Cloning And Functional Analysis Of Cyclic Nucleotide Gated Channels In Arabidopsis Thaliana

Soil salinity results in the decreasing yields of crops. Although scientists have kept on studying salt stress for several decades, how Na+influx into plants is still unclear so far. Early experiment results showed there were at least three pathways for Na+ influx into plants, two were mediated by proteins and could be distinguished by the sensitivity to Ca2+ and the last is mediated by apoplast. NSCCs may involve in Ca2+-sensitive Na+ influx among the discovered ion channels/transporters. In plants, CNGCs and GLRs may be the good candidates for NSCCs.There’re 20 CNGCs in Arabidopsis genome. They share the similar sequences and structures:6 transmembranes are at N terminal and the P-loop locates at the fifth and the sixth transmembranes, while the hydrophilic C terminal in cytoplasm has cyclic nucleotide binding domain (CNBD) and calmodulin binding domain (CaMBD) and regulated by binding with Ca2+or cNMP. There’re some evidences pointing out that CNGCs have taken part in salt tolerance. Expression levels of CNGC19 and CNGC20 in shoots were up regulated under salt stress compared with WT, and other CNGCs such as CNGC1, CNGC3 and CNGC4 were permeable to Na+ when expressed in heterologous expression systems.To investigate the relationships between salt tolerance and CNGCs, we cloned 9 CNGCs:CNGC2, CNGC4, CNGC5, CNGC6, CNGC7, CNGC8, CNGC10, CNGC13 and CNGC16 and tested Na+ permeability using yeast complementary. We found CNGC8, CNGC10 and CNGC13 permeable to Na+ in yeast. We also obtained T-DNA insertion mutants of these genes from TAIR and got the homozygous mutant lines.4-day old seedlings treated with 200 mM NaCl for 4days, cngc10 and cngc16 showed salt tolerance than WT. based on these results and the highest similarity between CNGC10 and CNGC13, we chose CNGC10 and CNGC13 for further studies.Hydrophobic profils of CNGC10 and CNGC13 were analyzed by TMpred. The prediction results showed they were quite close to the family members and both had 6 transmembranes, P-loop located at the fifth and sixth transmembranes. In the plasma of onion epidermis expressing CNGC10::GFP and CNGC13..GFP, green fluorescence could be detected mainly in the plasma. And this confirmed the prediction result that CNGC10 and CNGC13 were membrane targeted.Tissue relative expression of CNGC10 and CNGC13 were investigated in flowering plants. CNGC10 was mainly expressed in root and less in stem, flower and leaf, while CNGC13 had a strong expression level in flower and root, less in stem and leaf. More details were detected using promoter::GUS transgenitic plants. In young seedlings, blue stains could be detected in the whole plants except the root tips in both transgenitic plants. In mature plants of promoter10::GUS transgenitic plants, blue stains only could be detected in roots (root hairs, epidermis, cortex and endodermis) and little in flowers while in mature plants of promoter 13::GUS transgenitic plants blue stains could be detected in flowers and senescence leaves.To investigate the functions of CNGC10 and CNGC13 in plants, we got transgenic plants over-expressing CNGC10 or CNGC13 (OE) and the RNA expression levels of the T-DNA insertion mutants (KO) and the OE lines of the two genes were detected using qPCR. Results show CNGC10 or CNGC13 could be detected little in KO while the RNA levels were up regulated by 3 times or 7 times respectively in OE. Under salt stress, the expression level of CNGCIO and the promoter activity of CNGC10 were dramatically decreased, while CNGC13 was not response to salt stress. Germinating KO, OE and WT on 1/2 MS containing 200 mM NaCl, we found cngclO was more tolerance to Na+and OE was more sensetive. cngclO had higher survival rates than WT under salt stress and accumulated less Na+ and K+ in hydroponic plants after salt treatmeat. And the complementary lines based on cngclO background restored the sensitivity to salt stress. All genotypes of CNGC13 had no significant difference under salt stress.Then we tested ion permeability of CNGC10 and CNGC 13 more detail using yeast complementary experiments. CNGC10 was permeble to Na+, K+, but not Ca2+, while CNGC 13 was permeable to Na+, K+ and Ca2+ when tested in sodium sensitive strains B31 or potassium uptake defective strain CY162. Considering the CNBD and CaMBD, additional Ca+ or/and di-cAMP (a membrane permeable analogue as cAMP) was also tested, but little affection had found on the growth of the yeasts. Because of contradiction between the high similarity of two proteins (the differences were mainly at p-loops and the C-terminals) and the quite different permeabilities, we mutated the P-loop of CNGC10 and also exchanged the C-terminal of CNGC13 with CNGC10 and found permeabilities were affected more by C-terminal but not P-loop.Besides, we set up voltage clamp for further studying CNGCs. We expressed AtKAT1 in oocytes as positive control and recorded the sodium currents mediated by CNGC13 in oocytes in 100 mM NaCl (2 mM KC1) bath solution. We found CNGC13 had activity in very wide range of voltage and could mediate inwardly currents under hyperpolarized command and CNGC13 is not sensitive to voltage until hyperpolarized more than-100 mV. We confirmed the inwardly current was mediated by Na+ and K+ together through tail current analysis.Above all, although CNGC10 and CNGC13 shared the highest similarity and both permeable to Na+ in yeast, CNGC10 was negatively regulated salt tolerance in Arabidopsis while CNGC13 might not directly involved in salt tolerance due to their diffenent expression patterns.