Pyrimading The Knockout Of Two Pathways Of Cl~? Xylem Loading In Arabidopsis

Excessive Cl~?accumulation under salt stress is toxicity to plants.The molecular mechanism of how Cl~?transport is regulated in response to salt stress is largely unknown.Previous studies have shown that NPF2.4 and SLAH1/SLAH3 mediate xylem loading of Cl~?in the root,regulating the accumulation of Cl~?in the shoot in response to salt stress.Due to the lack of T-DNA insertion mutants for both the two genes,CRISPR/Cas9 was used to knock out the expression of NPF2.4 and SLAH1 in the current study.Furthermore,homozygous npf2.4 slah1 double mutants and npf2.4slah1 slah3 triple mutants were generated by crossing.The single mutants npf2.4(1,2,4)and slah1-2 were shown to have lower Cl~?accumulation in the shoot in soil-grown systems.More importantly,it was shown that the reduction of Cl~?accumulation was even larger in double mutants of npf2.4 slah1.When treated with200 mM NaCl in soil-grown system,the Cl~?content in the shoot of the double mutant npf2.4-2 slah1 and npf2.4-4 slah1 was reduced by 55%and 33%,respectively,compared to the control.The results showed that the regulation of Cl~?content in the shoot by knocking out the two Cl~?xylem loading pathways of NPF2.4 and SLAH1may have an addiditve effect.Subsequently,RNAseq will be performed using the double mutants and triple mutants to identify novel genes involved in the regulation of Cl~?accumulation in the shoot under salt stress.The gene expression of both NPF2.4 and SLAH1 was previously shown to be down-regulated by salt stress.However,NPF2.4 gene expression was shown strongly up-regulated by salt stress(75mM NaCl)in the background of the mutant(pyr1 pyl1 pyl2 pyl4 pyl5 pyl8,aba6m)lacking ABA receptors.This indicates that the expression of NPF2.4 is under tight control of ABA signaling pathway.In contrast,SLAH1 gene expression was still strongly inhibited in the background of aba6m,suggesting that inhibition of NaCl is independent of the ABA signaling pathway.In addition,one gene-editing mutant,npf2.4-4,showed abnormality in the growth and development,such as an increase in the size and weight of seeds,changes in leaf shape and chlorophyll concentration,serrated leaf margin,changes in the number of trichome bifurcations and delayed flowering time.This could be the reason of non-specific gene editing or disruption of unknown endogenous gene due to the insertion of the transformed construct,therefore needs to be further investigated.