| About 30%of the world's arable land is acidic soil,and the soil is being further acidified with being overused and overusing nitrogen.Aluminum is the main factor that the acidic soil restricts plant growth and crop yield,and the aluminum toxicity mainly acts on the root tip transition zone,by binding targets of cell wall,cell membrane and intracellular.Plants have evolved a variety of detoxification mechanisms to deal with aluminum toxicity in the process of long-term adaptation to acidic soils,including the secretion of organic acids and the internal detoxification.In Arabidopsis,malic acid is mainly secreted organic acid to detocify Al,which is through AtALMT1.AtALMT1 expression is induced by aluminum.The transcription factor STOP1 directly regulates the expression of AtALMT1,but the expression of STOP1 itself is constituent,and cannot explain the mechanism of AtALMT1 regulation,so there may be other genes involved in the regulation of STOP1 transcriptional level.In addition,the expression of AtALMT1 is induced by many factors,such as low pH,ABA,H2O2 and IAA,in addition to its response to aluminum.In this paper,we construct a report gene system of AtALMT1 promoter driving luciferase gene,and use this materials to perform EMS mutation to screen mutants.The functional analysis was carried out by means of heredity and physiology and biochemistry to further understand the mechanism of AtALMT1 expression regulation.The main results are as follows:(1)The construct of AtALMT1 promoter driving luciferase gene were constructed,and the mutant affecting LUC fluorescence was screened from the EMS mutation libray,and 10 strains were lightened,3 strains darkened and 2 strains were completely not bright mutants.Among them,8 mutants were named as rael,and 2 mutants were rae2,rae3,and 2 completely unlit mutants were the mutants of two conservative loci mutations in the STOP1.(2)The expression level of AtALTM1 in rae2,rae3 was decreased and rae2,rae3 were sensitive to aluminum.It was also found that the expression of STOP1 for AtALMT1 was very necessary,and AtALMT1 was almost not expressed after STOP1 mutation.The gene RAE1 was cloned by map-cloning and the second generation sequencing technique.RAE1 contains a F-box structure and 18 LRR repeating structures,which are the component of SCF type E3s.(3)The expression of RAE1 and GUS staining showed that RAE1 were expressed in root,leaf,stem,flower and fruit pod,mainly in the vascular bundle of the roots and tissues,and RAE1 in the root was induced by aluminum.The RAE1 expression was reduced in stop1.In vitro pull-down,vivo Co-IP and tobacco split-LUC experiments proved that RAE1 and STOP1 interacted,and RAE1 and STOP1 were located together in the nucleus.rae1-1 mutant proteins can also be combined with STOP1,suggesting that rae1-1 mutants are not functionally deficient through interaction with STOP1.We also found that STOP1 will be degradated through the ubiquitin proteasome pathway both in the absence of A1 and with Al,the IP results also prove that STOP1 will be ubiquitin modified,Aluminum treatment can reduce the degradation of STOP1.Conbining the accumulation of STOP1 protein in rael-1 mutants,it was proved that the degradation of STOP1 protein was mediated by RAE1 under the condition of Al and without Al.(4)In Arabidopsis,there is a RAE1 homologous gene RAL1,the AtALMTl expression in mutant ral1 increas.RAL1 expression pattern is similar to RAE1,but the expression of RAL1 in the stele and crown of root is different from RAE1.RAL1 is located in nucleus and interacts with STOP1,and the mechanism of RAL1 and RAE1 to degrade STOP1 needs being further studied.There were two homologous genes(OsRAE1.1 and OsRAE1.2)of RAE1 in rice.OsRAE1.1 and OsRAE1.2 expression are similar to RAE1.They were induced by Al,mutant art1 in the expression of OsRAE1.1 was reduced by half,but unlike Arabidopsis,in art1 OsRAE1.1 expression can still be induced.OsRAE1.1 was able to interact ART 1,whether like Arabidopsis OsRAE1 has a degradation function needs to be further studied.In brief,this study cloned a F-box gene RAE1,regulates STOP1 proteins degradation through the ubiquitin-26S proteame passway.Expression of downsteam genes of STOP1-regulated were increased.rael was more tolerant to Aluminum.On the other hand,the cell cycle gene ATR(ataxia telangiectasia and rad3 related)in response to aluminum toxicity was researched.Previous research suggests that ATR may inhibit the elongation of the root by detecting DNA damage caused by aluminum toxicity.In order to explore whether the ATR-mediated root-length inhibition mechanism is suitable for all aluminum-sensitive mutants,the double mutants of als3atry star 1atr,almt1atr and stop1atr are constructed,and the phenotype is analyzed.The results show that ATR mutation can rescue the sensitivity of star1 and als3 to aluminum toxicity respectively,but it can not rescure the sensitivity of atalmtl and stopl to aluminum toxicity.The results suggest that atr can rescue the mutant phynotype only affected by internal aluminum toxicity,but it can't recover the phynotype of the mutant affected by external aluminum toxicity. |
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