Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor

Authors: DOSHIM, RUPAK - University Of California; MCGRATH, AARON - University Of California; Pineros, Miguel; SZEWCZYK, PAUL - University Of California; GARZA, DENISSE - University Of California; KOCHIAN, LEON - Former ARS Employee; CHANG, GEOFFREY - University Of California

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/1/2017
Publication Date: 12/21/2017
Citation: Doshim, R., McGrath, A., Pineros, M., Szewczyk, P., Garza, D., Kochian, L., Chang, G. 2017. Functional characterization and discovery of modulators of SbMATE, the agronomically important aluminium tolerance transporter from Sorghum bicolor. Scientific Reports. 7(17996). https://doi.org/10.1038/s41598-017-18146-8.
DOI: https://doi.org/10.1038/s41598-017-18146-8

Interpretive Summary: Over 20% of the US land area and approximately 50% of the world's arable lands are acidic (pH < 5). On these acid soils, aluminum (Al) toxicity is the primary factor limiting agricultural productivity, as toxic Al results in damaged and stunted plant root systems, ultimately resulting in a reduction of crop yields. The release of Al detoxifying organic acids from the root apex in response to Al-stress constitutes a widespread Al-resistance mechanism by which plant roots are able to ameliorate the toxic levels of Al surrounding the growing root. Several Al tolerance genes encoding for membrane proteins involved in the transport of these organic acids out of the root have been identified and cloned. In sorghum, the major Al tolerance gene, SbMATE, encodes an Al-activated root citrate transporter. Our study describes a detailed characterization of the SbMATE, an agronomically important aluminium tolerance transporter from the cereal crop Sorghum bicolor. SbMATE mediates efflux of the anionic form of citrate into the soil rhizosphere, chelating aluminum ions and imparting Al-resistance based at the root tip. As such, Al tolerance conferred by SbMATE is important for sorghum production and food security on acid soils, many of which are located in developing countries. Through this study we have obtained a broader understanding of SbMATE's functionality by studying its transport characteristics in a series of biochemical and biophysical assays. We also demonstrate that the SbMATE protein can be highly expressed using the yeast Pichia pastoris and, a use the purified SbMATE transporter as an antigen for discovery of single-domain antibodies, which upon binding alters its transport characteristics. Our study broaden the understanding of SbMATEs important role in Al tolerance, and prompts further investigations regarding the physiological role of this family of transporters in plants extending that of other MATE transporter systems found throughout other kingdom.

Technical Abstract: About 50% of the world's arable land is strongly acidic (soil pH < 5). The low pH of these soils solubilizes root-toxic ionic aluminium (Al3+) species from clay minerals, driving the evolution of various counteractive adaptations in cultivated crops. The food crop Sorghum bicolor, for example, upregulates the membrane-embedded transporter protein SbMATE in its roots. SbMATE mediates efflux of the anionic form of the organic acid, citrate, into the soil rhizosphere, chelating Al3+ ions and thereby imparting Al-resistance based on excluding Al+3 from the growing root tip. Here, we use electrophysiological, radiolabeled, and fluorescence-based transport assays in two heterologous expression systems to establish a broad substrate recognition profile of SbMATE, showing the transport of 14C- citrate anion, as well as the organic monovalent cation, ethidium, but not the divalent ethidium-derivative, propidium. The transport cycle is proton and/or sodium-driven, and shares certain molecular mechanisms with bacterial MATE-family transporters. We further complement our transport assays by directly measuring substrate binding to detergent-purified SbMATE protein. Finally, we use the functionally-folded, purified membrane protein as an antigen to discover high-affinity, native conformation-binding and transport function-altering nanobodies using an animal-free, mRNA/cDNA display technology. Our results demonstrate the utility of using Pichia pastoris as an efficient eukaryotic host to express large quantities of functional plant transporter proteins for in vitro characterization. The nanobody discovery approach is applicable to other low immunogenic plant proteins.