Molecular Mechanism Of RING-box Proteins ULS1 And ULS2 In Regulating Leaf Senescence And Stomatal Closure In Arabidopsis

Leaf senescence is an important process of plant growth and development,which is precisely regulated.This process is a highly complex dynamic process determined by genetic,developmental and environmental factors.In the process of leaf senescence,plants redeploy valuable nutritional resources from old leaves to active tissues,especially to reproductive systems,to ensure the stable breeding of plants.As a pivotal plant hormone,abscisic acid(ABA)is a positive signal of importance in regulation of leaf senescence.Although the mechanism of ABA biosynthesis and ABA signal transduction in regulating leaf senescence has been known,the contribution of ABA transport to leaf senescence and its regulatory mechanism remain elusive.There are about 500 types of RING-box proteins with potential ubiquitination activity in Arabidopsis,which have been classified into nine categories according to the characteristics of RING domain.The existence of various RING domains may represent a specific ubiquitination pathway and play a pivotal role in plant growth and hormone signal transduction.However,the function of most RING-box genes is unknown,and their roles in leaf senescence remain to be studied.This study identified two previously uncharacterized RING-box family genes UBIQUITIN LIGASE of SENESCENCE 1 and 2(ULS1 and ULS2)play roles in Arabidopsis senescence.The main conclusions are summarized as follows :1.Loss of ULS1 and ULS2 results in premature senescence of Arabidopsis leavesULS1 and ULS2 were highly expressed in senescence leaves,and found their sequences highly homologous.We obtained the T-DNA insertion mutants uls1 and uls2,and found that the single mutant did not have any growth and development phenotype,but the double mutant uls1/uls2 showed an obvious leaf senescence phenotype under normal condition.Introduction of either gene of ULS1 and ULS2 to the double mutant can rescue its leaf senescence phenotype.Based on real-time quantitative PCR and GUS staining,we further clarified that ULS1 and ULS2 are predominantly expressed in old leaves,which indicating that ULS1 and ULS2 function redundantly in regulating leaf senescence.2.ULS1 and ULS2 affect leaf senescence through plant hormone ABAThrough treatment with various plant hormones and dark conditions,we then found that the mature and unsenescent detached leaves of uls1/uls2 showed earlier senescence than those of WT,which was specifically promoted by exogenous ABA treatment.Meanwhile,the expression of senescence related genes was also significantly increased in the double mutant compared with that in WT.Moreover,we detected the content of free ABA in uls1/uls2 and WT leaves at different stages,and found that the ABA content in uls1/uls2 leaves was significantly higher than that in WT leaves at the late growth stage,indicating that ULS1 and ULS2 are presumably involved in ABA-induced leaf senescence.Furthermore,through transcriptome analysis of leaves at different stages of uls1/uls2 and WT,we found that the expression level of ABA transporter ABCG40 at each stage of uls1/uls2 was significantly higher than that of WT,which was also verified by RT-q PCR.Further quantitative proteomics of ubiquitination revealed significant differences in protein levels of many ABA-related genes including ABCG40 between WT and uls1/uls2.3.ULS1 and ULS2 regulate leaf senescence and stomatal closure through ABA transporter ABCG40To confirm the genetic relationship between ULS1/2 and ABCG40,we constructed the uls1/uls2/abcg40 triple mutant,and found it displayed a comparable leaf senescence phenotype with WT.In accordance,the triple mutant has less ABA content in leaves than uls1/uls2.Given that ULS1 and ULS2 are expressed in stomata and that ABCG40 is involved in regulating stomatal movement,we found that the stomatal aperture and leaf water loss rate of uls1/uls2 decreased significantly,which can be suppressed by abcg40 mutation.We also found that ULS1 and ULS2 did not affect seed germination and seedling response to ABA.In addition,the expression of ULS1 and ULS2 was found to be induced by ABA.In conclusion,we demonstrate that ULS1 and ULS2 function in feedback inhibition of ABCG40-dependent ABA transport during ABA-induced leaf senescence and stomatal closure.