Two homologous transcription factors have opposite roles in regulating accumulation of the soybean phytoalexin glyceollin I to provide resistance to Phytophthora. List of author contributions: NK designed the study, organized and supervised the research and wrote the manuscript. NK and MAJ interpreted the results. MAJ conducted UPLC-PDA, cloning, statistics, and hairy root transformations, MAJ and ML the EMSAs, MAJ, ML, and NK the RT-qPCR, BH the Y1H, RJP the RNA-seq library preparation and AMI processed the RNA-seq data.

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...

Phytoalexins are metabolites biosynthesized in plants in response to pathogen, environmental, and chemical stresses that often have potent bioactivities, rendering them promising for use as therapeutics or scaffolds for pharmaceutical development. Glyceollin I is an isoflavonoid phytoalexin from soybean that exhibits potent anticancer activities and is not economical to synthesize. Here, we tested a range of source tissues from soybean, in addition to chemical and biotic elicitors, to understand how to enhance the bioproduction of glyceollin I. Combining the inorganic chemical silver nitrate (AgNO3) with the wall glucan elicitor (WGE) from the soybean pathogen Phytophthora sojae had an additive effect on the elicitation of soybean seeds, resulting in a yield of up to 745.1 µg gt−1 glyceollin I. The additive elicitation suggested that the biotic and chemical elicitors acted largely by separate mechanisms. WGE caused a major accumulation of phytoalexin gene transcripts, whereas AgNO3 inhibited and enhanced the degradation of glyceollin I and 6″-O-malonyldaidzin, respectively.

Glyceollins are the major pathogen-and stress-inducible natural products (phytoalexins) of soybean that possess broad-spectrum anticancer and neuroprotective properties. Yet like other phytoalexins, glyceollins are difficult to obtain because they are typically biosynthesized only transiently and in low amounts in plant tissues. We recently identified acidity stress (pH 3.0 growth medium) as an elicitor that exerted prolonged (week-long) inductive effects on glyceollin biosynthesis and identified the NAC family TF gene GmNAC42-1 that activates glyceollin biosynthesis in response to acidity stress or WGE from the soybean pathogen Phytophthora sojae. GmNAC42-1 was annotated as an SAR gene and SAR genes were statistically overrepresented in the transcriptomic response to acidity stress suggesting that acidity stress triggers the systemic elicitation of glyceollin biosynthesis. Here, we demonstrate that acidity stress acts as a systemic elicitor when provided to soybean roots. Acidity stress preferentially elicited specific glyceollins in different soybean organs with exceptionally high yields of glyceollin I in root tissues.

To minimize the cost of biodiesel production, massively available biomass, rice straw was selected as starting material for fermentation by oleaginous yeast, Lipomyces starkeyi. Acid hydrolysis was carried out to obtain Rice Straw Hydrolysates (RSH). Maximum amount of glucose was liberated with 3.5% H 2 SO 4. Highest levels of biomass (~12 g/L) and lipid content (~36%) of L. starkeyi were obtained through fermentation of RSH produced with 3.5% H 2 SO 4. The optimum pH for biomass yield and lipid accumulation of L. starkeyi was 6.0. Periodic supplementation of fresh RSH increased lipid content and lipid yield approximately15% and 40%, respectively. The growth and lipid accumulation of L. starkeyi were enhanced when carbon and nitrogen sources were supplemented with RSH. Gas chromatographic analysis revealed that the lipid obtained from L. starkeyi cultivated with RSH was composed of saturated and unsaturated fatty acids. The fatty acid composition of this microbial lipid is similar to that of vegetable oils. The results reported herein indicate that rice straw might be a valuable alternative feedstock for microbial lipids production by L. starkeyi for biodiesel synthesis.