Functional Characterization Of Arabidopsis ADF5 In Responding To Drought Stress And Gelsolin-Like Domain 3 Of The Villin/Gelsolin/Fragmin Superfamily

Drought is a global problem in today's world and drought stress seriously affects plants growth and development,resulting in crop yield reduction and environmental degradation.Plant endogenous hormone abscisic acid?ABA?plays a very important role in the process of plant growth,development and protection against environmental stress.ABA participates in regulating plant stomatal closure to reduce transpiration and prevent moisture loss under drought conditions.Increasing researches have shown that microfilaments are involved in the regulation of stomatal opening and closing,which was evidenced by the facts that its dynamic disorder can cause disorders of stomatal opening and closing and actin cytoskeleton conformation transformation affects the speed of stomatal opening and closing.However,the mechanism of the dynamic changes of the actin cytoskeleton responding to the ABA signal is not clear.Besides,this paper also pays attention to the function of another actin binding protein?ABP?,gelsolin-like domain 3of villin/gelsolin/fragmin superfamily.The villin/gelsolin/fragmin superfamily is a major group of Ca²⁺-dependent actin-binding proteins involved in various cellular processes.Members of this superfamily typically possess three or six tandem gelsolin-like?G?domains,and each domain plays a distinct role in actin filament dynamics.Although the activities of most G domains have been characterized,the biochemical function of the G3 domain remains poorly understood.In this paper,we functionally characterized Arabidopsis ADF5 in responding to drought stress and gelsolin-like domain 3 of the villin/gelsolin/fragmin superfamily.1.ADF5 was a negative regulator of ABA signaling during seed germination and cotyledon greening.The results of qRT-PCR proved that the expression of ADF5 was up-regulated after inducing by ABA and drought,indicating that ADF5 participated in drought stress;functional defect of ADF5 resulted in insensitivity of seed germination and cotyledon greening to exogenous ABA in arabidopsis.2.ADF5 was involved in the response to drought stress through regulating the stomatal closing.The adf5 showed more serious wilting compared with WT in soil drought experiment;the water loss analysis of detached leaves showed that the water loss rate of adf5 is higher than WT;the stomatal closing experiment showed that the stomatal closure rate of adf5 is slower than WT and stomatal closure was impaired in adf5.It is speculated that the response of ADF5 to drought stress is mediated by regulating the stomatal closing.3.ADF5 but not ADF9 was involved in the process of seed germination,cotyledon greening and drought response mediated by ABA.Both ADF5 and ADF9 are the third subfamily of ATADFs in arabidopsis with high homology,but the adf9 and adf5×adf9 plants were similar to WT or adf5 respectively,in the process of seed germination,cotyledon greening and drought response mediated by ABA.4.ADF5 promoted the stomatal closing via regulating the configuration transformation of actin cytoskeleton.Living actin filaments observation of guard cells showed that the loss of ADF5led to the delay of actin filaments dynamic and configuration transformation,resulting in impaired stomatal closure.5.DPBF3 may be involved in plant responses to drought stress mediated by ABA through positively regulating ADF5 expression.The sequence analysis of the ADF5 promoter showed that the promoter contains multiple ABRE core motif,ACGT.The ADF5 expression analysis in the mutants of abf/arebs has showed that the loss of DPBF3 inhibited the up-regulation of ADF5induced by ABA.Yeast one hybrid assay proved that DPBF3 can combine with ADF5 promoter containing the ACGT element.Chromatin immune coprecipitation and transcriptional activation analysis proved that DPBF3 combines with ADF5 promoter and further promotes its transcriptional activation.6.DPBF3 may be involved in the response to drought stress through regulating the stomatal closing.The water loss analysis of detached leaves showed that the water loss rate of dpbf3 was higher than WT and the stomatal closing analysis showed that the stomatal closure rate of dpbf3was slower than WT.7.The main function of G3 domain is to regulate the dynamic changes of the actin filaments through affecting the activities of the villin/gelsolin/fragmin superfamily proteins.In this study,we carefully compared the detailed biochemical activities of ABP29?a new member of this family that contains the G1-G2 domains of lily ABP135?and ABP135_G1-G3?which contains the G1-G3domains of lily ABP135?and found that ABP135_G1-G3 exhibited greater actin severing and/or depolymerization and nucleating activities than ABP29 in the presence of high Ca²⁺levels?200and 10?M?and ABP135_G1-G3 had a weaker capping activity than ABP29 in the presence of low levels of Ca²⁺?41 nM?;ABP135_G1-G31-G3 promoted F-actin depolymerization,while ABP29 inhibited F-actin depolymerization,and they exerted opposing effects on the actin dynamics during dilution-mediated depolymerization.In summary,the study of ADF5 revealed that the ABF/AREB transcription factor DPBF3may regulate the expression of ADF5,further being involved in the response to drought stress through regulating the stomatal closing via affecting the dynamic rearrangement of actin cytoskeleton.Besides,the function comparison of ABP29 and ABP135_G1-G3 revealed that the main function of G3 domain is to regulate the dynamic changes of the actin filaments though affecting the activities of the villin/gelsolin/fragmin superfamily proteins.This study will provide theoretical guidance to improve the water use efficiency of plants and develop drought-resistant crops though revealing the regulation mechanism of stomatal movement and gene regulatory network of drought stress.