Functional Verification Of Suaeda Salsa SsClcc In Arabidopsis Thaliana

Suaeda salsa L.is an annual euhalophyte with high salt tolerance,which is related to its special salt-tolerant mechanism.The psecies can maintain Na⁺homeostasis through certain ion transporters?e.g.,SsSOS1 and SsNHX1?,but there are few reports in maintaining Cl^-homeostasis in the species.In the current study,the function of Suaeda salsa SsCLCc was verified in Arabidopsis thaliana in physiological and molecular levels to investigate whether the gene plays a role in maintaining Cl^-homeostasis.1.Subcellular localization of SsCLCc proteinAfter the epidermis of the tobacco leaves were treated with 5%saline solution,the vacuolar membrane separated from plasma membrane was observed under a two-photon laser scanning confocal microscope.The entire structures of epidermal cells transfected with pCAMBIA1300-35S-sGFP presented green fluorescence.Only the vacuolar membrane in epidermal cells of SsCLCc transgenic tobacco leaves presented green fluorescence,indicating that SsCLCc was localized on the vacuolar membrane.2.Screening of Arabidopsis AtCLCc mutants and SsCLCc transgenic homozygous plants of ArabidopsisAs S.salsa has no regeneration system,heterologous expression was used to verify the function of SsCLCc.The Arabidopsis clcc mutant SALK₁15644c was purchased from TAIR and identified by the double primer method.Meanwhile,the CDS region of SsCLCc was ligated to the expression vector pCAMBIA1300,and SsCLCc overexpression positive plants?OE?and complement lines?C?were obtained.Transgenic seedlings of Arabidopsis were screened by hygromycin-resistance method.After identification in molecular level,homozygous plants were obtained.3.Functional verification of SsCLCc in ArabidopsisWild-type WT,mutant SALK₁15644c,overexpression homozygous plants?1-200-OE1,4-59-OE2?,and complement lines?3-69-C1,18-17-C2?were sown in small pots filled with nutrient soil.After 3 weeks,seedlings of similar growth were transplanted.After they were treated with 0 and 100 mM NaCl for 7 days,physiological indexes of salt tolerance were measured.Salinity?100 mM NaCl?had no significant effect on the germination and germination potential in different Arabidopsis lines,but had significant effects on the taproot length and the number of lateral roots in Arabidopsis seedlings,i.e.,under 100mM NaCl treatment,the length of the taproot of the overexpression homozygous line was the longest,followed by the wild type and complement lines,while the value in the mutant line clcc was the shortest.The number of lateral roots and fresh dry weight were consistent with the result of root length.During seedling stage,salinity increased the content of Cl^-in the leaves and roots of different Arabidopsis lines after seedlings were treated with NaCl for 1 and 7 days.The content of Cl^-in the leaves and roots of overexpression lines was significantly higher than that of other plants,and the value in the mutant line was the lowest.The gene expression was upregulated under salinity.This indicates that SsCLCc may play a role in the process of Cl^-compartmentalization in vacuole.Salinity reduced the content of NO₃^-in the leaves and roots,but there was no significant difference in the content of NO₃^-among different Arabidopsis lines after seedlings were treated with NaCl for 1 and 7 days.This indicates that SsCLCc should not have the function of transporting NO₃^-.At 100 mM NaCl,the relative conductivity of the mutant plants was higher than that of other lines,and the overexpression line was the lowest.This indicates the membrane integrity of the mutant plants was severely damaged,while the membrane integrity of the overexpression plants can be well maintained.The above results indicate that S.salsa SsCLCc may participate in the compartmentalization of Cl^-and improving salt tolerance of transgenic plants.