Molecular characterisation of the Arabidopsis TT12 MATE protein and functional analysis of other plant MATE transporters

Abstract

Flavonoids are a diverse group of plant secondary metabolites that accumulate in a variety of plant tissues and play many roles in important processes including stress protection, defence and seed dispersal. To accomplish many of these functions, flavonoids accumulate at high concentrations. However, the denaturing properties of these phenolic substances make them toxic to the cytosol. Therefore, flavonoids undergo secretion into the extracellular space or accumulate in the vacuole. The knowledge of transport mechanisms involved in the accumulation of flavonoids is limited, even though the biosynthetic pathway is well known. Recent reports suggest that MATE proteins are involved in flavonoid transport and act as cation H+/Na+-antiporters. However, the identification of conserved amino acid residues or domains essential to a transport function remains poorly characterised, with no reports for plant MATEs. TT12 is a vacuolar flavonoid/H+-antiporter expressed exclusively in the seed coat endothelium actively involved in the transport of glucosylated anthocyanins (Marinova et al, 2007). In this work we describe strategies to (i) functionally identify important amino acid residues using TT12 as a model for plant MATE family, (ii) functionally characterise two novel MATE transporters in Lupinus albus implicated in citrate and isoflavonoids transport and (iii) identify novel flavonoid transporters within the 56 members of the Arabidopsis MATE family. (i) Since TT12 gene was well characterised in our laboratory and due to its easily scorable seed phenotype, we decided to use TT12 as a model to investigate which amino acids are crucial for proper functioning of plant MATE transporters. Given that transport critical residues for NorM from Vibrio parahaemolyticus and hMATE1 from Homo sapiens were identified and we found an amino acid residue homologous to them in TT12 (E290), we performed site-directed mutagenesis on E290 and nine others amino acid residues. In the range of experiments we searched specially for the mutated versions of TT12 which were unable to functionally complement the PA deficient seed phenotype of tt12 mutant. We describe that only a single amino acid (E290) failed to complement the tt12 seed phenotype and further show that this mutated version completely lacks transport activity on cyanidin 3-glucoside (the substrate for the native TT12). (ii) White lupins characteristically produce cluster roots under phosphate starvation. These cluster roots are known to excrete both citrate and isoflavonoids into the rhizosphere. Previous work within the group identified two full length cDNAs expressed in cluster roots. These were designated LaMATE1 and -2. We investigated if these cDNAs, when heterogously expressed in yeast, were able to transport either citrate or the isoflavonoid, genistein. We describe that LaMATE1 was unable to transport citrate but LaMATE2 transported genistein, when uptake experiments were conducted with microsomes. (iii) Using previously published data and in silico sequence analyses, we selected three candidate genes for novel flavonoid transporters. AtDTX35 (At4g25640) has been reported to be homologous to a tomato MATE which is upregulated in the tomato mutant, ANT1 encoding proteins involved in the anthocyanin biosynthesis (Mathews et al, 2003). We started our approach with downregulation of DTX35 gene with RNAi in PAP1 overexpressing line, which due to hyperaccumulation of anthocyanin exhibits purple pigmentation. Interestingly, numerous lines carrying the RNAi construct reverted from purple color to green. We describe that DTX35 was unable to complement tt12 seed phenotype and its promoter tissue activity is not specific in young seedlings. AtDTX31 (At1g12950) has been shown to be transcriptionally upregulated in Arabidopsis roots under salt treatment and displayed a similarity to proteins predominantly expressed in tomato fruits (Maathuis, 2006). To investigate if DTX31 is expressed in salt stress conditions, we performed sqPCR analyses and examined if DTX31 promoter activity is also tissue specific. We describe that DTX31 is highly expressed in the roots of Arabidopsis subjected to salt stress and its promoter‘s spatial activity in response to high concentration of salt. AtDTX28 (At5g44050) has been reported to be induced after UV-B treatment, which is a main reason for increase in the transcript abundance of genes, including MYB12 (the transcription factor) which are involved in the flavonol biosynthesis. We investigated if DTX28 expression in MYB12 overexpressing line, myb12 knock-out line and triple knock-out line myb11-myb12-myb111 is different in comparison to the wild-type line by sqPCR and HPLC analyses. We describe that DTX28 does not show higher transcript level in MYB12 overexpressing lines compared to the wild-type and no differences were observed in metabolic profiles between mutants and a wild-type.