Effects Of Autophagy On The Programmed Cell Death In Wheat Root Cells Under Waterlogging Stress

Autophagy is the main catabolic pathway for the survival of eukaryotes,which enables the cells to maintain homeostasis under stress conditions.Programmed cell death(PCD)is an orderly cell death process strictly controlled by genes.Previous studies in this laboratory showed that wheat(Triticum aestivum L.)accumulated reactive oxygen species(ROS)in root cells in the early stage of waterlogging stress,which in turn induced autophagy and PCD.However,it is unclear how autophagy affects PCD,whether the mechanisms of root cortex and stele cells responding to waterlogging stress are the same,and how the mechanisms of different wheat cultivar responding to waterlogging stress are different.In this study,the moisture-tolerant wheat cultivar Huamai 8 and moisture-sensitive wheat cultivar Huamai 9 were used as materials,using techniques such as cell biology and molecular biology to explore the mechanism of autophagy affected PCD of wheat roots under waterlogging stress,differences in the expression levels of autophagy-related genes,PCD levels and antioxidant capacity in roots cortex and stele cells,as well as differences in autophagy,PCD,ROS and antioxidant enzymes in wheat cultivar with different moisture-tolerant in response to waterlogging stress.The research results are as follows:1.With the extension of waterlogging time,the length,fresh weight,vitality and activity of the roots gradually decreased compared with the control,reaching the minimum value at 48 h.2.The expression levels of Ta ATG genes showed a trend of increasing and then decreasing in wheat root cells than those in the control group.The expression levels of Ta ATG8 a gene reached a peak at 12 h,while other genes all reached a peak at 24 h.3.Waterlogging stress induced morphological changes such as cell nucleus deformation and nucleocytoplasmic concentration,up-regulation the expression of metacaspase genes and the activity of caspase-like proteases,which reached a peak at24 h or 36 h.During 24 h-48 h of waterlogging stress,the PCD characteristics such as DNA laddering bands and TUNEL-positive nucleus appeared in root cells,so waterlogging stress induces the occurrence of PCD in wheat roots.4.Autophagy inducer rapamycin(RAPA)enhanced the activity of SOD and the expression levels of Ta ATG genes in root cells,but the expression levels of metacaspase genes,activity of caspase-like proteases,the degree of nuclear morphologic changes,and PCD(the number of DNA laddering bands and the percentof TUNEL-positive nucleus,the same below)levels were decreased.While the effect of inhibitor 3-Methyladenine(3-MA)on root cells is opposite to that of inducer.5.After waterlogging stress,the PCD related levels are higher than those in stele cells,while the expression levels of autophagy-related genes and antioxidant enzyme genes,the activity of antioxidant enzymes and hypoxic-related enzymes were lower than those in stele cells.This indicated that under waterlogging stress,the antioxidant capacity of root cortex cells was lower than that of stele cells,and the PCD level was higher than that in stele cells.6.The response of different moisture-tolerant wheat cultivars to waterlogging stress was systematically studied,those results showed that: the accumulation of ROS was lower,the expression levels of metacaspase genes,the changes of nuclear morphology,the activity of caspase-like proteases and the levels of PCD than those of Huamai 9,while the expression levels of autophagy and antioxidant enzyme genes,activity of antioxidant enzymes and hypoxic-related enzymes,the vitality of antisuperoxide anion,the growth potential,vitality,activity and the soluble protein content of Huamai 8 were higher than those of Huamai 9.