAVONE SYNTHASE2 (GmIFS2), however the GmADT family had not been characterized. Here, we present the identification of the nine member GmADT family. We determined that the GmADTs share sequences required for enzymatic activity and allosteric regulation with other characterized plant ADTs. Furthermore, the GmADTs are differentially expressed, and multiple members have dual substrate specificity, also acting as PREPHENATE DEHYDRATASES. All GmADT isoforms were detected in the stromules of chloroplasts, and they all interact with GmIFS2 in the cytosol. In addition, GmADT12A interacts with multiple other isoflavonoid metabolon members. These data substantiate the involvement of GmADT isoforms in the isoflavonoid metabolon

Genome-Wide Identification of Chalcone Reductase Gene Family in Soybean: Insight into Root-Specific GmCHRs and Phytophthora sojae Resistance

Soybean (Glycine max [L.] Merr) is one of the main grain legumes worldwide. Soybean farmers lose billions of dollars’ worth of yield annually due to root and stem rot disease caused by the oomycete Phytophthora sojae. Many strategies have been developed to combat the disease, however, these methods have proven ineffective in the long term. A more cost effective and durable approach is to select a trait naturally found in soybean that can increase resistance. One such trait is the increased production of phytoalexin glyceollins in soybean. Glyceollins are isoflavonoids, synthesized via the legume-specific branch of general phenylpropanoid pathway. The first key enzyme exclusively involved in glyceollin synthesis is chalcone reductase (CHR) which coacts with chalcone synthase for the production of isoliquiritigenin, the precursor for glyceollin biosynthesis. Here we report the identification of 14 putative CHR genes in soybean where 11 of them are predicted to be functional. Our results show that GmCHRs display tissue-specific gene expression, and that only root-specific GmCHRs are induced upon P. sojae infection. Among 4 root-specific GmCHRs, GmCHR2A is located near a QTL that is linked to P. sojae resistance suggesting GmCHR2A as a novel locus for partial resistance that can be utilized for resistance breeding

Identification of Differentially Expressed Genes in Brassinosteroid-Treated 'Brassica napus' Seedlings

Brassinosteroid-mediated gene expression changes have been reported primarily in 'Arabidopsis thaliana' but not in its close relative Brassica napus. To obtain an initial idea of the molecular changes induced by long-term exposure to 24-epibrassinolide (EBR) in 'B. napus' seedlings, we used the differential display-reverse transcription PCR technique. Six differentially expressed cDNAs were isolated and characterized. These encode a mitochondrial transcription termination factor (mTERF)-related protein, glycine-rich protein 22 (GRP22), myrosinase, 3-ketoacyl-CoA thiolase, and a copia-like polyprotein. The first four were upregulated in EBR-treated seedlings while the latter was expressed at higher levels in untreated seedlings. Transcripts of mTERF-related protein, GRP22, and myrosinase were present at higher levels in treated seedlings under nonstress conditions, whereas those of 3-ketoacyl-CoA thiolase rose to higher levels in treated seedlings during exposure to heat stress. The results of the present study indicate that EBR treatment in 'B. napus' leads to substantial changes in the expression levels of genes involved in a variety of physiologic responses. The results provide a useful framework for further research into EBR-mediated molecular changes in 'B. napus', which will also add to our understanding of how brassinosteroids mediate stress tolerance in this agriculturally important oil crop

Identification of differentially-expressed genes in brassinosteroid-treated Brassica napus seedlings

Brassinosteroid-mediated gene expression changes have been reported primarily in Arabidopsis thaliana but not in its close relative Brassica napus. To obtain an initial idea of the molecular changes induced by longterm exposure to 24-epibrassinolide (EBR) in B. napus seedlings, we used the differential display–reverse transcription PCR technique. Six differentially expressed cDNAs were isolated and characterized. These encode a mitochondrial transcription termination factor (mTERF)-related protein, glycine-rich protein 22 (GRP22), myrosinase, 3-ketoacyl-CoA thiolase, and a copia-like polyprotein. The first four were upregulated in EBR-treated seedlings while the latter was expressed at higher levels in untreated seedlings. Transcripts of mTERF-related protein, GRP22, and myrosinase were present at higher levels in treated seedlings under nonstress conditions, whereas those of 3-ketoacyl-CoA thiolase rose to higher levels in treated seedlings during exposure to heat stress. The results of the present study indicate that EBR treatment in B. napus leads to substantial changes in the expression levels of genes involved in a variety of physiologic responses. The results provide a useful framework for further research into EBR-mediated molecular changes in B. napus, which will also add to our understanding of how brassinosteroids mediate stress tolerance in this agriculturally important oil crop

Functional characterization of Cinnamate 4-hydroxylase gene family in soybean (Glycine max).

Cinnamate 4-hydroxylase (C4H) is the first key cytochrome P450 monooxygenase (P450) enzyme in the phenylpropanoid pathway. It belongs to the CYP73 family of P450 superfamily, and catalyzes the conversion of trans-cinnamic acid to p-coumaric acid. Since p-coumaric acid serves as the precursor for the synthesis of a wide variety of metabolites involved in plant development and stress resistance, alteration in the expression of soybean C4H genes is expected to affect the downstream metabolite levels, and its ability to respond to stress. In this study, we identified four C4H genes in the soybean genome that are distributed into both class I and class II CYP73 family. GmC4H2, GmC4H14 and GmC4H20 displayed tissue- and developmental stage-specific gene expression patterns with their transcript accumulation at the highest level in root tissues. GmC4H10 appears to be a pseudogene as its transcript was not detected in any soybean tissues. Furthermore, protein homology modelling revealed substrate docking only for GmC4H2, GmC4H14 and GmC4H20. To demonstrate the function of GmC4Hs, we modified a cloning vector for the heterologous expression of P450s in yeast, and used it for microsomal protein production and enzyme assay. Our results confirmed that GmC4H2, GmC4H14 and GmC4H20 contain the ability to hydroxylate trans-cinnamic acid with varying efficiencies