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Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae. Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi:H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica. However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi. A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr179 in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr179, Asp178, is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr179 then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr179 in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

Natural alloplasmic cytoplasmic male sterile (CMS) clones of industrial chicory were obtained after crossing wild chicory with selected breeding lines. We investigated the CMS stability of 10 clones in various environmental growing conditions. CMS was stable under cool growing conditions in most of them. Fertility restoration, based on pollen production scores, was observed in all clones after a period of hot temperatures. The early flower bud stage was sensitive, resulting in fertile flowers 12-17 days after exposure to high temperatures. Experiments under controlled growing conditions at 15°C demonstrated that a heat shock of 2 days at 25 or 30°C was sufficient to restore fertility. Sterile flowers were formed when plants were again grown at lower temperatures. Significant differences between individual clones were observed, indicating the potential of genetic selection to obtain stable CMS parent lines. K E Y W O R D Salloplasmic male sterility, cytoplasmic male sterility, fertility, heat shock, pollen production

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