Characterization of a genistein transporter in cluster roots of white lupin

White lupin (Lupinus albus L.) has developed a highly efficient strategy to mobilize nutrients from soil, in particular when P is scarcely available. This strategy is based on the modification of the root architecture with the formation of cluster roots. From these structures, a huge quantity of root exudates (organic acids and flavonoids) are released. These exudates mobilize sparingly available nutrients in soils via complexation, ligand exchange and, in the case of flavonoids, reduction . These secondary metabolites can also influence the biological characteristics of the rhizosphere, affecting the presence and activity of the microorganisms. Hence acquiring a better comprehension of physiological, biochemical and molecular mechanisms that regulates roots exudation processes is fundamental for the improvement of the mineral nutrition of plants. It is well known that in P-deficient conditions, in the mix of flavonoids released by white lupin, genistein is the one mainly rappresented and it is exuded mainly from the early stages of the proteoid roots; nevertheless up to now there are no data on proteins involved in this processes even if in some plant species transmembrane flavonoid transport via MATE or ABC transporters has been already characterized. The aim of this work is the physiological, biochemical and molecular characterization of a protein putatively involved in the release of genistein from white lupin plants. Via cDNA-AFLP a gene sequence sharing a high similarity with known MATE proteins involved in flavonoid transport had been isolated. This gene was called LaMATE2 and analyses of its expression shown that it is upregulated in P-deficient white lupin roots. To investigate the role of LaMATE2 in genistein exudation, a RNA interference approach was used with the aim to knock down the expression of this gene and to analyses its effect on the exudate release activity of the transformed roots. qPCR results confirmed that the LaMATE2 expression was reduced and HPLC-analyses of the composition of root exudates revealed that these roots released less genistein. To biochemically characterize the activity of LaMATE2, the full-length cDNA of the gene was clone and expressed in Saccharomyces cerevisiae. The membrane vesicles isolated from transformed yeast were then used to perform transport assay of 3H-genistein. Microsomes isolated from yeast cells that express LaMATE2, compared to the control, accumulated a higher amount of the flavonoid in short time (30 s) and long time of the reaction (10 min). These data demonstrate that LaMATE2 protein is involved in the transport of genistein. Finally, it has been demostrated to be localized on the plasma membrane of Arabidopsis transformed protoplast