Cloning And Functional Analyses Of AtSLAH3Homologous Genes In Maize And Tomato And Screening Of Regulatory Components Of AtSLAH3

Slow anion channels(SLAC/SLAH)are a class of anion channel proteins in plants and microorganisms.SLAC/SLAH mediate efflux of various anions such as chloride and nitrate.In Arabidopsis,the SLAC/SLAH family contains five members,including AtSLAC1(SLOW ANION CHANNEL ASSOCIATED1),and AtSLAH1 to 4(SLAC1 Homologue 1 to 4).The function of AtSLAC/SLAH involves plant growth,development,and stress adaptations.Previous study of our group has shown that AtSLAH3 is involved in the responses to high ammonium stress and high potassium stress in Arabidopsis.Through bioinformatics analysis,we found that the homologous genes of AtSLAH3 is ubiquitously exist in plants.In order to investigate the function and potential application of AtSLAH3,we utilized monocotyledonous plant maize and dicotyledonous plant tomato as materials and cloned 11 homologous genes of AtSLAH3 from maize(ZmSLAH3-Like,ZmSLAH3L)and 7 homologous genes of AtSLAH3 from tomato(SlSLAH3-Like,SlSLAHL).The paralog genes of AtSLAH3 in maize and tomato were heterogeneously expressed in slah3-5 mutant background.We found that the expression of ZmSLAH3L2,ZmSLAH3L3,ZmSLAH3L4,ZmSLAH3L5,ZmSLAH3L6,ZmSLAH3L7,ZmSLAH3L8,ZmSLAH3L9,ZmSLAH3L10,ZmSLAH3L11,SlSLAH3L1,SlSLAH3L3,SlSLAH3L4,or SlSLAH3L6 can partially or fully recover the phenotype of slah3-5 under high ammonium stress condition.In addition,the expression of ZmSLAH3L8,ZmSLAH3L10,ZmSLAH3L11,SlSLAH3L1,or SlSLAH3L6 can recover the phenotype of slah3-5 under high potassium stress condition.The genetic complementation experiments thus indicate the conserved functions of AtSLAH3 homologous genes in maize and tomato.We next ordered and identified a number of maize mutant lines that bear point mutations in ZmSLAH3 L causing stop condon.By analyzing the phylogenetic tree and the expression patterns,we preliminarily identified that ZmSLAH3L6 and ZmSLAH3L8 are the closest paralogs of AtSLAH3 in maize.Interestingly,we found a natural point mutation in the amino acid 510 of ZmSLAH3L8.The conserved F(Phenylalanine)is substituted by S(Serine),which potentially turns ZmSLAH3L8 into a continuously activated channel.To investigate the functions of ZmSLAH3L8,the overexpression and electrophysiological vectors of ZmSLAH3L8 have been constructed for further studies.Leucine-rich repeat receptor-like protein kinase(LRR-RLK)are a group of cell surface-localized proteins,mediating signal transduction from external ligand molecules into intracellular signaling pathways through phosphorylation cascades.Previous studies have shown a novel regulatory pattern in the Arabidopsis that LRRRLK can directly phosphorylate and activate ion channels in response to various signals.In order to test whether AtSLAH3 is also regulated by a similar pattern,we cloned 161 LRR-RLK in a yeast system.By using AtSLAH3 as the bait protein,we screened the AtSLAH3-inertacting proteins via a mbSUS yeast system.We found that seven LRRRLK including RLK4,RLK6,RLK106,RLK108,RLK122,RLK156 and RLK169 are able to interact with AtSLAH3.The detailed mechanisms that how RLK regulate AtSLAH3 will be further investigated in future studies.