Potassium Regulates PnPME1 Gene Of P.notoginseng(Burk.) F.H. Chen Under Cadmium Stress Through Brassinolide Signaling Pathway

Cadmium(Cd)is a non-essential element in plants.Its excessive accumulation will lead to slow plant growth and development,inhibition of photosynthesis,oxidative damage to cells and damage to DNA synthesis and replication.Panax notoginseng(Burk.)F.H.Chen is a precious Chinese medicinal material in Yunnan Province.Its special active ingredient,P.notoginseng saponins,can be used to treat cardiovascular and cerebrovascular diseases and bruises.However,due to the high overlap of the plantation area of P.notoginseng and non-ferrous metal mines,P.notoginseng suffered from serious heavy metal Cd pollution,which not only affected its efficacy and quality,but also endangered public health.In this study,by studying the cell wall pectin metabolism in the roots of P.notoginseng under Cd stress,it was found that the accumulation of Cd in the roots of P.notoginseng was closely related to pectin,and that pectin metabolism was regulated by potassium(K)and brassinolide.Based on the differentially expressed genes found in the transcriptome sequencing of P.notoginseng under Cd stress,a new PME gene PnPME1 was cloned from the roots of P.notoginseng by RACE amplification technology(rapid amplification of cDNA ends)and subjected to a series of bioinformatics analysis.The expression characteristics of PnPME1 gene in the roots of P.notoginseng were analyzed using real-time quantitative PCR technology.A transient expression vector pCAMBIA1300-mCherry-PnPME1 was constructed,and the onion epidermal cells were mediated by Agrobacterium tumefaciens.The fluorescence of PnPME1 was observed under a laser confocal microscope to determine its subcellular localization.In addition,the PnPME1 gene was overexpressed into Nicotiana benthamiana,and the function of PnPME1 was studied and verified.The main work and results of this study are as follows:(1)Pectin metabolism experiments on the cell wall of P.notoginseng showed that Cd stress can significantly increase the activity of pectin methyl esterase in the main root,rhizome and rootlet of P.notoginseng and reduce the degree of pectin methyl esterification,which promotes the increase of pectin content and makes a large amount of Cd incorporates in pectin.Under Cd stress,the external addition of brassinolide can further strengthen the pectin metabolism in the root of P.notoginseng and increase the accumulation of Cd.The addition of K can reduce the activity of pectin methyl esterase and increase the degree of pectin methyl esterification,so that the content of pectin is reduced,and the accumulation of Cd in pectin is reduced,which is consistent with the results of exogenous addition of brassinazole(BRz).The addition of K under the simultaneous application of cadmium and brassinolide also reduced pectin metabolism and Cd content.In addition,by measuring the expression of brassinolide pathway related genes and the content of brassinolide in the root of P.notoginseng,it was found that exogenous application of K under Cd stress can reduce the expression of brassinolide synthesis related genes and reduce the brassinolide content.(2)The full-length cDNA and gene sequence of the PnPME1 gene at the root of P.notoginseng were cloned.It was found that the total length of the PnPME1 gene was1,749 bp,including a 1,563 bp ORF(Open Reading Frame),which encodes 520 amino acids.Bioinformatics analysis revealed that PnPME1 is a secreted protein containing an N-terminal signal peptide.The isoelectric point is 9.34.It is hydrophilic and does not contain a transmembrane structure.The secondary structure of PnPME1 containsα-helix(H),β-sheet(E)and irregular curl(L).Multiple sequence alignment showed that PnPME1 belonged to the PMEs family,and its three-dimensional structure was most similar to that of Daucus carota subsp.Sativus PME,showing a cylindrical shape.RT-qPCR results showed that exogenous addition of brassinolide could further increase the expression of PnPME1 under cadmium stress,while exogenous potassium and brassinolide inhibitors significantly down-regulated the expression of PnPME1 under Cd stress.After the fusion expression vector pCAMBIA1300-mCherry-PnPME1 was transferred into LBA4404 to infect onion epidermal cells,a clear red fluorescence appeared on the cell wall of onion epidermal cells under a confocal microscope.The results indicate that PnPME1 is a protein localized on the cell wall.(3)Transgenic tobacco was breed by constructing a plant over-standard vector pCAMBIA2300s-PnPME1,and transducing it into wild-type tobacco.RT-qPCR was used to detect the expression of PnPME1 in transgenic tobacco,and it was found that it could be stably and highly expressed in tobacco.By observing the phenotypes of wild-type and transgenic tobacco,it was found that transgenic tobacco showed stronger growth ability under cadmium stress,K and exogenous brassinolide had a certain regulation and improvement effect on the growth of tobacco lines under Cd stress.The pectin metabolism and Cd content of wild-type and transgenic tobacco were measured.It was found that the transgenic tobacco had higher uronic acid and total sugar content than the wild-type tobacco,which was related to the high expression of PnPME1 gene in transgenic tobacco.And pectin metabolism in the roots of tobacco is regulated by exogenous K and brassinolide.The regulation of exogenous K and brassinolide on PME activity and the degree of pectin methyl esterification was consistent with the trend in P.notoginseng.Transgenic tobacco accumulates more Cd in the root pectin than wild-type tobacco,but their binding and accumulation of Cd are regulated by exogenous K and exogenous brassinolide.