Functional Study Of The TWEETY Family Homologous Genes In Arabidopsis Thaliana

Chloride is an essential micronutrient for plant growth and has several functions in plant metabolism.Cl' is not only an indispensable cofactor in enzyme activation and photosynthesis,but also acts as a counterion for cation transport,which plays an important role in stabilizing membrane potential,maintaining charge balance,regulating osmotic stress and stomatal movement.Fertilizer application and seawater erosion can easily deposit excessive chloride ions in the soil.The high concentration of Cl-in the tissue lead to atrophy and necrosis of the leaves,which limits the growth of various sugar plants.Therefore,there must be a series of transport mechanisms in plants to maintain the homeostasis of chloride ions.In this paper,several homologous genes of the large-conductivity chloride channel TWEETY family in Arabidopsis were selected as candidate genes for chloride transporters,and their functions in chloride transport were investigated.The main findings are as follows:(1)A potential new chloride channel TWEETY family exists widely in plantsThrough the electrophysiological experiments of CHO cells,the homologous genes TTYH1,TTYH2 and TTYH3 of the TWEETY family in mammals were identified as novel Cl-Channels with large conductivity.This family has not been discovered in plants,so we selected the TWEETY family as a candidate gene for plant Cl-transporters.By proteomics,we found four homologous genes of the TWEETY family in Arabidopsis:AtTTYH1,AtTTYH2,AtTTYH3,and AtTTYH4.Through bioinformatics analysis,it was found that the TWEETY family of Arabidopsis thaliana had 37.11%sequence similarity with the mammalian TWEETY family.AtTTYH1,AtTTYH2 and AtTTYH4 are all predicted to be a multi-transmembrane protein with a 6transmembrane structure.After BLAST with the protein sequence of AtTTYH1,we found that the TWEETY family has a relatively broad distribution in plants.The presence of AtTTYH1 homologous protein was found in important cash crops such as soybean,tobacco and cotton.The functional research of the TWEETY family has important potential application value.(2)AtTTYH1 localizes to the plasma membrane,while AtTTYH2 and AtTTYH4 localize to the cytoplasmTo explore the subcellular localization of the TWEETY family in Arabidopsis thaliana,we constructed three recombinant plasmids with green fluorescent protein,AtTTYH1-GFP,AtTTYH2-GFP and AtTTYH4-GFP.After transfecting Arabidopsis protoplasts,it was observed that AtTTYH1 was expressed on the plasma membrane and colocalized with plasma membrane Marker SCAMP-RFP.AtTTYH2 and AtTTYH4 are similar to empty GFP in cells and localize in the cytoplasm.(3)AtTTYH1 is involved in plant salt stress adjustmentTo explore the function of the TWEETY family in Arabidopsis,we obtained TDNA insertion mutants of AtTTYH1,AtTTYH2 and AtTTYH4 from the laboratory seed bank,and obtained double mutants of ttyh1*ttyh4 and ttyh1*ttyh2 by hybridization.By treatment with different concentrations of NaCl,we found that the ttyh1mutant grew worse under 150mM NaCl conditions.The ttyh1 mutant and the ttyh1*ttyh4 double mutant phenotype a more severe growth inhibition phenotype under the same concentrations of KCl and NaCl.Thus,the phenotype sensitive to salt stress in the ttyh1 mutant is associated with excess Cl-in vivo,and this phenotype is due to ttyh1 mutations.We also observed the expression of AtTTYH1,AtTTYH2 and AtTTYH4 after NaCl 150mM treatment at different times.The results showed that the expression of the three genes increased significantly after NaCl treatment,and the results of GUS staining also confirmed this result.Therefore the TWEETY family may be associated with salt stress in plants.(4)AtTTYH1 participates in stomatal aperture adjustment in response to drought stressAtTTYH1 sensitive to salt stress and its localization on the plasma membrane,we preliminarily concluded that AtTTYH1 may be an outward Cl-transporter.Based on previous studies of chloride ions in plants,outward transporters are likely to be involved in stomatal movements in plants.In order to validate our hypothesis,we performed dehydration rate determination and drought treatment experiments on different mutants.Both the ttyhl mutant and the ttyh1*ttyh4 double mutant were found to exhibit a higher rate of water loss,almost 1.5 times that of the wild type.After drought treatment,the ttyhl mutant showed more obvious growth inhibition,the leaves dried up and turned yellow and even died.This confirms our previous hypothesis that AtTTYH1 may be an outward Cl-transporter.AtTTYH1 mutation leads to an increase in Cl-content in the body,the guard cells absorb water and expand,the stomatal opening increases.Therefore,the ttyhl mutant has an increased rate of water loss and is more sensitive to drought.In conclusion,a latent Cl-transporter localized on the plasma membrane was initially locked by studying the functions of several genes in the TWEETY family of Arabidopsis thaliana.AtTTYH1 is involved in the process of salt stress and drought stress in plants,and may regulate stomatal movement by regulating the efflux of Cl-in guard cells.This paper provides a new solution for plant salt stress and chloride ion transport research,enriches the theoretical basis of chloride transporter in plants,and has important guiding significance for agricultural production.