Functional Analysis Of GhCIPK6 And Its Mechanism In Regulating Sugar Homeostasis

As a second messenger,calcium plays vital roles in plant development and response to environment.A series of calcium sensors and adaptors are involved in the decoding of calcium signaling arising from the changing environment and plant physiological states.The calcium signaling transduction by CBL-CIPK pathway has been reported to participate in various biological processes,especially in the regulation of ions homeostasis.However,the research of CBL-CIPK pathway is limited to date due to the functional redundancy of CBL and CIPK families,thus potential function of CBL-CIPK pathway is largely undiscovered.Sugar is the most important primary metabolite,and its homeostasis requires precise regulation.In this study,a novel GhCBL2-GhCIPK6 pathway is reported to participate in sugar homeostasis in plants and the details are as follows:1.Identification of GhCIPK family members and analysis of their expression patternA total of 79 GhCIPK members were identified in Gossypium hirsutum acc.TM-1 reference genome by BLAST search with At CIPK protein sequence.Most of the GhCIPK members from A subgenome has a homologous copy in D subgenome.Phylogenetic analysis revealed that 8 members were divided into GhCIPK6 subfamily.The expression profile of GhCIPK family members in cotton leaves under different conditions revealed that GhCIPK6A3/D3 have the highest expression level,and hereafter are specified as GhCIPK6.In addition,GhCIPK6 was identified to be a circadian gene also with relative high expression levels in different tissues.2.GhCIPK6 is involved in the regulation of sugar homeostasis,plant development as well as stress resistanceGhCIPK6 regulates soluble sugar content and component in cotton.Overexpression of GhCIPK6 results in increased glucose and fructose contents with decreased sucrose content.The RNAi of GhCIPK6 results in decreased expression not only GhCIPK6 but also its homologues,and leads to decreased glucose content without influence on fructose and sucrose levels.However,the mutation of GhCIPK6A3/D3 has no influence on the sugar content and composition,suggesting the functional redundancy of GhCIPK6 subfamily.GhCIPK6 also promotes the cotton resistance to starvation and high level of exogenous glucose stress resistance in the cost of retarded development.3.GhCBL2 acts as a calcium sensor in the upstream of GhCIPK6GhCBL2 was screened out to interact with GhCIPK6 in yeast two-hybrid(Y2H)analysis.The interaction between GhCBL2 and GhCIPK6 was further verified by Pull-down,Bi FC and Luciferase Complementation Imaging(LCI)assay in vitro and in vivo.Overexpression of GhCBL2 results in significantly increased soluble sugar and glucose contents.The CRISPRCas9 mediated mutation of GhCBL2 can largely recover the sugar contents in GhCIPK6 overexpression plants,indicating that GhCBL2 involves in the pathway of GhCIPK6 mediated sugar homeostasis.4.Gh TST2 functions downstream of GhCIPK6 in sugar homeostasis regulationA potential tonoplast sugar transporter Gh TST2 was identified as the target of GhCIPK6 by phosphoproteomic analysis in GhCIPK6 transgenic plants.The interaction between GhCIPK6 and Gh TST2 was further verified by Y2 H,Pull-down and Bi FC analysis.The soluble sugar and glucose contents are significantly increased in Gh TST2 overexpression cotton.In contrast,mutation of Gh TST2 results in decreased glucose content.In addition,the mutation of Gh TST2 can largely recover the sugar contents in GhCIPK6 overexpression plants,indicating that the function of GhCIPK6 in sugar homeostasis depends on Gh TST2.5.The function and mechanism of GhCIPK6 in sugar homeostasis are conserved in different speciesOverexpression of GhCIPK6 can also largely increase the soluble sugar and glucose content in Arabidopsis,which is consistent with the phenotype in cotton.The double mutation of At TST1/2 can recover the sugar content in GhCIPK6 overexpression Arabidopsis to the wide type level,which indicates that the function of GhCIPK6 and the regulation mechanism by GhCIPK6 in sugar homeostasis are conserved in different species.6.GhCIPK6 has multiple functions in different biological processesGhCIPK6 was also found to be involved in other important biological processes,which are independent of the function in sugar homeostasis.Several kinds of mineral elements including Na,K,Ca and Mg decreased significantly in GhCIPK6 overexpression plants.In addition,GhCIPK6 leads to water loss in detached leaves but repressed the transpiration rate in the daytime.Moreover,Water Use Efficiency(WUE)is significantly increased in GhCIPK6 overexpression plants compared with wild type.The RNAi plants have no obvious difference in such processes,which might be due to the functional redundancy in GhCIPK6 subfamily members.Polygenic mutants should be created and further researches are needed to uncover such mechanisms.In summary,we identified a novel and conserved pathway that GhCBL2-GhCIPK6-Gh TST2 involves in sugar homeostasis.In addition,our results have great potential in crop quality and stress resistance improvement.