Physiological And Molecular Mechanisms Of Winter Wheat TaG6PDH And Ta6PGDH In Response To Cold Stress

Wheat(Triticum aestivum)is one of the important food crops in China.Its high yield and stable production are extremely important for national food security and sustainable agricultural development.Cold stress is a major abiotic stress that can seriously affect the growth and development of wheat,thereby reducing its yield.Northeast Agricultural University has independently cultivated the strong cold-tolerant winter wheat variety “Dongnongdongmai1”(Dn1)(returning rate ?85%),which can overcome the frigid climate conditions in Heilongjiang region.It is important to explore its cold resistance mechanism.The eukaryotic pentose phosphate pathway(PPP)is a major source of NADPH and provides a metabolic intermediate for biosynthesis.When a plant is subjected to abiotic stress,the PPP pathway maintains the redox balance of the cell.Therefore,this pathway is a key pathway for plants to resist abiotic stress.Glucose-6-phosphate dehydrogenase(G6PDH,EC.1.1.1.49)and 6-phosphogluconate dehydrogenase(6PGDH,EC.1.1.1.44)are key enzymes in the pentose phosphate pathway.In this study,Dn1 grown in field under natural cooling condition was used as the basic material and the gene expression levels and enzyme activities of TaG6PDH and Ta6PGDH in the tillering node and leaf were measured at 5°C,0°C,-10°C and-25°C.The TaG6PDH and Ta6PGDH genes were isolated from Dn1 and overexpressed in Arabidopsis to explore their physiological and molecular mechanisms in response to cold stress.The experimental results are as follows:(1)Bioinformatics analysis showed that the winter wheat TaG6PDH and Ta6PGDH proteins were localized in the cytoplasm and closely related to Uraltu wheat and goat grass.Both proteins contained a substrate binding site and a NADP+ binding site.(2)The expression levels of TaG6PDH and Ta6PGDH increased gradually in tillering node and leaf of winter wheat and the folds of gene expression level in tillering node was higher than that in leaf under cold stress.Exogenous ABA significantly increased the expression levels of TaG6PDH and Ta6PGDH under cold stress.(3)The activities of TaG6PDH and Ta6PGDH peaked at-10°C and decreased to the control level at-25°C in the winter wheat tillering node.Under cold stress,the activities of the two enzymes did not increase significantly in leaf,but the activities decreased sharply when the temperature dropped to-25°C.Exogenous ABA significantly increased the activities of TaG6PDH and Ta6PGDH under cold stress.(4)Overexpression vectors of TaG6PDH and Ta6PGDH were successfully constructed and Arabidopsis overexpressing plants were obtained.(5)Arabidopsis were subjected to indoor cold stress(4°C,0°C,-10°C),and overexpressing plants of TaG6PDH and Ta6PGDH were able to return to normal growth state after cold stress.The results of physiological indicators showed that the overexpressed plants had higher chlorophyll content,lower MDA content and injury rate than the wild type and mutant at the same low temperature.DAB and NBT staining results also showed that overexpressing plants were able to produce less reactive oxygen species(ROS)under cold stress,thereby reducing the damage of oxidative stress on plants.(6)The expression levels of TaG6PDH and Ta6PGDH in Arabidopsis overexpressing plants increased significantly at 0°C and-10°C under cold stress and the expression level of endogenous At6 PGDH gene in TaG6PDH overexpressing plants was significantly higher than that in wild type.Similarly,the expression level of endogenous At G6 PDH gene in Ta6PGDH overexpressing plants was also significantly higher than that in wild type under cold stress.(7)The activity of G6PDH in TaG6PDH overexpressing plants was significantly higher than that in wild type and mutant at 0 °C and-10 °C.Similarly,the 6PGDH activity in the Ta6PGDH overexpressing plants was also significantly higher than that of wild type and mutant.This study preliminarily confirmed that the winter wheat cytoplasmic TaG6PDH and Ta6PGDH responded positively to cold stress.The TaG6PDH and Ta6PGDH can enhance the low temperature tolerance of Arabidopsis.The experimental results provide a theoretical basis for the analysis of Dn1 cold resistance mechanism and also lay a theoretical foundation for winter wheat cold resistance molecular breeding.