Structural basis of redox modulation on chloroplast ATP synthase

In higher plants, chloroplast ATP synthase has a unique redox switch on its γ subunit that modulates enzyme activity to limit ATP hydrolysis at night. To understand the molecular details of the redox modulation, we used single-particle cryo-EM to determine the structures of spinach chloroplast ATP synthase in both reduced and oxidized states. The disulfide linkage of the oxidized γ subunit introduces a torsional constraint to stabilize the two β hairpin structures. Once reduced, free cysteines alleviate this constraint, resulting in a concerted motion of the enzyme complex and a smooth transition between rotary states to facilitate the ATP synthesis. We added an uncompetitive inhibitor, tentoxin, in the reduced sample to limit the flexibility of the enzyme and obtained high-resolution details. Our cryo-EM structures provide mechanistic insight into the redox modulation of the energy regulation activity of chloroplast ATP synthase.

Alpha-subunit of the Chloroplast ATP Synthase of Tomato Reinforces the Resistance to Grey Mold and Broad-spectrum Resistance in Transgenic Tobacco

Chloroplast ATP synthase (cpATPase) is responsible for ATP production during photosynthesis. Our previous studies showed the cpATPase CF1 alpha subunit (AtpA) is a key protein involved in Clonostachys rosea (C. rosea)-induced resistance to the fungus Botrytis cinerea (B. cinerea) in tomato. Here, we show the expression level of tomato’s cpATPase CF1 alpha subunit gene (atpA) was up-regulated by B. cinerea and C. rosea. The tomato atpA gene was then isolated, and transgenic tobacco lines were obtained. Compared with untransformed plants, the atpA-overexpressing tobacco showed an increased resistance to B. cinerea, characterized by reduced disease incidence, defense-associated hypersensitive response (HR)-like reactions, balanced reactive oxygen species, alleviated damage to chloroplast ultra-structure of leaf cell, elevated levels of ATP content and cpATPase activity, enhanced expression of carbon metabolism-, photosynthesis-, and defense-related genes. Incremental Ca2+ efflux and steady H+ efflux were observed in transgenic tobacco after their inoculation with B. cinerea. Additionally, overexpression of atpA gene conferred enhanced tolerance to salinity and resistance to the fungus Cladosporium fulvum. Thus, the α subunit of cpATPase is a key regulator that links signaling to cellular redox homeostasis, ATP biosynthesis, and gene expression of resistance traits to modulate immunity to pathogen infection, in the process providing broad-spectrum resistance in plants.

ECS-based investigation of chloroplast ATP synthase regulation

The chloroplast ATP synthase (CF1Fo) contains a specific feature to the green lineage: a γ-subunit redox domain which contains a cysteine couple and interacts with the torque-generating βDELSEED-loop. Based on the recently solved structure of this domain, it was proposed to function as a chock. In vitro, γ-disulfide formation slows down the activity of the CF1Fo at low transmembrane electrochemical proton gradient . Here, we utilize in vivo absorption spectroscopy measurements for functional CF1Fo activity characterization in Arabidopsis leaves. The spectroscopic method allows us to measure the present in dark-adapted leaves, and to identify its mitochondrial sources. Furthermore, we follow the fate of the extra generated by an illumination, including its osmotic and electric components, and from there we estimate the lifetime of the light-generated ATP. In contrast with a previous report [Joliot and Joliot, Biochim. Biophys. Acta, 1777 (2008) 676-683], the CF1Fo γ-subunit exists mostly in an oxidized form in the dark-adapted state. To study the redox regulation of the CF1Fo, we used thiol agent infiltration in WT and a mutant that does not form the γ-disulfide. The obtained -dependent CF1Fo activity profile in the two γ-redox states in vivo reconciles with previous biochemical in vitro findings [Junesch and Gräber, Biochim. Biophys. Acta, 893 (1987) 275-288]. The highest rates of ATP synthesis we measured in the two γ-redox state were similar at high . In the presence of the γ-dithiol, similar rates were obtained at a ~45 mV lower value compared to the oxidized state, which closely resembled the energetic gap of 0.7 ΔpH units reported in vitro.