Functions And Expression Regulation Of MATE Genes In Rice Bean (Vigna Umbellata)

Aluminum toxicity is one of the main factors limiting crop production on acidic soils. Plants have developed mechanisms in their own way for the better adaption to grow on acidic soils. To date, the physiological and molecular mechanisms of aluminum-activated organic acid secretion have well been documented. Rice bean (Vigna umbellata), as an Al tolerance plant species, is well adapted to acidic soils by means of secretion of citrate from root tips under the regulation of VuMATEl. VuMATEl, a gene responsible for Al-inducible citrate secretion from rice bean roots, is not expressed in the absence of Al stress which is distinct fromother known MATEs. Howerver, the process leading to VuMATE1expression involved in the citrate secretion remains unclear. This study sought to clarify the interplay between VuMATEl expression and citrate secretion in rice bean to elucidate the underlying regulatory mechanisms, as well as the potential functions of MATEs in rice bean. The main results were as followed:1. The role of VuMATE1expression in aluminium-inducible citrate secretion in rice bean (Vigna umbellata) rootsAluminium(Al)-activated citrate secretion plays an important role in Al resistance for a number of plant species such as rice bean. Here, we further characterized the regulation of VuMATEl, an aluminum-activated citrate transporter. Al stress induced VuMATEl expression, followed by the secretion of citrate. Citrate secretion was specific to Al stress, whereas VuMATE1expression was not, which could be explained by a combined regulation of VuMATE1expression and Al-specific activation of VuMATE1protein. Pretreatment with protein translation inhibitor suppressed VuMATE1expression, indicating that de novo synthesis of proteins is required for gene expression However, posttreatment with protein translation inhibitor inhibited citrate secretion, indicating that posttranscriptional regulation of VuMATEl is critical for citrate secretion. Protein kinase and phosphatase inhibitor studies showed that reversible phosphorylation was important not only for transcriptional regulation of VuMATE1expression but also for posttranslational regulation of VuMATE1protein activity. These results suggest that citrate secretion is dependent upon both transcriptional and posttranscriptional regulation of VuMATE1. Furthermore, VuMATEl promoter-β-glucuronidase fusion lines revealed that VuMATEl expression was restricted to the root apex and entirely Al induced, indicating the presence of cis-acting elements regulating root tip-specific and Al-inducible gene expression, which will be an important resource for genetic improvement of plant Al resistance.2. Isolation of root tip-specific and Al-inducible cis-elements in VuMATEl promoter regionThe2Kb VuMATE1promoter sequence was abtained using the genome-walker method and the potential transcription start site (TSS) A residue were determined by5’RACE technology. Lots of stress responsive cis-elements were identified in the VuMATE1promoter region using both the PLACE and PlantCARE databases, including the reported cis-element GGN(T/g/a/c)V(C/a/g)S(C/G) of ART1. To identify cis-acting elements regulating root tip-specific and Al-inducible of VuMATEl expression, deletion analysis in the promoter region of VuMATEl was conducted.9promoter deletions::GUS&one VuMATE1P2Kb::GUS plasmids were constructed and then transformed into Arabidopsis. The abtained transgenic lines included-1720P::GUS2lines,-1720P-Intron::GUS (intron deleted)2lines,-1330P::GUS10lines,1269P::GUS2lines,-1228P::GUS9lines,-624P::GUS8lines,-574P::GUS8lines,-254P::GUS3lines,-192P::GUS3lines&-115P::GUS6lines. Studies of GUS expression among all these transgenic lines under Al treatment indicated that a sequence between-1228and-574relative to the TSS of VuMATE1promoter was sufficient for root tip-specific and Al-inducible of VuMATEl expression. The yeast one-hybrid assay uncovered that promoter region Al&A2which contained the ART1cis-element could not interact with VuSTOP1transcription factor. However, A3promoter fragment which did not contain the ART1cis-element could interact with VuSTOP1. These results explored that the potential root tip-specific and Al-inducible cis-elements may be some novel elements dinstinct from the ART1cis-element. The cis-acting elements regulating root tip-specific and Al-inducible gene expression may be the same one or belong to different kinds of cis-elements. 3. Functional characterization of VuMATEl in iron efficiency in rice beanDepending on localization and expression pattern, citrate-transporting multidrug and toxic compound extrusion proteins have been demonstrated to play at least two physio logical roles i.e. detoxification of aluminum (Al) and improvement of iron (Fe) efficiency. VuMATE1, an Al-activated citrate transporter in rice bean (Vigna umbellata) was found to be constitutively expressed in the vasculature of mature root zone, implying that VuMATE1may be involved in Fe efficiency. Here, we found that VuMATE1was expressed in vasculature throughout the whole plant body in VuMATE1p2Kb::GUS transgenic, Arabidopsis lines, excepting root apex where vascular tissues are poorly developed. Analysis with VuMATE1P2Kb::GFP transgenic Arabidopsis lines revealed that VuMATEl was not expressed in root apex without Al stress, but could be greatly induced by Al stress; however, in the mature root zone VuMATE1was weakly expressed in root hairs, epidermis and vasculature, which was independent of Al stress. Unexpectedly, introduction of VuMATE1into frd3-1mutant that is defective in Fe translocation could not rescue the phenotype. Cell-specificity of localization of VuMATEl by immunostaining revealed that it was expressed at cells near xylem vessels, outmost layer of cortex, and epidermis in rice bean. In addition, neither was the secretion of citrate stimulated by Fe deficiency, nor the expression of VuMATEl. These results suggest that VuMATE1expression is evolved for the better adaptation of rice bean to grow on acidic soils.4. Cloning and functional investigation of VuMATE2in rice beanThe full length cDNA sequence of VuMATE2was2097bp containing a1605bp open reading frame (ORF) which encoded a534amino acid protein with12predicted transmembrane domains. The DNA sequence of VuMATE2spans5615bp,14extrons and13introns included. Phylogenetic analysis with other reported MATE proteins indicated that VuMATE2may play an additional role in the regulation of citrate exudation in rice bean apart from VuMATE1, as it showed little identity with VuMATEl but shared high homology with AtMATE and ZmMATEl and both belonged to different clusters. Subcellular-localization of VuMATE2in vivo revealed that it localized in plasma membrane. The expression level of VuMATE2which was constitutively expressed in root apex and showed low transcribed level in root mature region&leaf in rice bean was greatly induced under Al treatment within2h. Meanwhile, just the trivalent Al3+ion could induce a higher expression level of VuMATE2, while other metal ions (La3+, Cd2+, Cu2+) could not. Overexpression of VuMATE2in Arabidopsis wild type Col-0and complementation test in AtALMTl-KO/AtMATE-KD double mutant indicated that VuMATE2could increase the tolerance of transgenic plants to Al treatment. Taken together, VuMATE2may exert distinct roles in regulation of Aluminum tolerance in rice bean at early aluminum toxicity stage.