Study On The Response Of Arabidopsis Thaliana AtSIS To Combined Stress Of Salt And Drought

The compound stress composed of salt and drought and other adversities seriously affects the growth and development of plants,but the response mechanism of plants to compound stress is poorly understood.AtSIS encodes an inherently disordered protein,which participates in the response to a variety of single stresses and is mainly located in guard cells.In this paper,wild type,AtSIS overexpression and AtSIS deletion mutants were treated with salt,drought and combined stress in Arabidopsis thaliana seedling stage to observe the stress phenotype,growth index and physiological index,and analyze the physiological responses of Arabidopsis AtSIS to salt,drought and combined stress.In order to clarify the regulation of Arabidopsis AtSIS on stomatal movement,the plants were subjected to CaCl₂,ABA and H₂O₂ stress,and to speculate the molecular mechanism of AtSIS response to abiotic stress by detecting the transcriptional levels of AtSIS and related genes.The main results are as follows:The four-week-old strains were treated with NaCl,mannitol and combined stress.After 14 days,the phenotype observation showed that the leaves of the deletion mutants turned yellow,dehydrated and shrunk.Compared with the wild type,the growth status was significantly worse;the rosette leaf diameter,plant weight and root length growth of the AtSIS overexpression were higher than the wild type;the chlorophyll,soluble sugar and proline content of the AtSIS overexpression were higher than wild type,SOD,POD and CAT antioxidant enzyme activities of deletion mutant were lower than wild type,and malondialdehyde and hydrogen peroxide content of deletion mutant were higher than the wild type,which showed that AtSIS increased the tolerance of Arabidopsis to salt,drought and dual salt and drought stress by affecting the above physiological indicators.The fully expanded rosette leaves of four-week-old strains in the same leaf position and growing vigorously were taken and placed in the stomatal opening solution and stomatal closing solution.We found that under NaCl,mannitol and combined stress,the stomatal aperture of the AtSIS overexpression were significantly lower than that of the deletion mutant,indicating that Arabidopsis AtSIS promoted stomata closure and inhibited stomata opening thus enhancing the degree of stoma closure;Under CaCl₂,H₂O₂ and ABA treatments,there was a significant difference in the change rate of the stomatal opening of each plant,indicating that AtSIS may be an important component in the regulation of stomata movement path by ABA,H₂O₂ and Ca²⁺.The above results indicated that Arabidopsis AtSIS didn't completely rely on the ABA signaling pathway to regulate stomatal movement,and it enhanced the degree of stomatal closure by promoting stomatal closure and inhibiting stomatal opening,thus improved plant resistance to stress.The transcription results of Arabidopsis AtSIS,ABA synthesis genes,salt-drought stress response genes,antioxidant enzyme genes,and stomatal movement genes under dual salt and drought short-term stress showed that the alternative splices of AtSIS,AtSIS.1 and AtSIS.2,the expression of AtSIS were significantly induced;AtSIS negatively regulated the expression of the ABA synthesis genes AtNAP and NCED3;AtSIS promoted the expression of HKT1 and NHX1 that maintain the balance of Na⁺/K⁺,inhibited the expression of ABA signal response gene RAB18;AtSIS promoted the expression of antioxidant enzyme genes CAT2 and CSD2;AtSIS promoted the expression of stomatal movement genes RBOHD and SnrK2.6.To sum up,Arabidopsis AtSIS regulated plants' physiological response by changing physiological indicators such as proline,SOD enzyme activity and malondialdehyde;AtSIS enhanced the degree of stoma closure by promoting stomata closure and inhibiting stomata opening;and AtSIS negatively regulated the expression of the ABA synthesis genes AtNAP and NCED3,promoted the expression of salt stress response genes HKT1,NHX1 and antioxidant enzyme genes CAT2 and CSD2,inhibited the expression of ABA signal response gene RAB18,promoted the expression of stomatal movement genes RBOHD and SnrK2.6,making Arabidopsis AtSIS improve plant tolerance to stress.Under salt and drought dual stress treatment,the changes in the growth indicators of each strain and POD and CAT antioxidant enzyme activities,and in stomatal aperture,and in genes(AtNAP,NCED3,HKT1,NHX1,CAT2,RBOHD and SnrK2.6),were all not the superimposition of the changes of individual stress treatments,and the final phenotypic changes were not the superimposition of the individual stress effects,so the response of Arabidopsis AtSIS to salt-drought combined stress can not be judged by the superposition effect of salt and drought stress alone.