Investigation On Mechanisms Of Amino Acid Transporter AAT1 Regulating Root Elongation In Arabidopsis Thaliana Under Salt Stress

Roots play an extremely important role in plant growth and development.Root growth is regulated by many adverse factors such as high salinity and drought.Under salt stress,the growth of the primary roots is inhibited,and the whole plant development is disrupted.The contents of some amino acids in plants changed significantly under saline condition,which affected the development of primary roots.Amino acids are essential for life processes.Amino acid transporters transport amino acids to each part of plants and have a strong effect on plant growth and development.However,it has not been reported whether amino acid transporters play a role in regulating the development of primary roots under salt stress,and the underlying mechanisms are unclear.We found that elongation of the main roots from of the amino acid transporter AAT1?Amino acid transporter 1?gene deletion mutants aat1-1 and aat1-2 was more sensitive than that of wild type?WT?under salt stress.We further studied the expression of AAT1 in tissues,subcellular localization of AAT1 protein,and its role and mechanism in primary root growth under salt stress.The main results are as follows:Permanent transgenic plants expressiong 35S::AAT1-YFP showed that AAT1 was located in the plasma membrane.Gene AAT1 was strongly expressed in the leave,hypocotyl,root and pod by GUS staining.The expression of AAT1 was induced by salt stress.It was found that the length of meristematic zones of aat1-1 and aat1-2 in roots was shorter than that of WT,and the number of meristematic cells was less than that of WT,but there was no significant difference in cell length between WT and the mutants under salt stress.This indicates that disruption of AAT1 inhibites the division of root meristematic cells and shortened the length of meristematic zones under salt stress.The phenotypic differences in root length between aat1 and WT treated with NaCl could be reduced by exogenous application of low concentrations of auxin.Moreover,the primary root growth of aat1treated with NPA?Naphthyl phthalamic acid?was similar to that after imposure to NaCl.qRT-PCR was used to analyze the expression levels of PIN1?PIN-FORMED1?,PIN2,PIN3,PIN4,PIN7 and AUX1?AUXIN1?under NaCl treatment.The results showed that the expression levels of PIN4,PIN7 and AUX1 of aat1 mutants were significantly lower than those of WT under salt stress.The mutant aat1 was crossed with transgenic plants harboring DR5::DR5-GFP or PIN-GFP plants,and their offsprings were obtained,and the roots were observed after treatment with 100 mM NaCl.The GFP fluorescence results showed that loss of function of AAT1 gene caused clear suppression of the expression of auxin output carrier PIN4 and PIN7 under NaCl treatment,further changing the distribution pattern of auxin,and reduced the content of auxin in vivo,thus inhibited the elongation of primary roots.The expression of CYCB1;1?CYCLINB1;1?was analyzed by qRT-PCR.It was found that the CYCB1;1 transcripts of aat1 were fewer than those of WT after treatment with NaCl.GUS staining results reflecting the expression of CYCB1;1 were similar to the qRT-PCR results.These data indicate that knockout of AAT1 generesults in the down-regulation of CYCB1;1 expression,thereby inhibiting the differentiation of meristematic cells under salt stress.The promoter region of AAT1 was analyzed.It was found that transcription factors MYB73 and MYB77 can bind to the promoter of AAT1.The expression levels of MYB73 and MYB77 were studied under NaCl treatment.The results revealed that the transcription levels of MYB73 in aat1 mutant were lower than those of WT after treatment with 100 mM NaCl for 12 h.There was no significant difference in the expression of MYB73 at other treatment time points.However,the expression of MYB77 in aat1 mutant was significantly lower than that in WT after treated with NaCl for 3 h.Using yeast monohybrids and EMSA techniques,we demonstrated that transcription factor MYB77 but not MYB73 could bind to the upstream region of AAT1 promoter,suggesting that MYB77 might be involved in regulating the transcription of AAT1 under salt stress.The mutant aat1 is not sensitive to high concentrations of amino acids.The survival rate of the mutant treated with high concentration of Histidine?His?,Serine?Ser?,Isoleucine?Ile?,Alanine?Ala?,or Proline?Pro?was significantly higher than that of WT,indicating that AAT1 participates in the transport of these amino acids.It was found that proline could partially restore the root elongation of aat1 under salt treatment by exogenous application of appropriate concentration of proline.Meanwhile,the proline content of aat1under NaCl treatment was significantly lower than that of WT,indicating that AAT1 might be involved in transporting proline under salt stress.DAB staining,NBT staining and fluorescence probe method were used to measure the contents of H₂O₂ and O₂.^-in aat1 and WT in the presence of NaCl.It was observed that the levels of H₂O₂ and O₂.^-in aat1 were higher than those in WT,indicating that reactive oxygen species are of importance in mediating AAT1 regulation of elongation of primary roots in Arabidopsis under salt stress.