Regulatory Mechanism Study Of Protein Phosphatase ABI1 In Arabidopsis

The plant hormone abscisic acid(ABA)plays a critical role in plant growth and development,such as seed maturation and dormancy,germination,root growth,stomatal movement and plant resistance to biotic and abiotic stresses.So far,the research on the ABA signaling pathway has mainly focused on the isolation and identification of new signaling components,however,the interactions and cross-regulations among the currently identified components remain largely unknown.In this study,we investigated the degradation mechanism of ABA INSENSITIVE 1(ABI1),a negative regulator of ABA signaling pathway,and furthermore,we studied how ABI1 interacts with and regulates the activities of BAK1.Thus,this study contributes to a better understaning of how plants respond to drought,pathogen invasion and other stresses.First,we demonstrated that ABI1 is degraded by the ubiquitin/26S proteasome system.Two U-box E3 ligases PUB 12 and PUB 13,interact with ABI1,and consistently ABI1 is modified by ubiquitination.This process depends on the interactions between ABI1 and ABA receptors PYR/PYL/RCARs.When the Gly180 of ABI1 was mutated to Asp180(the mutation of abil-1,a dominant mutantion of ABI1),the ABI-G180D could not be degraded,consistent with the ABA insensitive phenotype of the abil-1 mutant.In addition,the expression of PUB12 and PUB13 is induced by ABA,and the pub12-2,publ3 and pub12-2 pub13 mutants were insensitive to ABA in terms of seed germination and root growth of seedlings.The ABA responsive genes were less induced in the pub mutants upon ABA treatment.These results indicate that ABA signaling is impaired in the pub mutants.The pub mutants lost water more quickly and therefore they were more sensitive to drought stress than the wild type plants.In terms of stomatal movement,ABA-induced stomatal closing and ABA-inhibited stomatal opening in light were both blocked in the pub mutants.Moreover,in the guard cells,ABA treatment failed to induce ROS accumulation in pub mutants.Together,this study revealed that Ubiquitin/26S proteasome system is involved in ABA signaling by regulating the stability of ABI1.Second,previous research has found that the degradation of flg22 receptor FLS2 also was regulated by PUB12/13,and this process relies on BAK1.So,we investigated the relationship between ABI1 and BAK1,and found that ABI1 interacts with BAK1,ABI1 inhibits the kinase activity of BAK1.Whereas BAK1 dose not affect the phosphatase activity of ABI1 nor its ability to interact with PYR/PYL/RCARs ABA receptors upon ABA treatment.These data suggest that ABI1 functions upstream of BAK1.In the soil,the bakl mutants lost water more quickly,and were more sensitive to drought stress than the wild type.The stomatal movement of bak1 was insensitive to ABA as well.In addition,genetic analysis revealed that the bakl-4 ghrl double mutant showed similar phenotype as the ghrl in response to drought stress,suggesting that BAK1 and GHR1 regulate stomatal movement in the same pathway..To summarize,this study revealed the molecular mechanism by which ABI1,a negative regulator of ABA signaling pathway,is degraded.In addition,we also discovered the interaction between ABI1 and BAK1,and how these two components act together in regulation stomatal movement.This study therefore provides novel in sight into the action mechanisms of ABI1,and a well sheds light on the complex regulatory mechanisms of ABA signaling pathway.