Molecular Mechanism Of Functional Module Of CBL1/9-CIPK1-PYLs For Negative Feedback Regulation Of Drought Stress In Arabidopsis Thaliana

Drought poses a serious threat to agricultural production and human activities,and global crop production losses due to drought have increased to approximately$30 billion in the past decade.As the global population increases,water demand will also increase.The geographical distribution of rainfall in China shows the norm of less rainfall in the north and more in the east and less in the west,and the northern region has long faced the great challenge of soil drought,which accounts for more than half of the total arable land area in the country.Therefore,it is crucial to explore high-yielding plants with efficient water utilization to ensure food security to alleviate the threat of drought to agricultural production.Elucidating the molecular mechanisms of drought resistance in plants and enriching the drought regulatory network of plants are major challenges for the development of modern agriculture.Abscisic acid（ABA）,an important stress hormone,is one of the important regulatory hormones for plants to cope with the stress response.ABA is perceived by ABA receptors Pyrabactin resistance 1（PYR1）and PYR1-like（PYL）proteins（PYLs）in plants,which initiate downstream signaling regulatory networks.Post-transcriptional regulations of ABA receptors are important factors in activating ABA signaling and have been the subject of numerous studies.Calcium(Ca²⁺)is one of the common second messengers in eukaryotes,and studies in Ca²⁺regulatory network have been a difficult and hot topic in plant cell signal transduction.Ca²⁺signals generated by external stimuli are sensed by Ca²⁺sensors,and in plants,Ca²⁺sensors Calcineurin B-like proteins（CBLs）and their interacting protein kinases（CIPKs）are involved in the regulation of complex processes such as plant growth and development and environmental response.Under drought stress,ABA-mediated cytoplasmic Ca²⁺oscillations via Cyclic nucleotide gated channels（CNGCs）also play an important role in plant response to drought.However,However,it is not clear whether Ca²⁺signaling can feedback regulate ABA signaling.In this study,using a series of physiological and cell biological experiments,we found that the Ca²⁺sensor CBL1/9-CIPK1 negatively regulates ABA signaling and drought stress response by phosphorylating ABA receptor PYLs.In addition,our results settled a long-standing controversy that cbl1/9 and cipk23 mutants exhibit a drought-tolerant phenotype,although the CBL1/9-CIPK23 complex activates the S-type anion channel-associated 1（SLAC1）.tolerant phenotype of cbl1/9 is the consequence of negative regulation of ABA receptors.CBL1/9-CIPK may preferentially regulate the activity of ABA receptors to negatively regulate drought stress.Together,these studies suggest that the negative regulation of PYLs activity by CBL1/9-CIPK1 is a key mechanism for optimal plant growth and response to drought stress.The main results of this study are as follows:1.Since cipk1 mutants have been reported to be sensitive to exogenous ABA in the Ws background,based on the possible differences in plant phenotypes among different ecotypes,we first used reverse genetics to find that mutations in CIPK1 gene in the Col background also led to ABA sensitivity in Arabidopsis.By expressing CIPK1 promoter-driven GUS,we found that CIPK1 is highly expressed in stomata,and further confirmed that CIPK1 may be a negative regulator of Arabidopsis in response to ABA signaling and drought stress through the soil drought assays and stomatal closure measurements.We also investigated the Ca²⁺receptor CBL1/9,which interacts with CIPK1,and found that the CBL1/9-CIPK1 complex is involved in the negative regulation of ABA signaling and drought stress response.2.Potential interactions between CIPK1 and ABA receptors were identified by yeast two-hybrid assays,and subsequent bimolecular fluorescence complementation（Bi FC）assays revealed that interactions between CIPK1 and PYLs were present in the nucleus,cytoplasm,and cell membrane.The physical interaction between CIPK1 and ABA receptors PYR1/PYL1/PYL2/PYL3/PYL4/PYL5/PYL6 was further confirmed using split luciferase complementation（LCI）assays,in vitro Pull-down assays,and Co-IP assays.It was also observed that the interactions between CIPK1 and PYLs occurred only at the plasma membrane when CBL1 was expressed using tobacco leaf chloroplast protoplasts.3.In vitro phosphorylation experiments were used to confirm that CIPK1 is able to regulate the activity of PYLs by phosphorylating PYLs at the conserved Ser129 site of PYL4.In vitro experiments using recombinant ABA signaling pathway as well as in vitro phosphatase activity revealed that CIPK1 phosphorylation-modified PYLs were inactive and could not inhibit ABI1 phosphatase activity in the presence of ABA.Yeast two-hybrid as well as in vitro Pull-down assays showed that phosphorylation affected the interaction of PYLs with ABI1.In vivo analysis of phosphorylated PYL4 was also performed and showed that phosphorylation did not alter the localization of PYL4 and only resulted in inactivation of PYL4.PYL4 and PYL4^S129A transgenic plant ABA receptors restored ABA tolerance and drought sensitivity in the pyr1pyl2458（12458）pentamutant with restoration of the growth-deficient phenotype,whereas the phosphomimic of PYL4^S129D plants exhibited a similar phenotype to that of 12458plants.Combined in vivo and in vitro results suggest that CIPK1 phosphorylated PYLs are inactive PYLs.4.Using LCI experiments,it was verified that Sn RK2.6 does not interact with CIPK1 and was not involved in the regulation of ABA receptors by CIPK1.Next,by exogenous addition of ABA,it was found that ABA directly affects the physical interaction between CIPK1 and PYLs using Co-IP experiments as well as phosphorylation experiments,which in turn affects the phosphorylation of PYLs by CIPK1.Finally,genetic experiments were performed by constructing 12458/cipk1 mutants to show that CIPK1 is located upstream of PYLs to phosphorylate PYLs for function.Based on the above study,we propose a new mechanism for Ca²⁺signaling to negatively regulate ABA signaling.Under normal conditions,the Ca²⁺sensor CBL1/9-CIPK1 negatively regulates ABA signaling by phosphorylating the ABA receptor PYLs at the conserved PYL4Ser129 to ensure normal plant growth and development.Under drought stress,ABA directly inhibits the interaction between CIPK1 and PYLs,and the active ABA receptor transmits drought signals to ensure plants survive in drought adversity.This feedback mechanism of Ca²⁺signaling on ABA signaling plays an important role in the balance of plant growth and adaptation to the environment,and analyzing the interrelationship between Ca²⁺signaling and ABA signaling regulatory network could provide better theoretical support for crop drought resistance breeding.