Activity Of Chloroplast ATP Synthase Research Articles

Regulation of Chloroplast ATP Synthase Modulates Photoprotection in the CAM Plant Vanilla planifolia.

Generally, regulation of cyclic electron flow (CEF) and chloroplast ATP synthase play key roles in photoprotection for photosystems I and II (PSI and PSII) in C3 and C4 plants, especially when CO2 assimilation is restricted. However, how CAM plants protect PSI and PSII when CO2 assimilation is restricted is largely known. In the present study, we measured PSI, PSII, and electrochromic shift signals in the CAM plant Vanilla planifolia. The quantum yields of PSI and PSII photochemistry largely decreased in the afternoon compared to in the morning, indicating that CO2 assimilation was strongly restricted in the afternoon. Meanwhile, non-photochemical quenching (NPQ) in PSII and the donor side limitation of PSI (Y(ND)) significantly increased to protect PSI and PSII. Under such conditions, proton gradient (∆pH) across the thylakoid membranes largely increased and CEF was slightly stimulated, indicating that the increased ∆pH was not caused by the regulation of CEF. In contrast, the activity of chloroplast ATP synthase (gH+) largely decreased in the afternoon. At a given proton flux, the decreasing gH+ increased ∆pH and thus contributed to the enhancement of NPQ and Y(ND). Therefore, in the CAM plant V. planifolia, the ∆pH-dependent photoprotective mechanism is mainly regulated by the regulation of gH+ rather than CEF when CO2 assimilation is restricted.

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.

Balancing light use efficiency and photoprotection in tobacco leaves grown at different light regimes

In this study, we have compared photosynthetic regulation in tobacco leaves (Nicotiana tabacum L.) grown under high light (HL) and low light (LL) by measuring chlorophyll fluorescence, P700 redox state and electrochromic shift signals. Under high light, HL-plants had much higher linear electron flow and cyclic electron flow (CEF) around photosystem I (PSI) than LL-plants. Meanwhile, HL-plants showed significantly lower proton gradient (ΔpH) across the thylakoid membranes, owing to the increased activity of chloroplast ATP synthase. The relationships between ΔpH, non-photochemical quenching (NPQ) and PSI donor side limitation [Y(ND)] differed between HL- and LL-plants. At a given ΔpH, HL-plants displayed higher NPQ and Y(ND). Furthermore, at a given CEF, LL-plants showed higher ΔpH and Y(ND). These results indicate that HL-plants down-regulate ΔpH to increase light use efficiency by enhancing the activity of chloroplast ATP synthase. In contrast, LL-plants decrease the activity of chloroplast ATP synthase to up-regulate ΔpH and to favor photoprotection. Our findings suggest that the coordination of CEF and chloroplast ATP synthase balances light use efficiency and photoprotection at different growth light conditions.

The decline in photosynthetic rate upon transfer from high to low light is linked to the slow kinetics of chloroplast ATP synthase in Bletilla striata.

Upon a sudden transition from high to low light, the rate of CO2 assimilation (AN) in some plants first decreases to a low level before gradually becoming stable. However, the underlying mechanisms remain controversial. The activity of chloroplast ATP synthase (gH+) is usually depressed under high light when compared with low light. Therefore, we hypothesize that upon a sudden transfer from high to low light, the relatively low gH+ restricts ATP synthesis and thus causes a reduction in AN. To test this hypothesis, we measured gas exchange, chlorophyll fluorescence, P700 redox state, and electrochromic shift signals in Bletilla striata (Orchidaceae). After the transition from saturating to lower irradiance, AN and ETRII decreased first to a low level and then gradually increased to a stable value. Within the first seconds after transfer from high to low light, gH+ was maintained at low levels. During further exposure to low light, gH+ gradually increased to a stable value. Interestingly, a tight positive relationship was found between gH+ and ETRII. These results suggested that upon a sudden transition from high to low light, AN was restricted by gH+ at the step of ATP synthesis. Taken together, we propose that the decline in AN upon sudden transfer from high to low light is linked to the slow kinetics of chloroplast ATP synthase.

In vivo regulation of thylakoid proton motive force in immature leaves.

