| Aluminum(Al)toxicity is a major limiting factor for crop production on acid soils,which make up over 30%of the world's arable soils and up to 70%of the potential arable land.To grow on Al-toxic soils,some plant species have evolved resistance mechanisms that enable them to tolerate toxic levels of Al.ART1/STOP1 is a C2H2-type zinc-finger transcription factor,which is required for Al tolerance through regulation of downstream Al tolerance genes in both rice and Arabidopsis.ART1 was constitutively expressed,while genes regulated by ART1 were upregulated by Al,indicating posttranscriptional regulation of ART 1,such as interaction with other factors,was required to activate ART1.In order to find out the mechanism of transcriptional control by ART1 and to illustrate the proteins interacting with ART 1,a yeast two-hybrid system screening was performed.Tartary buckwheat(Fagopyrum tataricum)shows a high level of aluminum(Al)tolerance and accumulation.However,the molecular mechanisms for Al detoxification and accumulation are still poorly understood.To begin to elucidate the molecular basis of Al tolerance and accumulation,we used the Illumina high-throughput mRNA sequencing(RNA-seq)technology to conduct a genome-wide transcriptome analysis on both tip and basal segments of the roots exposed to Al,and studied the functions of ART1 and ALS1 homologs in buckwheat.Results were as followed:(1)A normalized cDNA libarary with total clone number be 1.5×105 CFU and average length of insertions be larger than 1 kb was constructed.By yeast Two-Hybrid system screening,we obtained 22 candidate interaction proteins.According to the gene description,10 cadidate proteins were chosen for interaction testing in yeast,with ART1 be prey and these 10 proteins be baits.Results showed that OsVOZl,which is a transcription factor with one zinc finger,interact strongly with ART1 in yeast,and OsVOZ2,OsVOZl's homolog,showed transcription activation itself.Split-LUC assay confirmed the interaction between OsVOZ1/OsVOZ2 and ART1 in vivo in tobacco epidermis cells.In rice,neither OsVOZ2 nor OsVOZlFs expression was induced by Al.In order to find out if OsVOZI and OsVOZ2 function in Al resistance in rice,Al tolerace of osvozl;osvoz2 and osvoz2;OsVOZ1-Riwas investigated.Results came out to be that osvozl and osvoz2 showed similar Al tolerance as WT;osvoz2;OsVOZ1-Ri exhibited enhansed Al tolerance than WT.RT-PCR analysis showed that expression of ART1 was upregulated by Al in osvoz2;OsVOZ1-Ri,indicating that OsVOZl and OsVOZ2 possibaly act as the negative effector to ART 1 in rice.Double mutant atvoz1atvoz2 in Araidopsis also exhibited enhanced Al tolerance,implying that VOZ1 and VOZ2 are functionaly concerved in both rice and Arabidopsis.(2)Nearly 40,000 high quality transcript contigs were de novo assembled for tartary buckwheat,providing a reference platform for future research work in this plant species.Expression date showed that expression of genes involved in the defense of cell wall toxicity and oxidative stress was preferentially induced by Al stress.It is also revealed that organic acid metabolism was unlikely to be a rate-limiting step for the Al-induced secretion of organic acids in buckwheat.We identified two citrate transporter genes,FtFRDL1 and FtFRDL2,which belong to MATE family and FRD3 subfamily and were highly induced by Al,indicating that they were potentially involved in the release of citrate into the xylem.In addition,three of four conserved Al-tolerance genes were found to be duplicated in tartary buckwheat and display diverse expression patterns.(3)We identified two homologs of ART1,namely FtARL1 and FtARL2(ART1-Like)in buckwheat.Similar to ART1/STOP1,FtARL1 and FtARL2 contains four highly conserved C2H2 domain.Phylogenetic analysis indicated that both FtARLl and FtARL2 are closer to Arabidopsis STOP1 than to rice ART1.Real-time RT-PCR analysis showed that FtARLl was constitutively expressed in roots like ART 1/STOP 1,while FtARL2 was slightly upregulated by Al.To examine the subcellular localization,we transiently introduced a gene fused between FtARLl/FtARL2 and YFP in tobacco epidermis cells.FtARL2 was localized to nuclei,while the YFP-FtARLl/FtARLl-YFP yellow fluorescence was observed in both nuclei and cytoplasm.To study the gene function,we introduced FtARLl into Arabidopsis stopl mutant under the regulation of STOP1 promoter.Results showed that FtARLl could completely recover H+ sensitive phenotype of stopl by greatly recovering the H+tolerance genes that are suppressed in stopl,whereas Al sensitivity of stopl was only partially rescued through partially recovering AtALMT1;AtMATE and ALS3.(4)There were two ALS1 homologs found in buckwheat.FtALOLl(ALS One-Like 1)is closer to rice OsALS1 whereas FtALOL2 is closer to Arabidopsis AtALS1.Expression analysis showed that FtALOL2 expression was induced by Al stress specifically and subtly,10 ?M Al can fully induce FtALOL2's expression.FtALOL2 was downregulated by A1 and upregulated by Cu.To study the cellular localization of FtALOL1,we expressed GUS driven by 2 kb promoter of FtALOL1 in buckwheat root.GUS was detected mainly in the stele of buckwheat root.N-terminal FtALOLl/FtALOL2 fusions of YFP were introduced into epidermis cells of tobacco leaf,the yellow florescence signal from YFP-FtALOL1 was observed in plasma membrane,while the YFP signal from YFP-FtALOL2 showed misaggregates in tobacco cells.Heterogenous expression of FtALOL2 resulted in increased Al sensitivity in yeast,implying that FtALOL2 is probably a transporter for Al.FtALOL1 could not recover Al sensitive phenotype of als1-1 als1-1;FtALOL2 lines even showd higher Al sensitivity than alsl-1.In conclusion,OsVOZ1 and OsVOZ2 that can interact with ART1 possibaly act as the negative effector to ART1 in rice,and they are functionaly redundant.VOZ1 and VOZ2 are functionaly concerved in both rice and Arabidopsis.There are two copies of ART1 and ALS1 genes in tartary buckwheat and display diverse expression patterns and subscellular localization,which might play an important role in the high Al tolerance of buckwheat. |
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