Molecular Physiology Of Rice Bean VuFDH1 And VuNAC1 In Response To Al Stress

Aluminum toxicity is considered the main factor limiting crop production in acid soils. Originating from tropical and subtropical regions, rice bean (Vigna umbellata) is well adapted to acidic soils. Previous study have characterized some differentially expressed genes in early stage of rice bean tips in response to Al via Suppression Subtractive Hybridization (SSH) method. It was found that those functionally annotated genes related to’signal transduction and transcription’and’metabolism and energy’ were predominantly up-regulated, among which transcription factor NAC (VuNAC1) and formate dehydrogenase metabolism genes (VuFDHl) and so on are overrepresentated. Therefore, we studied these genes with respect to Al tolerance through Al phenotype analysis of transgenic lines overexpressing these candidate genes, and found VuNACl and VuFDH1 conferred Al tolerance to transgenic lines. We functionally characterized the two genes with respect to Al and/or H+tolerance in order to find out new clues of aluminum resistance mechanism. The results were summarized as following:(1) The full-length cDNA of VuFDH1 gene was amplified via RACE-PCR technology and the promoter sequence was isolated based on Genome Walking technology. Screening of various potential substrates for the VuFDH1 protein demonstrated that it functions as a formate dehydrogenase. Quantitative reverse transcription-PCR and histochemical analysis showed that the expression of VuFDH1 is induced in rice bean root tips by Al or H+ stresses. Fluorescence microscopic observation of VuFDH1-GFP in transgenic Arabidopsis plants indicated that VuFDH1 is localized in the mitochondria. We also found that Al rapidly induces the accumulation of formate in rice bean root tips. These results indicate that formate production may be involved in Al-induced root growth inhibition. We next found that overexpression of VuFDH1 in tobacco results in decreased sensitivity to Al and H+ stress. Compared with wild type (WT), transgenic overexpression tobacco lines produced less formate in response to Al stress. Further analysis of these overexpression lines revealed no changes (versus WT) with respect to NtMATE and NtALS3 expression, suggesting that the increased Al tolerance of these lines is not associated with an increased efflux of Al chelating substances. Thus, the increased Al tolerance of VuFDH1 overexpression lines is likely attributable to their decreased Al-induced formate production. Taken together, our findings advance understanding of higher plant Al toxicity mechanisms, and suggest a possible new route towards the improvement of plant performance in acidic soils, where Al toxicity and H+ stress co-exist.(2) The full-length cDNA of VuNACl gene was amplified via RACE-PCR technology and the promoter sequence was isolated based on Genome Walking technology. qRT-PCR analysis showed that VuNACl was significantly up-regulated by Al stress in rice bean root. The expression of VuNACl in rice bean root tip is dependent on Al treatment time and Al concentrations. Fluorescence microscopic observation of VuNACl-GFP in transgenic Arabidopsis plants indicated that VuFDH1 is localized in the nucleus. In addition, transcription activation experiments in yeast demonstrated that VuNAC1 had transcriptional activation potential. Tissue localization analysis showed that the expression of VuNAC1 in root tip was induced by Al stress. Compared with wild type of Arabidopsis, VuNAC1 overexpression plants increased its tolerance to Al stress. Further analysis of these overexpression lines revealed no changes (versus WT) with respect to the expression of aluminum tolerance genes, In addition, overexpression of VuNACl in Arabidopsis did not increase the secretion of malic acid and the root Al content. These results suggested that the increased Al tolerance of these lines is not associated with the classical mechanisms of aluminum tolerance. Therefore, we carried out the RNA-sequencing of wild type and VuNACl overexpression lines with or without Al treatment, trying to find out the underlying basis of VuNACl-meidated Al tolerance. After aluminun treatment, there are 128 up-regulated genes and 69 down regulated genes that are unique in VuNACl overexpression lines. In silico analysis revealed that there are many cis-elements in the promoters of these specific up-regulated genes that can physically interact with NAC transcription factors. Moreover, our results showed that VuNAC1 overexpression lines can reduce the aluminum content of cell wall and increase the aluminum content of cell sap, indcating that VuNAC1 confers Al tolerance through modification of cell wall property.