Mechanistic connections between the proton motive force and ATP homeostasis in higher plant photosynthesis under dynamic environmental conditions

Through photosynthesis, plants can capture light energy from the sun for the conversion to a more stable high-energy form, ATP and NADPH. These products are then used to fuel an array of metabolic processes including the biosynthesis of sugars and complex carbohydrates. Yet, the abundant source of solar energy used in the process is highly varied and fluctuates throughout the day, directly impacting the photosynthetic apparatus and carbon assimilation. This dissertation focuses on several mechanisms by which plants are able to respond to the dynamic environmental pressures through modulation of the proton motive force (pmf) and ATP homeostasis.ATP is the primary energy currency in cells and is synthesized in plastids by the chloroplast ATP synthase. However, unlike other stromal thiol-regulated enzymes that incrementally become redox-activated in response to light, chloroplast ATP synthase acts more like an on-off switch, only requiring minimal irradiance to become fully active. Previous work suggested that the rapid sensitivity to light could be explained by the relative redox potentials of the regulatory thiols on the γ-subunit of ATP synthase. This work uncovered a new, unexpected component, NADPH thioredoxin reductase C (NTRC) that controls thiol regulation specifically under low light intensities. Mutants lacking NTRC show strong photosynthetic phenotypes, e.g., increased nonphotochemical quenching and inhibition of linear electron flow, at low irradiances, consistent with an inability to activate ATP synthase resulting in a buildup of the thylakoid pmf. We predict both NTRC and the canonical ferredoxin-thioredoxin reductase system co-regulate the thiol state of ATP synthase at specific light intensities using different reducing potentials (NADPH versus ferredoxin) that allow for added flexibility.Photosynthesis copes with, and adapts to, fluctuating environments using a wide range of mechanisms. While most of the research has been devoted to the processes occurring inside the plastid, work described here on the nucleotide triphosphate transporter (NTT) illuminates an additional mechanism of augmenting and balancing ATP. Previous work suggested that the chloroplast transporter, NTT, acted primarily as an importer of ATP during the night cycle, presumably under non-photosynthesizing conditions. However, isolated intact chloroplasts from both spinach and Arabidopsis thaliana export ATP at rapid rates that can constitute a large fraction of that generated by the light reactions. Furthermore, these findings suggest that earlier results of minimal rates of ATP transport were based on suboptimal assay conditions and incorrect characterization of T-DNA knockout lines, rendering NTT essential for seed germination. Work on double NTT knock-down lines (NTTdKD) have decreased gene expression levels of ntt1 and ntt2 and show strong photosynthetic responses, particularly in the pH and energy-dependent quenching response (qE) with related accumulation of the pmf under fluctuating light and/or decreased CO2 levels. These results indicate a greater role for NTT in balancing ATP levels between the stromal and cytosolic pools than previously thought.