Identification And Functional Analysis Of Early Aluminum-responsive Genes In Rice Bean(Vigna Umbellata) Root Tips

Rice bean (Vigna umbellata) is a leguminous species well adapted to acid soil. Previous studies uncovered that Al induced secrete citrate from the root apex of rice bean is delayed several hours, suggesting de novo protein synthesis is required for the secretion. However, the process leading to the gene expression involved in the secretion remains not fully understood. VuMATEl that mediating Al induced citrate secretion is not transcribed in the root apex in the absence of Al stress, however, induced greatly by Al stress.Therefore, identification of early responsive genes associated with A1stress should provide valuable information on how the secretion of citrate from rice bean roots is regulated. Severe A1toxicity that induced inhibition of root elongation usually associates with generation of the secondary stress, which is difficult to distinguish primary targets of Al toxicity. In this study, to find new components regulating citrate secretion and probably molecular mechanism associated with A1tolerance and toxicity in rice bean, we characterized some differentially genes in ealy stage of rice bean tips in response to Al by Suppression Subtractive Hybridization (SSH). Meanwihle, a gene involved in regulation of citrate secretion potentially, VuSTOPl, was further studied. The results were summarized as following:(1) Successfully constructed forward and reverse SSH libraries from rice bean apex after treatment with5μM-and25μM concentrations of AlCl3for4h. In combination with reverse Northern blots and real-time PCR identified815differentianlly expressed ESTs from libiraires, and represented394unigenes.The unigenes were functionally categorized according to the GO biological process, and classified into10functional categories, including’Signal transduction and transcription’,’Transport’,’Stress/defense response’,’Metabolism and energy’,’Cell wall synthesis and organization’,’Cell division and cytoskeleton’,’Protein translation, processing and degradation’,’DNA and RNA processing’,’Unknown’and ’No hit’. Among functionally annotated genes, those related to ’metabolism and energy’,’signal transduction and transcription’ and ’transport’ was predominantly up-regulated, whereas those associated with ’protein translation, processing and degradation’was predominantly down-regulated. Comparative analysis of transcriptional profiles highlighted up-reglated genes involvoing citrate sectretion and regulation play important roles in rice bean in response to low and high concentrations of Al. The changes of energy metabolism and protein synthesis are probably autonomous adaptive mechanism in rice bean under Al stress and mainly related to A1tolerance. High concentrations of Al that induced inhibition of root elongation significantly may be associated with down-regulated expression of genes whose function in transport of water and mineral elements, as well as cell division.(2) Based on identified EST sequence, we amplified a orthologous gene of. STOP1like from rice bean by Rapid-amplification of cDNA ends (RACE) PCR, and designated VuSTOP1. Sequence analysis indicated that VuSTOPl cDNA has a total length of1772bp, and can be cut to two transcripts. VuSTOP1.1with an open reading frame of1497bp is predicted to encode498amino acid residues, while VuSTOP1.2with an open reading frame of1263bp is predicted to encode420amino acid residues. Amino acid alignment and phylogenetic tree showed that VuSTOP1contains four conserved zinc-finger domains that were highly homologous to those found in previously identified functional STOP1-like proteins. On the evolutionary relationships, the closest relationship is common bean, followed by soybean. Southern blots analysis showed VuSTOP1is a single gene in rice bean. Real-time PCR analysis showed VuSTOP1.1had similar expression pattern with VuSTOP1.2. Both of them were mainly expressed in root of rice bean, in wich expreesion abundance in root apex (0-1cm) was higher than basal root (1-2cm), but very low in leaf. In addition, A1and protein synthesis inhibitors (Cycloheximide) treatment could induce significantly increased expression levels of VuSTOP1.1and VuSTOP1.2, while low pH and cadmium treatment slightly up-regulated their expression. The transient expression assays with GFP fusion protein in onion and Nicotina benthamiana epidermal cell showed VuSTOP1located in nucleus. Transcriptional activation assay in yeast indicated VuSTOPl had two transcriptional activation domain locating on the N-terminal (1-78amino acid residues) and C-terminal (389-498amino acid residues).Complementation assays by introducing VuSTOPl into an Arabidopsis stop1mutant under the regulation of the AtSTOPl promoter showed VuSTOPl could greatly recover H+sensitive phenotype of stopl mutant though recovering transcription of H+tolerant genes that are suppressed in stopl mutant, whereas only partially Al sensitive phenotype of stop1mutant was recoved by transcription activation of AtMATE and ALS3.