The NAC family transcription factor GmNAC42–1 regulates biosynthesis of the anticancer and neuroprotective glyceollins in soybean

Abstract

Background Glyceollins are isoflavonoid-derived pathogen-inducible defense metabolites (phytoalexins) from soybean ( Glycine max L. Merr) that have important roles in providing defense against pathogens. They also have impressive anticancer and neuroprotective activities in mammals. Despite their potential usefulness as therapeutics, glyceollins are not economical to synthesize and are biosynthesized only transiently and in low amounts in response to specific stresses. Engineering the regulation of glyceollin biosynthesis may be a promising approach to enhance their bioproduction, yet the transcription factors (TFs) that regulate their biosynthesis have remained elusive. To address this, we first aimed to identify novel abiotic stresses that enhance or suppress the elicitation of glyceollins and then used a comparative transcriptomics approach to search for TF gene candidates that may positively regulate glyceollin biosynthesis. Results Acidity stress (pH 3.0 medium) and dehydration exerted prolonged (week-long) inductive or suppressive effects on glyceollin biosynthesis, respectively. RNA-seq found that all known biosynthetic genes were oppositely regulated by acidity stress and dehydration, but known isoflavonoid TFs were not. Systemic acquired resistance (SAR) genes were highly enriched in the geneset. We chose to functionally characterize the NAC (NAM/ATAF1/2/CUC2)-family TF GmNAC42–1 that was annotated as an SAR gene and a homolog of the Arabidopsis thaliana (Arabidopsis) indole alkaloid phytoalexin regulator ANAC042 . Overexpressing and silencing GmNAC42–1 in elicited soybean hairy roots dramatically enhanced and suppressed the amounts of glyceollin metabolites and biosynthesis gene mRNAs, respectively. Yet, overexpressing GmNAC42–1 in non-elicited hairy roots failed to stimulate the expressions of all biosynthesis genes. Thus, GmNAC42–1 was necessary but not sufficient to activate all biosynthesis genes on its own, suggesting an important role in the glyceollin gene regulatory network (GRN). The GmNAC42–1 protein directly bound the promoters of biosynthesis genes IFS2 and G4DT in the yeast one-hybrid (Y1H) system. Conclusions Acidity stress is a novel elicitor and dehydration is a suppressor of glyceollin biosynthesis. The TF gene GmNAC42–1 is an essential positive regulator of glyceollin biosynthesis. Overexpressing GmNAC42–1 in hairy roots can be used to increase glyceollin yields > 10-fold upon elicitation. Thus, manipulating the expressions of glyceollin TFs is an effective strategy for enhancing the bioproduction of glyceollins in soybean. Electronic supplementary material The online version of this article...

Complex Gene Regulation Underlying Mineral Nutrient Homeostasis in Soybean Root Response to Acidity Stress

“…(Table S3). This is consistent with the recent report that these genes were up-regulated in soybean variety Harosoy 63 after a 9-day pH3.0 treatment [35]. Therefore, it suggests that overlays between acidity-stress responses and pathogen responses are conserved in soybean.…”

“…The other pH stat pathway is the GABA shunt pathway, which consists of GDH, GAD, and GABA-T and also contributes greatly to the H + homeostasis in plant cells [58,91,92]. Expression of genes involving in these two pH-stat pathways was significantly suppressed in Arabidopsis stop1 mutants, strongly suggesting that they play important roles in Arabidopsis low-pH-tolerance [35]. In the present study, GmGDH1 in the GABA shunt pathway, and several other genes (i.e., GmME1-1 , GmPCD1 , GmPCD2-1/2 , GmADH1-1/2/3 ) in the other biochemical pH stat pathway were significantly up-regulated under acid conditions (Table 2).…”

“…With the aid of genome wide transcriptomic analysis and functional characterization of sets of genes, molecular mechanisms underlying complex responses of plants to acidity stress have been elucidated [33,34,35]. A microarray assay on wheat ( Triticum aestivum ) identified 1057 differentially expressed genes (DEGs) after two hours of acid treatment, in which rhizosphere alkalification was suggested to be the earliest responses to acidity stress [34].…”

“…Interestingly, a recent RNA-seq analysis in soybean found a total of 3242 genes were up-regulated by pH3.0 medium for 9 days. Among them, 27 genes were identified to be involved in the biosynthesis of glycollins, which are isoflavonoid-derived pathogen-inducible defense metabolites [35]. Moreover, genes function in systemic acquired resistance (SAR), which is a component of the plant immune system to pathogen, were also significantly up-regulated [35].…”

“…Among them, 27 genes were identified to be involved in the biosynthesis of glycollins, which are isoflavonoid-derived pathogen-inducible defense metabolites [35]. Moreover, genes function in systemic acquired resistance (SAR), which is a component of the plant immune system to pathogen, were also significantly up-regulated [35]. These results indicated that acidity stress is an elicitor of plant defensive responses to pathogens.…”