Table_3_Genome-Wide Analysis of the Glucose-6-Phosphate Dehydrogenase Family in Soybean and Functional Identification of GmG6PDH2 Involvement in Salt Stress.DOC

Glucose-6-phosphate dehydrogenase (G6PDH) is known as a critical enzyme responsible for nicotinamide adenine dinucleotide phosphate (NADPH) generation in the pentose phosphate pathway (PPP), and has an essential function in modulating redox homeostasis and stress responsiveness. In the present work, we characterized the nine members of the G6PDH gene family in soybean. Phylogenic analysis and transit peptide prediction showed that these soybean G6PDHs are divided into plastidic (P) and cytosolic (Cy) isoforms. The subcellular locations of five GmG6PDHs were further verified by confocal microscopy in Arabidopsis mesophyll protoplasts. The respective GmG6PDH genes had distinct expression patterns in various soybean tissues and at different times during seed development. Among them, the Cy-G6PDHs were strongly expressed in roots, developing seeds and nodules, while the transcripts of P-G6PDHs were mainly detected in green tissues. In addition, the activities and transcripts of GmG6PDHs were dramatically stimulated by different stress treatments, including salt, osmotic and alkali. Notably, the expression levels of a cytosolic isoform (GmG6PDH2) were extraordinarily high under salt stress and correlated well with the G6PDH enzyme activities, possibly implying a crucial factor for soybean responses to salinity. Enzymatic assay of recombinant GmG6PDH2 proteins expressed in Escherichia coli showed that the enzyme encoded by GmG6PDH2 had functional NADP+-dependent G6PDH activity. Further analysis indicated overexpression of GmG6PDH2 gene could significantly enhance the resistance of transgenic soybean to salt stress by coordinating with the redox states of ascorbic acid and glutathione pool to suppress reactive oxygen species generation. Together, these results indicate that GmG6PDH2 might be the major isoform for NADPH production in PPP, which is involved in the modulation of cellular AsA-GSH cycle to prevent the oxidative damage induced by high salinity.

葡萄糖-6-磷酸脱氢酶（Glucose-6-phosphate dehydrogenase, G6PDH）是戊糖磷酸途径（pentose phosphate pathway, PPP）中负责合成烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）的关键酶，在调控氧化还原稳态与应激响应中发挥不可或缺的重要功能。本研究对大豆G6PDH基因家族的9个成员进行了鉴定与表征。系统发育分析与转运肽预测结果表明，这些大豆G6PDH可分为质体型（plastidic, P）与胞质型（cytosolic, Cy）两类同工型。研究进一步通过拟南芥叶肉原生质体的共聚焦显微镜成像，验证了5个GmG6PDH蛋白的亚细胞定位。各GmG6PDH基因在大豆不同组织及种子发育的不同阶段均呈现独特的表达谱。其中胞质型G6PDHs（Cy-G6PDHs）在根、发育中种子与根瘤中呈高表达，而质体型G6PDHs（P-G6PDHs）的转录本主要在绿色组织中被检测到。此外，盐胁迫、渗透胁迫与碱胁迫等多种逆境处理均可显著诱导GmG6PDHs的酶活性与转录本积累。值得注意的是，胞质型同工型GmG6PDH2在盐胁迫下的表达量显著升高，且与G6PDH酶活性呈显著正相关，提示其可能是大豆响应盐胁迫的关键调控因子。在大肠杆菌（Escherichia coli）中异源重组表达的GmG6PDH2蛋白的酶学实验证实，该基因编码的酶具备依赖NADP+的功能性G6PDH活性。进一步分析显示，过表达GmG6PDH2可通过调控抗坏血酸-谷胱甘肽库的氧化还原状态，抑制活性氧（reactive oxygen species, ROS）的生成，从而显著提升转基因大豆的耐盐性。综上，本研究结果表明，GmG6PDH2可能是戊糖磷酸途径中负责NADPH合成的主要同工型，通过调控细胞内的AsA-GSH循环以抵御高盐胁迫引发的氧化损伤。