A Multidrug and Toxic Compound Extrusion Transporter, RgMATE6, Facilitates Vacuolar Transport of Acteoside in Rehmannia glutinosa

Acteoside (ACT), a prominent compound of the hydroxytyrosol-type phenylethanol glycoside (HPG) class, is present in plants and holds significant potential for food and pharmaceutical applications. However, the limited production of ACT in plants restricts its broader utilization. Although the biosynthetic pathways of ACT are increasingly understood, its transport mechanisms within plants remain unclear. RgMATE6, a vacuolar-type Multidrug and Toxic Compound Extrusion (MATE) transporter identified in <i>Rehmannia glutinosa</i> (a plant known for ACT p roduction), was selected for investigation. This study aims to elucidate the role of RgMATE6 in ACT transport and its impact on ACT biosynthesis. Our study utilized a multidisciplinary approach, including in silico analysis to predict substrate specificity, quantitative real-time PCR (qRT-PCR) to quantify gene expression, HPLC to measure HPG levels, vacuolar membrane vesicle uptake assays to validate RgMATE6 transport activity in vitro, and genetic transformation in <i>R. glutinosa</i> to assess its functional roles in vivo. In silico analysis identified RgMATE6 as a phenolic compound transporter, and correlation analysis revealed a strong positive association between the HPG accumulation and <i>RgMATE6</i> expression in <i>R. glutinosa</i>. Functional validation through vacuolar membrane vesicle uptake assays in <i>Nicotiana benthamiana</i> confirmed RgMATE6's role as an HPG transporter, demonstrating a significant preference for ACT. Overexpression and repression experiments in <i>R. glutinosa</i> further demonstrated that RgMATE6 facilitates ACT import into vacuoles and enhances its production. Additionally, tissue-specific expression analysis revealed the coordinated expression patterns between <i>RgMATE6</i> and six ACT biosynthetic genes in the transgenic plants. RgMATE6 facilitates the transport and accumulation of ACT within vacuoles, and its expression might synergize with ACT biosynthesis. These findings establish a framework for improving ACT and other HPG production through targeted manipulation of plant MATE transporters.