Cloning And Functional Analysis Of Aluminum Resistance Genes RAE2 And RAE3 In Arabidopsis Thaliana

About 35%of the arable land in the world was acid soil.The area of acid soil is still increasing year by year with the rapid development of industry and the aggravation of human activities.The toxic ion form of aluminum increased in acid soil destroys cell wall components,suppresses DNA synthesis and mitosis,results in the growth inhibition of plant root system and affects the absorption of water and nutrients by root.Therefore,aluminum toxicity is the main limiting factor of crop production in acid soil.Plants evolved a series of aluminum resistant mechanisms in the process of adapting to the environment.The secretion of malic acid chelates aluminum ion mediated by malate transporter AtALMT1(Aluminum-activated malate transporter 1)in Arabidopsis thaliana is one of the main mechanisms of aluminum resistance.Different form the constitutive expression of wheat TaALMT1,the expression of AtALMT1 was induced by aluminum in Arabidopsis thaliana.C2H2 zinc finger structure transcription factor STOP1(Sensitive to proton rhizotoxicity 1)plays a key role in the aluminum resistance process.It is mainly through the regulation of downstream gene expression,including A tALMT1,to achieve aluminum detoxification.Although the expression of downstream genes of STOP1 is elevated by aluminum toxicity induction,the expression of STOP1 itself is not affected by aluminum toxicity,which suggests that STOP1 is regulated after transcription or translation.The transgenic line of AtALMT1 promoter fused with luciferase gene LUC was constructed in our laboratory.After mutagenesis with EMS,a mutagenetic screening library was established.It is hoped that the new aluminum resistance gene can be cloned and analyzed by screening mutants with stronger or weaker LUC fluorescence,and finally the posttranscriptional regulation mechanism of STOP1 is clarified.In this paper,we mainly studied mutant rae2 and rae3 with decreased LUC signal which screened from mutagenesis library,then analyzed the expression of aluminum resistance genes and physiologically aluminum sensitive phenotype.Cloning the target genes of the mutants and analyzing their function.The main results are as follows:(1)The LUC signal was attenuated and the expression of reporter gene LUC was decreased in rae3 mutant compared with WT.The expression of aluminum resistant genes,A tALMT1,AtMATE,ALS3 and RAE1,that regulated by STOP1 were reduced in rae3 compared with WT under both minus and plus aluminum conditions.Consistently,GUS activity in rae3 was less than that in WT in transgenic background of pA tALMT1:GUS under either minus or plus aluminum conditions.While the expression level of AtSTAR1 and ALS1,which regulated by STOP1 almost the same in rae3 and WT under either minus or plus aluminum conditions.These results suggested that RAE3 might affect the expression of STOP 1 downstream genes through the modulation of STOP1.We compared and analyzed the malate secretion rate between WT and rae3 because of the reduced expression level of AtALMT1.The result showed that the malate secretion rate in rae3 mutant was less than that in WT under both minus and plus aluminum conditions.Meanwhile,the root aluminum content in rae3 was higher than that in WT under plus aluminum condition.We then evaluated the physiological phenotype using soaked gel medium with a series of aluminum concentration gradient.The result indicated that rae3 was more sensitive to aluminum toxicity compared with WT,but less sensitive than Atalmt1.STOP1AtALMT1 mediated malate secretion induced root growth inhibition under low Pi condition,so we compared the responses of WT and rae3 to low Pi.The result showed that rae3 was insensitive to low Pi compared with WT and Atalmt1.Genetic analysis of rae3 found that the attenuated LUC signal was controlled by a single recessive gene.The whole genome sequencing result showed that RAE3 gene located on chromosome 5.According to the sequencing results,dCAPS markers were developed and linkage analysis was carried out.The results showed that RAE3 encoded HPR1,which belongs to a core component of THO/TREX complex.The mutation site of rae3 is the last base of the third intron,which leads to the third intron incorrectly recognized by splicing factor and thus retained,resulting in extra 32 amino acids in the structure of HPR1 protein in the mutant.In order to confirm that HPR1 is RAE3,we constructed pHPR1:HPR1-Flag complementation lines to perform complementation test.The results showed that the LUC signal and the expression of aluminum resistance genes in the downstream of STOP1 were rescued to WT level.Accordingly,the aluminum sensitive phenotype of mutant rae3 was also restored by the complementation lines.We found that HPR1 was expressed all over