In chloroplast, proton motive force (pmf) is critical for ATP synthesis and photoprotection. To prevent photoinhibition of photosynthetic apparatus, proton gradient (ΔpH) across the thylakoid membranes needs to be built up to minimize the production of reactive oxygen species (ROS) in thylakoid membranes. However, the regulation of thylakoid pmf in immature leaves is little known. In this study, we compared photosynthetic electron sinks, P700 redox state, non-photochemical quenching (NPQ), and electrochromic shift (ECS) signal in immature and mature leaves of a cultivar of Camellia. The immature leaves displayed lower linear electron flow and cyclic electron flow, but higher levels of NPQ and P700 oxidation ratio under high light. Meanwhile, we found that pmf and ΔpH were higher in the immature leaves. Furthermore, the immature leaves showed significantly lower thylakoid proton conductivity than mature leaves. These results strongly indicated that immature leaves can build up enough ΔpH by modulating proton efflux from the lumenal side to the stromal side of thylakoid membranes, which is essential to prevent photoinhibition via thermal energy dissipation and photosynthetic control of electron transfer. This study highlights that the activity of chloroplast ATP synthase is a key safety valve for photoprotection in immature leaves.

Crosstalk between chloroplast thioredoxin systems in regulation of photosynthesis.

Thioredoxins (TRXs) mediate light-dependent activation of primary photosynthetic reactions in plant chloroplasts by reducing disulphide bridges in redox-regulated enzymes. Of the two plastid TRX systems, the ferredoxin-TRX system consists of ferredoxin-thioredoxin reductase (FTR) and multiple TRXs, while the NADPH-dependent thioredoxin reductase (NTRC) contains a complete TRX system in a single polypeptide. Using Arabidopsis plants overexpressing or lacking a functional NTRC, we have investigated the redundancy and interaction between the NTRC and Fd-TRX systems in regulation of photosynthesis in vivo. Overexpression of NTRC raised the CO2 fixation rate and lowered non-photochemical quenching and acceptor side limitation of PSI in low light conditions by enhancing the activation of chloroplast ATP synthase and TRX-regulated enzymes in Calvin-Benson cycle (CBC). Overexpression of NTRC with an inactivated NTR or TRX domain partly recovered the phenotype of knockout plants, suggesting crosstalk between the plastid TRX systems. NTRC interacted in planta with fructose-1,6-bisphosphatase, phosphoribulokinase and CF1 γ subunit of the ATP synthase and with several chloroplast TRXs. These findings indicate that NTRC-mediated regulation of the CBC and ATP synthesis occurs both directly and through interaction with the ferredoxin-TRX system and is crucial when availability of light is limiting photosynthesis.

Nucleotide binding to noncatalytic sites is essential for ATP-dependent stimulation and ADP-dependent inactivation of the chloroplast ATP synthase.

Light-dependent binding of labeled ADP and ATP to noncatalytic sites of chloroplast ATP synthase and the effect of light-exposed thylakoid membrane preincubation with ADP or ATP on ATPase activity were studied. ADP binding during the preincubation was shown to inactivate the chloroplast ATPase, whereas ATP binding caused its activation. The rate and equilibrium constants of ATPase inactivation and activation were close to those of ADP and ATP binding to a noncatalytic site, with K (d) values of 38 and 33μM, respectively. It is suggested that ADP- or ATP-binding to one of the noncatalytic sites affects the ATPase activity of chloroplast ATP synthase through a mechanism that modulates tightness of ADP binding to a catalytic site.

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca2+, whereas the Ca2+-ATPase activity of reduced thylakoids is very low. The Mg2+-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg2+-ATPase activity. The Mg2+-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75-80% of those of untreated thylakoids. The Mg2+-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the gamma subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the epsilon subunit, but only partially overcomes inhibition by Mg2+ and ADP during assay.

