| With changes in the global climate environment,drought has emerged as a key abiotic stress limiting plant growth and development,which seriously affects plant productivity and the yields of crops.To adapt to the changing environment,plants have evolved a number of response mechanisms against drought stress,among which guard cells constituting stomata in plant leaves can influence stomatal aperture through changing their volume,which affects the exchange of gases and water between the plant and the outside world,and ultimately influences the plant’s ability to withstand drought stress.The volume of guard cells is regulated by light,temperature,pathogenic bacteria and various plant hormones,among which the plant hormone abscisic acid(ABA)responds to drought stress,and its elevated levels lead to suppression of guard cell volume,resulting in a significant increase in plant drought tolerance.There are many functional genes involved in the ABA signal transduction process.The exploration of the functional genes involved in the regulation of ABA signal transduction can help to provide important theoretical significance for the improvement of drought resistance in plants.To adapt to different geographic environments,Arabidopsis(Arabidopsis thaliana)has evolved many different accessions through natural variation,among which Columbia(Col)and Landsberg erecta(Ler)are most widely used in plant physiological and molecular genetic studies.However,whether Col-0 and Ler-0 differentially regulate plant drought stress,and whether the specific mechanisms of drought stress regulation differ between the two have not been reported,and further exploratory studies are needed.Cysteine-rich receptor-like kinases(CRKs)are widely involved in a variety of abiotic stresses.In this study,we used the Arabidopsis accessions Col-0 and Ler-0 as experimental materials,to investigate the specific functions and the mechanism of action of CRK4,a member of the CRKs,in differentially regulating ABA-mediated drought stress responses in Col-0 and Ler-0.In this study,we first determined the stomatal movement and drought phenotypes of Col0 and Ler-0.It was found that Col-0 and Ler-0 differentially regulated ABA-mediated stomatal movement and drought stress response.Compared to Col-0,Ler-0 was significantly less sensitive to ABA in stomatal movement and the drought tolerance of Ler-0 was also significantly weakened.Subsequent phenotypes of the F1 and F2 populations between Col-0 and Ler-0 revealed that the F1 population showed similar ABA sensitivity and drought tolerance as that of Col-0,and the separation ratio of ABA hypersensitive to ABA hyposensitive plants in the F2 population was 2.89:1.It can be concluded that Col-0 and Ler-0 differentially regulate the ABA-mediated stomatal movement under the control of recessive single genes.The genomes of Col-0 and Ler-0 were analysed and compared,and it was found that CRK4 and CRK14 were partially or completely absent in Ler-0,and that these two genes were likely to be involved in the regulation of ABA-mediated drought stress response.Subsequently,the crk4 mutant and crk14 mutant in the Col-0 background were characterised for their drought phenotypes,respectively.The results showed that CRK4 loss-of-function exhibited ABA sensitivity and drought phenotypes similar to those of Ler-0,whereas CRK14 loss-of-function exhibited ABA sensitivity and drought phenotypes opposite to those of Ler-0,which suggests that CRK4 is likely to be involved in the differential regulation of ABAmediated drought stress in the Col-0 and Ler-0.The CRK4 overexpression vector was transformed into Ler-0,obtained the CRK4OELer plants.The related phenotypic experimrnts showed that the CRK4OE-Ler plants were able to partially restore the Ler-0 phenotypes of weak sensitivity to ABA and reduced drought tolerance to the Col-0 level.To further demonstrate CRK4 can act as a key gene involved in the differential regulation of stomatal movement and drought stress response by Col-0 and Ler-0,we also characterized the drought phenotypes of Ler-0crk4-1,a cross between Ler-0 and the crk4-1 mutant.We found that the Ler-0crk4-1 plants remained similar to Ler-0 in both ABA sensitivity and plant drought tolerance.This all suggests that CRK4 plays an important role in the differential regulation stomatal movement and drought stress response between Col-0 and Ler-0.To explore the regulatory mechanism of CRK4 involved in the drought difference between Col-0 and Ler-0,we screened the yeast library for the CRK4 interalating protein Ubox E3 ligases(PUB 13),and verified the interaction between the two through Pull-down and CoIP experiments.Subsequently,a series of biochemical experiments were carried out,which showed that showed that PUB 13 protein abundance in Col-0 was significantly higher than that in the crk4 mutant,suggesting that CRK4 loss-of-function reduces the protein stability of PUB 13,and that CRK4 can play a role in the post-translational regulation of PUB 13.Meanwhile,The stomatal movement and drought phenotypic experiments also showed that the ABA sensitivity and whole plant drought tolerance ofpub13crk4-1,a cross between pub13 and the crk4-1 mutant,was more similar to that of the pub 13 mutant.Thus CRK4 acts epistatically of PUB13 in stomatal movement and drought stress response,CRK4 was located in the upstream of the genetics of PUB13 to exert its function.In addition,related biochemical experiments showed that the protein abundance of ABA-insensitive 1(ABI1)in Ler-0,crk4-1 mutant,pub13 mutant,and pub13crk4-1 plants were significantly higher than that of Col-0.CRK4 can regulate the protein level of ABI1 through PUB 13.In summary,this study verified the important role played by CRK4 in the differential regulation of stomatal movement and drought stress response by Col-0 and Ler-0,and elucidated the molecular mechanism by which CRK4 affects the stability of PUB 13,thereby regulating the ABI1 protein level,and consequently the stomatal movement and drought stress response. |
PDF Full Text Request