Identification And Preliminary Functional Characterisation Of Genes Regulating Root To Shoot Transport Of Chloride In Arabidopsis

Chloride ions(Cl^-)are common to be observed in the natural environment.As a micro-nutrient necessary for plant growth and development,Cl^-participats in the regulation of pivotal biological processes such as photosynthesis,stomatal movement and cell turgor maintenance.However,under salt stress conditions,the excessive accumulation of Cl^-in plants often causes Cl^-toxification(especially for Cl^-sensitive plants).On the other hand,when Cl^-is insufficient for plants,the expansion of plant leaf cells is positively correlated with the accumulation of Cl^-.In our group,through the use of CRISPR/Cas9 editing and genetic pyramiding,the two known key pathways(NPF2.4 and SLAH1/3)that mediate xylem loading of Cl^-in Arabidopsis were knocked out.In this study,the transcriptom of the npf2.4 slah1 slah3 mutant was studied,through which the candidate genes involved in the long-distance transportation of Cl^-from root to shoot were identified and preliminarily characterized.It is found that the accumulation of Cl^-in the shoot of hydroponically grown npf2.4 slah1 slah3 decreased to 60%of wild type.Moreover,the cell size of mutant leaf epidermal cells increased significantly when compared with control under the condition of Cl^-deficiency.Transcriptomics analysis identified 20 genes that were differentially expressed in npf2.4slah1 slah3 compared to wild-type.The candidate genes such as ICln,NPF2.5 and AT4G27654 encoding membrane proteins were expressed heterologously in yeast to unveil their transport activity to Cl^-.Using Quantitative PCR to study the transcriptional responses of candidate genes to Cl^-deficiency and Cl^-stress,it was found that ALMT12and CCC1 were significantly induced under Cl^-deficiency conditions,however,they didn't respond to Cl^-stress.It is therefore suggested that ALMT12 and CCC1 may regulates the accumulation of Cl^-in reponse to low Cl^-conditions.At the same time,it was found that NPF2.3 and NPF2.5 did not respond to Cl^-stress in the wild-type background,but were significantly induced in the mutant background,suggesting that they might compensate the lost function of NPF2.4 and SLAH1/3 to regulate Cl^-transport.In future,more in-depth functional analysis of ALMT12,CCC1,NPF2.3 and NPF2.5 is expected to reveal new mechanisms underlying the regulation of Cl^-uptake,transport and accumulation.