The RAE1-STOP1-Gl2-RHD6 Module Regulates The ALMT1-Dependent Aluminum Resistance In Arabidopsis

Soil acidification is one of the main factors limiting agricultural production.In acid soil,Aluminum(Al)is one of the most critical factors to inhibit the growth of plant roots.In the long-term natural evolution,plants have evolved different mechanisms to adapt to Al stress,including external exclusion and internal resistance.In most types of plants,the external exclusion mechanism occupies a dominant position in the Al resistance.In Arabidopsis,ALMT1-dependent malate exudation of roots plays a crucial role in regulating plant Al resistance.However,the transcription factors STOP1,CAMTA2 and WRKY46 can directly regulate the expression of ALMT1 under Al stress,which proves that the regulation of Al stress resistance in plants was a complex network.Therefore,it was essential to study the regulatory mechanism for explaining the molecular mechanism of plant resistance to Al stress.bHLH transcription factors are the second largest family of transcriptional regulatory factors in plants,and they play an essential role in plant growth.The study has found that under Al stress,STOP1 can regulate the expression of bHLH transcription regulator family member bHLH83(RHD6,Root Hair Defective 6).Our previous research found that rhd6-1 was more severely poisoned by Al than wild type(WT),but the molecular regulation mechanism of RHD6 is unclear.Therefore,in this study,the molecular mechanism of RHD6 regulating aluminum stress tolerance was studied by combining techniques.The research results are as follow:(1)RHD6 is involved in the regulation of Al resistance through mediating the ALMTl-dependent malate exudation from roots,which is regulated by STOP1The root growth inhibition of rhd6-1,rhd6-3,rhd6-4 and RHD6-OE transgenic materials were analyzed under Al stress.It was found that the mutants were more poisoned to Al stress than WT,RHD6-OE were less poisoned to Al stress than the WT.Next,toluidine blue staining was used to analyze the deposition of callose in the root under A1 stress,and it was found that the root of the mutant had accumulated more callose than the WT.These results indicate that RHD6 was involved in the regulation of Al stress.Through different metal ions and pH conditions,it was found that the regulation of the RHD6 on root growth inhibition was specific to Al stress.Studies have found that RHD6OE could reduce the Al stress of stop1,and the results showed that the regulation of RHD6 on root growth inhibition under A1 stress was controlled by STOP1..By analyzing the expression and promoter expression activity of RHD6 in WT and stop1,it was found that STOP1 was involved in regulating the expression of RHD6 under Al stress.Combined with genetic research,it was found that RHD6 overexpression can compensate for the strong sensitivity of stopl to A1 stress,indicating that STOP1 regulates root growth inhibition under A1 stress through RHD6.By comparing malate exudation in the roots of WT and rhd6-1 under Al stress,it was found that RHD6 was involved in the regulation of malate exudation under A1 stress.Combining qRT-PCR and GUS histochemical assay,it was found that RHD6 was involved in the regulation of ALMT1 expression and ALMT1 promoter expression under Al stress.Combined with genetic research,it was found that RHD6 regulates root growth inhibition under Al stress by regulating the expression of ALMT1.Using yeast one-hybrid,Dual-Luciferase system and Chromatin Immunoprecipitation(ChIP),it was found that RHD6 can regulate the expression of promoter of ALMT1 by combining with G-box and GCG-box cis-acting elements.(2)GL2 negatively regulates Al resistance by suppressing RHD6 expressionUsing qRT-PCR and GUS histochemical assay,it was found that the expression of RHD6 in gl2-5 was significantly higher than that in WT under A1 stress.Compared with WT,the inhibition degree of mutant root growth was significantly reduced under Al stress.It was also found that gl2-5 mutants accumulated more callose than WT under Al stress.Meanwhile,it was found that compared with gl2-8 and RHD6-OE,gl2-8/RHD6-OE showed a significantly lower sensitivity to Al stress by combining genetic means to obtain gl2-8/RHD6-OE hybrid material.These results showed that the regulation of RHD6dependent Al stress on root growth inhibition was negatively regulated by GL2.It was found that A1 stress significantly inhibited the expression of GL2 in roots at transcription and protein levels,and it was specific to Al stress by analyzing the expression of GL2 in roots under A1 stress.Meanwhile,it was found that GL2 can also directly bind to the L1-Like cis-acting element in the promoter of ALMT1 to regulate the expression activity of the ALMT1 promoter negatively.(3)GL2,STOP1 and RHD6 form a protein complex to coregulate the expression of ALMT1Through the combination of qRT-PCR,genetics and cytology,we found that STOP1 and GL2 inhibit each other.Because of the mutual regulation between STOP1,GL2 and RHD6,we found that the protein complex formed by yeast two-hybrid,Bimolecular Fluorescent Complimentary(BiFC)and Co-Immunoprecipitation(Co-IP)techniques.These results showed that STOP1 and GL2 mutually inhibit each other in terms of binding to the ALMTl promoter,GL2 inhibited the activation of RHD6 to the ALMT1 promoter,and STOP1 promoted the binding of RHD6 to the ALMT1 promoter.These results showed that GL2,STOP1 and RHD6 form a protein complex to coregulate the expression of ALMT1.(4)RAE1 affects the stability of RHD6 protein while RHD6 represses the expression of RAE1Using the transient expression system of protoplasts in the epidermal cells of Arabidopsis leaves,it was found that MG132 can inhibit the degradation of RHD6 by RAE1,indicating that RAE1 degrades RHD6 through 26S proteasome ubiquitination.Compared with 35Sp:RHD6,the expression of RHD6 protein in rae1/35Sp:RHD6 was significantly increased under Al stress.This result showed that RAE1 affects the stability of RHD6 protein.Furthermore,it was found that RAE1 inhibited the transcriptional activation of the ALMT1 promoter by RHD6.Combining yeast two-hybrid,BiFC and CoIP methods,it was found that there was protein interaction between RAE1 and RHD6.Then the use of qRT-PCR and GUS histochemical assay found that RHD6 can regulate RAE1 and promoter expression activity.Meanwhile,it was found that RHD6 can directly bind to the promoter of RAE1 by combining with E-box cis-acting elements.This result indicates that RHD6 has feedback regulation on RAE1.Moreover,the study found that RHD6 inhibited the degradation of STOP1 protein by RAE1.(5)RAE1 and GL2 promote each other at the translational and transcriptional level,respectivelyIt was found that RAE1 promoted the expression of GL2 protein.Meanwhile,it was found that the GL2 protein expression of Arabidopsis rae1/GL2p:GL2-GFP under Al stress was lower than that of GL2p:GL2-GFP,which further proved that RAE1 could promote the expression of GL2 protein.The study found that GL2 promoted the expression of RAE1 and the expression of promoter activity under A1 stress by combining qRT-PCR and GUS histochemical assay,.It was found that GL2 can directly bind to the promoter of RAE1 to regulate it was expression by using yeast one-hybrid,Dualluciferase expression system and ChIP technology.These results showed that GL2 and RAE1 had a mutual promotion relationship under Al stress.