Analysis Of Aluminum-insensitive Arabidopsis Mutants And Gene Mapping

The imbalance of soil nutrient elements in facility cultivation of China and the trend of soil acidification is a serious problem.Soil acidity increases soluble aluminum(Al)that inhibits plant growth and physiological functions.The excretion of malate from the roots can prevent plants from A1 toxicity because it can detoxify Al by formation of Al-activated malate transporter(ALMT1)in plants plays a.critical role in Al regulated malate release and Al tolerance in response to Al.To investigate complex regulatory mechanisms of Al-inducible expression of ALMT1,through analysis of Al-insensitive Arabidopsis mutants,this research tried to identify the genes involved in Al signal transduction and transcription factor of AtALMT1 expression.The main results were as follows:Seven Al-insensitive Arabidopsis mutants were screened,they were carrying abnormal response of Al-inducible AtALMT1 expression through a preliminary screening of EMS-mutagenized Arabidopsis carrying AtALMT1 promoter::GFP.The phenotype of mutants was observed under the Al-stress condition,all mutants more or less were inhibited compare with the wild type(WT)in root growth or hypocotyl length:MD1 showed significantly inhibition in hypocotyl length,while MD4,MD5,MD6 and MD7 displayed significantly inhibition in root growth;MD2 and MD8 could not been observed obviously inhibitory growth.Through characterization of the malate release and expression of AtALMT1 together with Al-stress related genes,such as CAMTA2,STOP1,ALS3 and MATE in the seven mutants.Except MD5,malate excretion of mutants was significantly reduced;AtALMT1 expression also decreased in different degrees.Meanwhile,according to previous studies,Al-induced AtALMT1 expression in Arabidopsis can be classified into two stages,early phase and late phase(Tokizawa et al.,2015).In this study,the screened Al-insensitive mutants can also be classified:MD1,MD2,MD5 and MD8 involved in late phase of regulation;MD4,MD6,and MD7 involved in early phase of regulation.Mutants carrying the obvious character with Al-insensitive were selected for AtALMTl expression detection under the IAA and ABA treatment.MD4,MD6 and MD7 related to IAA-inducible expression;MD1,MD4 and MD6 related to ABA-inducible expression.Furthermore,transcriptome profiling of the mutants under the Al stress,revealed that some genes like ALS3,MATE,PLT3,SULTR3 were involved in signal transduction and transcription factor activity,they were obviously difference repressed expression in MD4 and MD5 compared to the wild type and mutants.According to character separation of root length and hypocotyl length in Arabidopsis,the F2 and BC population of Al-insensitive mutants were screened which generated by artificial hybridization for based map clonig,combined with AtALMTl expression and related molecular markers,the mutation sites of 5 mutants were rough mapping.After molecular marker linkage analysis,MD1 at the first part of chromosome 4,physical map distance was 5.2 Mbp(Mega base pair);MD4,MD6,and MD7 were located on different molecular markers in the second half of the chromosome 1,physical map distance was 20.9 Mbp and 27.6Mbp respectively;MD5 has been found two mutation sites at the end position of chromosome 2 and at the middle position of chromosome 5,physical map distance was 14.7 Mbp and 15.9 Mbp respectively.According to InDel molecular marker and PCR-based fine mapping,the mutation sites of MD1 on chromosome 4 were narrowed down between 5.15 Mbp to 6.14 Mbp;the mutation sites of MD5 on chromosome 2 were narrowed down between 5.15 Mbp to 6.14 Mbp.Combining genome sequence analysis of mutant by NGS assisted in identifying the 4 and 14 candidate causative genes respectively in MD1 and MD5.Subsequently,the homozygous T-DNA insertion strains of these candidate genes were screened and investigated their Al-inhibited phenotype and AtALMT1 expression under the Al-stress,experimental results showed MEKK3,CRY1 and PGM were initially involved in the regulation of AtALMT1 expression.According to the above research results,a model of Al-responsive AtALMT1 expression in mutants was made that clarifying 7 screened mutants related to early and late phase of Al-induced AtALMTl expression.This research results will be the foundation of molecular biology and genetics experiments for further exploring the mechanism of Al-stress in plants.