The Research Of Reactive Oxygen Species Regulation The Fe Deficiency Response In Malus Xiaojinensis

Iron?Fe?,an essential micronutrient required in plant,is an integral part of the mitochondrial and chloroplast electron transport chains.Fe deficiency results in an imbalance of cellular redox and change of ROS content,which plays an critical role in growth and development.Until now,it is not clear how ROS mediates Fe deficiency,so it is necessary to identify the role of ROS in response to Fe deficiency in Fe-efficient woody plant.We analyzed the ROS,H₂O₂ content and ROS scavenging enzyme activity both in the iron efficient genotype Malus xiaojinensis and the inefficient genotype Malus baccata under iron deficiency.Fe deficiency adaptive responses were detected by exposure to diphenyleneiodonium?DPI?at 9 hours and exposure to H₂O₂ at 9 days under iron deficiency.Screening the ROP gene which in consistent with ROS content from ROPs gene family in M.xiaojinensis and M.baccata under Fe deficiency.The main results were as follows:The ROS content and the activity of ROS scavenging enzymes of roots,like CAT,increased dramatically at 9 hours in M.xiaojinensis but there is no significance in roots of M.baccata under iron deficiency.ROS scavenging enzymes activity levels quantified in roots like CAT,POD and SOD,exposed to Fe deficiency were higher at day 9 in M.xiaojinensis than those in M.baccata,while the ROS content was lower in M.xiaojinensis than in M.baccata.Fe deficiency adaptive responses were markedly repressed by exposure to diphenyleneiodonium?DPI?,which can block ROS production.DPI application strongly inhibited total CAT,POD,SOD enzymes activity and the Fe deficiency-induced reductase activity and proton extrusion at 9 hour under iron deficiency.H₂O₂ application experiments were performed in order to reveal the roles of ROS in the response of M.xiaojinensis to prolong Fe deficiency?9 d?stress.Through the clustering analysis of MdROP gene family,we discovered that MdROP1,MdROP8,MdROP9 clustered together with AtROP6.More over,MdROPl was the nearest gene with AtROP6.Futher,we found that the expression of MdROPl gene increased at 9 hours in roots of M.xiaojinensis,but that was not affected in roots of M.baccata,at day 9.Gene expression in roots of M.baccata were higher than that in M.xiaojinensis during Fe deficiency.which was in consistent with ROS content.M.xiaojinensis's and M.baccata's MdROP1 genes were aligned and had 594 bp and 198 amino acid sequences in their ORFs.The pSuper1300::MxROP1::GFP vector was constructed and used for tomato protoplast transformation and GM wild type?WT?Arabidopsis plants.MxROP1 was located at cytomembrane,The ROS content,chlorophyll content,root amount and FCR activity in overexpression MxROP1?OE-1 and OE-2?plants were increased than those in WT plant under Fe deficiency and the OE-1,OE-2 plant leaves,which were greener than WT.Overexpression MxROPl up-regulated the expression of AtRBOHD,AtRBOHF and Fe absorption related genes.It showed that MxROP1 promoted the ROS production and Fe deficiency-response and enhanced the roots uptake Fe ability.Overall,this study makes a primary exploration on the mechanism in which ROS mediates iron deficiency stress,and ROS plays a positive or negative role in the different phrases during iron deficiency.M.xiaojinensis and M.baccata showed different iron uptake effects to respond iron deficiency:the efficiency of early ROS production and late ROS scavenging were different.Under iron deficiency stress,the ROS content increase along with the elevated expression of MdROPl.MxROP1 induced ROS production and the expression of iron-related genes,then enhanced Fe deficiency responses.