Response Of MhCLC-c1 And MhCLC-e2 To NaCl And Nitrogen Deficiency Stress In Malus Hupehensis

Apple is the fruit crop with the largest cultivated area and the highest yield in northern China.Soil salinization and inappropriate application of N fertilizer are major factor to apple production.Soil salinization is primarily due to NaCl.Under NaCl stress,in addition to Na⁺,Cl^-also plays an important role.Chloride channels are anionic transport proteins,which not only regulate Cl^-transport,but also mediate other anions（such as NO₃^-）transport.In this study,MhCLC-c1 and MhCLC-e2,were amplified from Malus hupehensis on the basis of 9CLC gene family members in apple.Their transgenic apple calli and MhCLC-c1overexpressed Arabidopsis thaliana were obtained.And then response of MhCLC-c1 and MhCLC-e2 to NaCl and nitrogen deficiency stress was investigated.The main findings are as follows:1.Bioinformatics analysis of CLC gene in appleIn this study,nine CLC genes were identified in the Malus×domestica genome,which were located on 6 chromosomes,and the number of exons ranged from 4 to 22.Md CLCs promoters existed many cis-acting elements associated with abiotic stress and hormones.Md CLCs were located in plasma membrane and categorized into two subclasses,with subclass I containing conserved regions Gx Gx PE,GKx GPxx H and Pxx Gx LF except Md CLC-c2 whereas subclass II not.There were 5 to 11 transmembrane regions in 9Md CLCs.2.MhCLC-c1 responds to NaCl stress but not to nitrogen deficiencyThe MhCLC-c1,chloride channel gene,was cloned from Malus hupehensis c DNA.MhCLC-c1 encoded 778 amino acids.we constructed overexpression and antisense suppression vector of MhCLC-c1 and transformed them into apple calli and Arabidopsis thaliana.Under NaCl stress,compared with wild-type,the H₂O₂ and MDA content,cell relative mortality and Cl^-content in MhCLC-c1 overexpressing lines significantly decreased,while for silencing lines,the opposite results were obtained.Subcellular localization demonstrated that MhCLC-c1 protein was localized to plasma membrane.These results indicate that under NaCl stress,Malus may decrease intracellular Cl^-content by upregulating the expression of MhCLC-c1,which could alleviate NaCl stress damage.Under nitrogen deficiency,there was no significant phenotype difference between MhCLC-c1 transgenic apple calli and wild-type,indicating that MhCLC-c1 does not respond to nitrogen deficiency.3.MhCLC-e2 responds to NaCl stress and nitrogen deficiencyThe MhCLC-e2,chloride channel gene,was cloned from Malus hupehensis c DNA.MhCLC-e2 encoded 786 amino acids.Three MhCLC-e2 overexpressing apple calli were obtained.Under NaCl stress,the growth and fresh weight of three MhCLC-e2 overexpressing apple calli were worse than those of the wild-type,indicating that MhCLC-e2 overexpressing apple calli were sensitive to NaCl.Under nitrogen deficiency,the growth of three MhCLC-e2overexpressing apple calli were better than the wild-type.Nitrate nitrogen,nitrous nitrogen,ammonium nitrogen and total nitrogen contents were lower than the wild-type,while amino acid content was higher than the wild-type.These results suggest that MhCLC-e2 may promote apple calli growth by influencing nitrogen content under nitrogen deficiency.