N‐terminal deletion of the γ subunit affects the stabilization and activity of chloroplast ATP synthase

Five truncation mutants of chloroplast ATP synthase gamma subunit from spinach (Spinacia oleracea) lacking 8, 12, 16, 20 or 60 N-terminal amino acids were generated by PCR by a mutagenesis method. The recombinant gamma genes were overexpressed in Escherichia coli and assembled with alphabeta subunits into a native complex. The wild-type (WT) alphabetagamma assembly i.e. alphabetagammaWT exhibited high (Mg2+)-dependent and (Ca2+)-dependent ATP hydrolytic activity. Deletions of eight residues of the gamma subunit N-terminus caused a decrease in rates of ATP hydrolysis to 30% of that of the alphabetaWT assembly. Furthermore, only approximately 6% of ATP hydrolytic activity was retained with the sequential deletions of gamma subunit up to 20 residues compared with the activity of the alphabetaWT assembly. The inhibitory effect of the epsilon subunit on ATP hydrolysis of these alphabetagamma assemblies varied to a large extent. These observations indicate that the N-terminus of the gamma subunit is very important, together with other regions of the gamma subunit, in stabilization of the enzyme complex or during cooperative catalysis. In addition, the in vitro binding assay showed that the gamma subunit N-terminus is not a crucial region in binding of the epsilon subunit.

Conformational change of the chloroplast ATP synthase on the enzyme activation process detected by the trypsin sensitivity of the γ subunit

ΔμH + is known to stimulate the enzyme activity of chloroplast ATP synthase in addition to its important role as energy supply for ATP synthesis. In the present study, we focused on the relationship between the proton translocation via the membrane sector of ATP synthase, F o, and the conformational change of the central stalk subunit γ. The conformational change of CF 1 mainly at the γ subunit was induced by the proton flow via F o in the absence of substrates. The effects of inhibitors on CF o or CF 1 for this conformational change were also examined. The observed conformational change was partially suppressed by ADP binding. From these results, we propose the ΔμH +-dependent conformational change of CF 1 on the enzyme activation process, which is affected by both ADP binding to the catalytic sites and proton flow via F o portion.

Molecular devices of chloroplast F 1-ATP synthase for the regulation

In chloroplasts, synthesis of ATP is energetically coupled with the utilization of a proton gradient formed by photosynthetic electron transport. The involved enzyme, the chloroplast ATP synthase, can potentially hydrolyze ATP when the magnitude of the transmembrane electrochemical potential difference of protons (ΔμH +) is small, e.g. at low light intensity or in the dark. To prevent this wasteful consumption of ATP, the activity of chloroplast ATP synthase is regulated as the occasion may demand. As regulation systems ΔμH + activation, thiol modulation, tight binding of ADP and the role of the intrinsic inhibitory subunit ε is well documented. In this article, we discuss recent progress in understanding of the regulation system of the chloroplast ATP synthase at the molecular level.

Mutation in cysteine bridge domain of the g-subunit affects light regulation of the ATP synthase in Arabidopsis

The catalytic regulation of the chloroplast ATP synthase is considerably more intricate than tits counterpart ATP synthase of mitochondria and bacteria. The light-dependent activation of chloroplast ATP synthase involves several steps including redox modulation of a cysteine bridge in g -subunit which is unique to the chloroplast enzyme. However, it is unclear exactly what role redox modulation plays in the overall regulation of the chloroplast enzyme. In order to investigate the significance of redox modulation we used site-directed mutagenesis and plant transformation to change the regulatory bridge domain within g -subunit in Arabidopsis. We designed four mutations in the redox active domain designated as C199S, C205S, C199S/C205S and deletion. These were transformed into wild type Arabidopsis (Columbia ecotype) plants. The expression levels of transgenes and original genes in T0 plants were identified by RT-PCR following by restriction digestion. The results showed the transgenes were extensively overexpressed behind a constitutive 35S promoter, compared with the endogenous g -subunit gene, indicating that g -subunit in ATP synthase of transgenic plants originated predominately from the transgenes. We monitored the activation and redox modulation of the chloroplast ATP synthase by measuring D A518 relaxation kinetics of the dark-adapted transgenic mutant plants after a 3 s of preillumination with 45 µmol quanta m-2 s-1 red light and 4 min of re-dark adaptation. The D A518 relaxation kinetics of the transgenic mutant plants confirmed interference with redox modulation in transgenic plants. Our ongoing work is focusing on TMR-maleimide labeling of cysteine residues to quantify the extent of substitution of the thiol group in g -subunit of the transformed plants and investigating the physiological consequences of the loss of redox modulation.