The Molecular Analysis Of WRKY28-and WRKY29-regulated Aluminum Resistance In Arabidopsis

Aluminum(Al)is the third most abundant element in the crust.When the soil pH is lower than 5.5,A1 in the soil will change from the non-free state to the higher activity free state,which will have a serious toxic effect on the root system of the plant.The secretion of root organic acids plays a key role in the process of plant resistance to Al.The organic acids secreted by the root system,such as malic acid and citric acid can chelate Al3+ ions to form non-toxic compounds that do not enter into the roots,thus making anti-Al.Malic acid transporter ALMT1 and citric acid transporter MATE play an important role in this process.Transcription factor is a kind of protein that can significantly regulate plant growth and development,and plays an important role in plant defense and stress response.The WRKY transcription factor is one of the largest families of transcriptional regulatory factors in plants.WRKY is mainly involved in plant biological stress,such as the defense of pathogens and non-biological stresses such as damage,drought,salinity,heat,cold and osmotic pressure.So far,the study of WRKY in the regulation of Al resistance is less.Recent studies have found that WRKY46 in Arabidopsis and WRKY22 in rice,respectively,regulate ALMT1 and the rice citric acid transporter OsFRDL4,thus regulating their Al resistance.We previously have found that Al stress induced the expression of WRKY28 and WRKY29 in roots and T-DNA insertion mutant wrky28 and wrky29 showed an increase in the sensitivity of Al.In order to further clarify the regulatory mechanism of WRKY28 and WRKY29 in Al resistance,physiological,biochemical,cellular,genetic and molecular approaches were combined in this study.Key results are shown as follows(1)Using QRT-PCR analysis,we found that both T-DNA insertion mutants were gain-of-function mutants.Furthermore,the WRKY28 and WRKY29 overexpression transgenic lines were constructed.Compared with the wild type,both overexpression lines showed higher sensitivity to Al stress.(2)Combined with different metal ions(La3+,Cd2+,Cu2+)stress and different pH stress treatment,we found that WRKY28 and WRKY29 were not involved in the regulation of La3+,Cd2+ and low pH stress,but were involved in the regulation of Cu2+stress.This indicates that WRKY28 and WRKY29 have no specificity in the regulation of Al tolerance,and also participate in the regulation of other metal ion stress.(3)QRT-PCR analysis showed that Al stress significantly induced the expression of WRKY28 and WRKY29 in the roots,and the expression increased gradually with the extension of stress time.To better understand the cellular expression pattern of WRKY28 and WRKY29 in the root tips under Al stress,we constructed and obtained the WRKY28pro::GFP and WRKY29pro::GFP transgenic lines.It was found that WRKY28 and WRKY29 were mainly expressed in root apex cells,and Al stress induced the expression of these two genes in apical cells.(4)Qualitative analysis with Morin staining and quantitative analysis with graphite furnace-atomic absorption spectrophotometer showed that more Al accumulated in the root tip of WRKY28 and WRKY29 overexpression lines.(5)Compared with the wild type,the secretion rate of malic acid in WRKY28 and WRKY29 was significantly reduced by Al stress.Using QRT-PCR analysis,the expression of ALMT1 in the overexpressed strain was significantly reduced.This indicates that the regulation of Al tolerance by WRKY28 and WRKY29 is regulated by the secretion of malic acid in the roots mediated by ALMT1.The results of yeast one hybridization showed that WRKY28 and WRKY29 could not directly bind to the ALMT1 promoter region to regulate its expression.But the transient expression analysis in Arabidopsis leaf protoplast showed that in the presence of Al stress,WRKY28 and WRKY29 can directly bind to the ALMT1 promoter regions to suppress its expression,and when both WRKY28 and WRKY29 existed,the suppression was higher.Further analysis with yeast two-hybrid and bimolecular fluorescence complementation(BiFC)showed a protein-protein interaction existed between WRKY28 and WRKY29.(6)Under Al stress,the expression of WRKY28,WRKY29 and WRKY46 was inhibited by STOP1,and WRKY28 and WRKY29 can inhibit the expression of WRKY46.In addition,we also found that,compared with the wild type,the expression of MATE in both WRKY28 and WRKY29 and ALS3 and ALS1 in WRKY29 overexpression lines was decreased.This indicates that,other potential mechanism exists to regulate the WRKY28-mediated and WRKY29-mediated Al resistance in addition to ALMT1-mediated malate exudation.Further study is In conclusion,this study reveals that WRKY28 and WRKY29 are involved in the regulation of Al tolerance through ALMT1-mediated malate exudation from roots,but this regulation is not specific to Al.And the Al-induced expression of WRKY28,WRKY29 and WRKY46 was inhibited by STOP1.