A Study On The Mechanisms Of Pectin Demethylated Modification In Responses To Copper Toxicity Of Citrus Root Cell Wall

Copper(Cu)is one of the essential micronutrients for the growth and development of higher plants.Due to the unreasonable application of copper-containing agrochemicals,the excessive Cu in soil and leaves of citrus orchards in southern China was common.Excessive Cu inhibited the growth of citrus plants and reduced the yield and quality of citrus fruits.The root cell wall is the first physical barrier of citrus in response to soil Cu toxicity.Pectin is the primary binding site of Cu on the cell wall of citrus roots.It has been demonstrated that pectin demethylation mediated by PME affects root cell wall Cu fixation.However,the mechanism of pectin demethylation in response to Cu toxicity in citrus root cell walls is unclear.In this study,the seedlings of Citrus grandis(C.grandis)and Citrus sinensis(C.sinensis)were treated with Cu at concentrations of 0.5μM(as control),100 μM(low concentration),300 μM(medium concentration)and 500 μM(high concentration)under p H about 4.3 ～ 4.5.The main content of the present study included:(1)The effects of excessive Cu treatment on the type and content of pectin in citrus root cell walls;(2)The effects of excessive Cu treatment on methyl esterification of citrus root cell wall pectin;(3)The effects of excessive Cu treatment on the expression of PME gene family in citrus roots.The main results are as follows:(1)Compared with the control,300 and 500 μM Cu treatments significantly decreased the biomass of both citrus seedlings,but significantly increased lateral root cell wall content.Under Cu treatment,the TP content of the citrus lateral root cell wall was increased,while no significant difference of WSP content was observed.The contents of CSP and NSP were significantly increased by 500 μM Cu treatment.Moreover,the contents of CSP and NSP of C.grandis were significantly higher than that of C.sinensis.At the same time,with the increase of Cu concentration,the Cu content in CSP and NSP increases significantly,and more Cu accumulates in NSP.(2)The content of LMP in the lateral root cell wall of two citrus species treated with excess Cu was much greater than that of HMP.Compared with the control,the content of LMP was significantly increased at 300 and 500 μM Cu treatments.With the increase of Cu concentration,PME activity in citrus lateral root increased significantly while DPM decreased significantly.At the same Cu concentration,the PME activity of C.grandis was significantly higher than that of C.sinensis,but the esterification degree was significantly lower than that of C.sinensis.(3)According to the published Arabidopsis PMEs sequences,49 PME genes in C.grandis and 53 PME genes in C.sinensis were identified.Bioinformatics analysis showed that PME family proteins were oversecreted to the cell wall in both citrus species.PCR Array analysis showed that 7 PME genes of C.grandis and 4 PME genes of C.sinensis might be the potential genes responding to Cu toxicity under long-term Cu treatment.RT-q PCR analysis showed that the expression of Cgpro PME1,Cgpro PME8,Cgpro PME22,Cspro PME4 and Cs Pro PME22 were triggered by Cu concentration or Cu stress duration under short-term Cu treatment.A relatively higher expression of pro PME22 might contribute to a higher Cu tolerance of C.grandis compared to that of C.sinensis.In conclusion,this study revealed the demethylation mechanism of citrus root cell wall pectin in response to Cu toxicity according to the pectin types and content,pectin methyl esterase activity,pectin esterification degree,and relative expression level of PME genes.The comparison of citrus species showed that the Cu tolerance of C.grandis was stronger than that of C.sinensis,which was related to a lower degree of pectin esterification in the root cell wall.The results of this study will enrich the mechanisms of Cu tolerance in citrus and provide a theoretical basis for revealing the strategies of the citrus stress response.