roots,stems,leaves,flowers and siliques by analyzing the expression level of HPR1 in different tissues.Tissue specific localization pattern indicated that HPR1 was mainly distributed in roots,young leaves and vascular tissues.GUS staining of transverse section of root tip showed that HPR1 was mainly expressed in meristem region.Since HPR1 has been demonstrated to be involved in the regulation of nucleocytoplasmic RNA export in Arabidopsis,we want to know whether mRNA export of Al-resistance genes is also regulated by HPR1.Therefore,we compared the expression of aluminum resistance genes in the nucleus of WT and mutant hpr1.The result showed that the mRNA level of STOP1 in the mutant was more than twice as much as WT.It indicated that HPR1 was involved in the regulation of STOP1 mRNA export from nucleus to cytoplasm.Then,we detected the levels of STOP1 protein in WT and the mutant of pSTOP1:STOP1-3HA transgenic background,and the results showed that the level of STOP1 protein in hpr1 was significantly lower than that in WT.Consistently,GUS activity in hpr1-7 mutant was also lower than that in WT underpSTOP1:STOP1-GUS transgenic background.In order to confirm this result,we performed the transient expression of plasmid pSTOP1:STOP1-3HA in Arabidopsis thaliana protoplasts.The result showed that the STOP1 protein level in mutant protoplasts was obviously lower than that in WT protoplasts.Above findings suggest that the decreased expression of STOP1-dowstream genes in hpr1 mutants might be caused by reduced expression of STOP1 protein.Our laboratory recently reported the regulation mechanism that RAE1 was involved in STOP1 protein degradation.It was found that the level of STOP1 protein in mutant rae1 was significantly higher than that in WT.To prove the above hypothesis,we introduced rae1 mutation into hprl and found that attenuated LUC signaling,down regulated LUC expression,decreased expression of STOP1 downstream genes,aluminum sensitive phenotype,low Pi insensitive phenotype in mutant hpr1 could all be partially recovered.(2)The LUC signal was significantly reduced and the expression of reporter gene LUC was decreased in rea1 mutant compared with WT.The expression of STOP1 downstream genes,A tALMT1,ALS3 and RAE1,were reduced in rae2 compared with WT under both minus and plus aluminum conditions.However,the expression of AtMATE,which also regulated by STOP1,was down-regulated in rae2 only under plus aluminum condition.The GUS activity in rae1 was lower than that in WT in the transgenic background of pAtALMT1:GUS under both minus and plus aluminum conditions.rae2 was more sensitive to aluminum toxicity and was more resistant to low Pi compared with WT.rae2 not only showed serrated leaf phenotype but also displayed increased number of rosette leaves.Genetic linkage analysis of rae2 showed that the reduced LUC signal was controlled by a single recessive gene.The result of whole genome sequencing combined with linkage analysis showed that RAE2 encoded TEX1,a component of THO/TREX complex.The mutation site of rae2 was located in the eighth exon,TGG mutated to TAG,leading to the premature termination of RAE2 protein.RAE2 protein contains 6 WD40 repeat domains and the mutation site of rae2 was located in the fourth WD40 repeat domain.To confirm that TEX1 is RAE2,we carried out complementation test.The results showed that the LUC signal and the expression of aluminum resistance genes regulated by STOP1 in rae2 were restored by the complementation lines.Accordingly,the aluminum sensitive phenotype of mutant rae2 was also restored by the complementation lines.The subcellular localization of RAE2 was in nucleus.RAE2 was expressed at a certain level in different tissues such as roots,stems,leaves,flowers and siliques.The expression of RAE2 was not induced by aluminum.Tissue-specific localization pattern showed that RAE2 was mainly expressed in root tip and young leaves.mRNA analysis of aluminum resistance genes in nucleus indicated that RAE2 was not involved in the process of mRNA export from nucleus to cytoplasm.While STOP1 protein level was decreased in rae2 compared with that in WT under both minus and plus aluminum conditions.Similarly,in order to confirm that the decreased expression of STOP1 downstream genes in mutant rae2 may be due to reduced STOP1 protein level,we introduced rael into rae2.The decreased LUC signal,aluminum sensitive phenotype and resistant phenotype to low Pi in rae2 could be partially recovered by rae1 mutation.In conclusion,we cloned RAE3 and RAE2 genes through forward genetic method.RAE3 regulates STOP1 mRNA export from nucleus to cytoplasm and affects STOP1 protein level,thus regulating the expression of STOP1 downstream genes.RAE2 affects STOP1 protein level through unknown pathway and thus regulating the expression of STOP1 downstream genes.