Isolation And Functional Characterization Of Suppressors Of Lks1 In Arabidopsis

As one of the essential macronutrients,potassium(K)plays crucial roles in plant growth and devdopment.Plants absorb K+ from soil through K+ transporters and K+ channels.In Arabidopsis,the Shaker inward K+ channel AKT1 has been considered as one of the most important components involving in root K+ uptake especially under low K+(LK)conditions.Our privous studies showed that the channel activity of AKT1 is regulated by CBL1/9-CIPK23 complexes,as well as by the channel regulatory subunit AtKC1.In this dissertation work,to further explore the detailed mechanism of CBL1/9-CIPK23-AKT1 pathway and identify new components involved in Arabidopsis response to LK stress,suppressors of lks1(cipk23)were screened and identified.A mutant population from EMS-mutagenized lks1-2 plants was screened for sls(suppressor of lks1),and two suppressor mutants(sls1 and sls2)were isolated.The sls1 mutant suppressed the leaf chlorosis phenotype of lks1 under LK conditions.Map-based cloning revealed a point mutation in AtKC1 of sls1,and the mutation resulted in an amino acid substitution(G322D)in the S6 region of AtKC1.The G322D substitution generated a gain-of-function mutation AtKC1D that enhanced K+ uptake capacity and K+ content thus lead to LK tolerance in Arabidopsis,Structural prediction suggested that G322 is highly conserved in K+channels and may function as the gating hinge of plant Shaker K+ channels.Electrophysiological analyses revealed that,compared with AtKC1 wild-type,AtKC1D showed enhanced inhibition of AKT1 activity and strongly reduced K+leakage through AKT1 under LK conditions.In addition,phenotype analysis revealed distinct phenotypes of lks1 and atkcl mutants in different LK assays,but the lks1 atkcl double mutant always showed a LK-sensitive phenotype similar to that of aktl.This study revealed a link between CIPK23-mediated activation and AtKC1-mediated modification in AKT1 regulation.CIPK23 and AtKC1 exhibit distinct effects,and they synergically modulate AKT1 function in Arabidopsis responses to low K+ stress.The sls2 mutant also rescued the leaf chlorosis phenotype of lks1 under LK conditions.There was a point mutation in AKT1 gene in sls2,and the mutation led to an amino acid substitution(D408N)in the CNBD domain of AKT1 C-terminal.The AKTID408N showed K+ transport ability in yeast and could be activated by CBL1-CIPK23 in Xenopus oocytes.The mutant AKT1D408N exhibited significant LK-tolerant phenotype and higher K+ content than that of wild-type plants.In summary,two suppressors of lks1,AtKC1D and AKT1D408N,were identified and functionally characterized.These two point mutations both endow Arabidopsis with enhanced K+ uptake activity and the tolerance to LK stress.The presented results suggest that CIPK23 and AtKC1 may act synergistically to balance K+ uptake/leakage and to modulate AKT1-mediated LK responses in Arabidopsis.The results also indicate that the variations in the key amino acid sequencess of K+channels might be useful for improving crop potassium use efficiency.