Proof of concept for a novel functional screening system for plant sucrose effluxers

Abstract

Membrane transporters play pivotal roles in facilitating sucrose transport in plants and their activities have been shown to impact plant growth rates and crop yield. In contrast to the well-defined mechanism of sucrose influx across plasma membranes, less is known about sucrose efflux mechanisms and the membrane proteins supporting this function. A major impediment blocking progress in this key area of plant science is the absence of a functional screening system for genes encoding sucrose effluxers. Here we report a novel yeast system for screening sucrose effluxers based on sucrose release from yeast cells genetically modified to synthesize, but not to metabolize, sucrose. Inhibiting sucrose metabolism was achieved using yeast strains, SEY 6210 and YSL4-6, carrying mutations in genes encoding invertase and maltase, respectively. Genes encoding essential components of sucrose biosynthesis, sucrose phosphate synthase (SPS) and sucrose phosphate phosphatase (SPP), were used to transform the two yeast hosts to make strains SuPy (from SEY6210) and Ysu (from YSL4-6). Cultures of SuPy15 cells were found to be capable of synthesizing sucrose when supplied with various compounds as the sole carbon source, including non-fermentable sugars and non-sugar substrates. A proof of concept of the screening system was demonstrated by transforming SuPy15 with sucrose transporter genes known to encode plasma membrane proteins that mediate sucrose efflux. The robustness of the yeast SuPy15 system as a novel platform to screen putative plant sucrose effluxers is discussed.