GmGPDH12,a mitochondrial FAD-GPDH from soybean, increases salt and osmotic stress resistance by modulating redox state and respiration

In plants,glycerol-3-phosphate dehydrogenase(GPDH)catalyzes the interconversion of glycerol-3-phosphate(G3P)and dihydroxyacetone phosphate(DHAP)coupled to the reduction/oxidation of the nicotinamide adenine dinucleotide(NADH)pool,and plays a central role in glycerolipid metabolism and stress response.Previous studies have focused mainly on the NAD+-dependent GPDH isoforms,neglecting the role of flavin adenine dinucleotide(FAD)-dependent GPDHs.We isolated and characterized three mitochondrialtargeted FAD-GPDHs in soybean,of which one isoform(GmGPDH12)showed a significant transcriptional response to NaCl and mannitol treatments,suggesting the existence of a major FAD-GPDH isoform acting in soybean responses to salt and osmotic stress.An enzyme kinetic assay showed that the purified GmGPDH12 protein possessed the capacity to oxidize G3P to DHAP in the presence of FAD.Overexpression and RNA interference of GmGPDH12 in soybean hairy roots resulted in elevated tolerance and sensitivity to salt and osmotic stress,respectively.G3P contents were significantly lower in GmGPDH12-overexpressing hair roots and higher in knockdown hair roots,indicating that GmGPDH12 was essential for G3P catabolism.A significant perturbation in redox status of NADH,ascorbic acid(ASA)and glutathione(GSH)pools was observed in GmGPDH12-knockdown plants under stress conditions.The impaired redox balance was manifested by higher reactive oxygen species generation and consequent cell damage or death;however,overexpressing plants showed the opposite results for these traits.GmGPDH12 overexpression contributed to maintaining constant respiration rates under salt or osmotic stress by regulating mRNA levels of key mitochondrial respiratory enzymes.This study provides new evidence for the roles of mitochondria-localized GmGPDH12 in conferring resistance to salt or osmotic stress by maintaining cellular redox homeostasis,protecting cells and respiration from oxidative injury.

Enhanced production of seed oil with improved fatty acid composition by overexpressing NAD^(+)-dependent glycerol-3-phosphate dehydrogenase in soybean

There is growing interest in expanding the production of soybean oils(mainly triacylglycerol, or TAG) to meet rising feed demand and address global energy concerns. We report that a plastidlocalized glycerol-3-phosphate dehydrogenase(GPDH), encoded by GmGPDHp1 gene, catalyzes the formation of glycerol-3-phosphate(G3 P), an obligate substrate required for TAG biosynthesis.Overexpression of GmGPDHp1 increases soybean seed oil content with high levels of unsaturated fatty acids(FAs), especially oleic acid(C18:1), without detectably affecting growth or seed protein content or seed weight. Based on the lipidomic analyses, we found that the increase in G3 P content led to an elevated diacylglycerol(DAG) pool, in which the Kennedy pathwayderived DAG was mostly increased, followed by PC-derived DAG, thereby promoting the synthesis of TAG containing relatively high proportion of C18:1. The increased G3 P levels induced several transcriptional alterations of genes involved in the glycerolipid pathways. In particular, genes encoding the enzymes responsible for de novo glycerolipid synthesis were largely upregulated in the transgenic lines, in-line with the identified biochemical phenotype. These results reveal a key role for GmGPDHp1-mediated G3 P metabolism in enhancing TAG synthesis and demonstrate a strategy to modify the FA compositions of soybean oils for improved nutrition and biofuel.