Kaori Kohzuma

An Arabidopsis Mutant with High Cyclic Electron Flow around Photosystem I (hcef) Involving the NADPH Dehydrogenase Complex

Analysis of a mutant, hcef1, in chloroplast fructose-1,6-bisphosphatase demonstrates that C3 plants are capable of high steady state fluxes of cyclic electron flow around photosystem I, which is important for chloroplast energy balance and involves the NAD(P)H dehydrogenase, but not the PGR5, pathway. Cyclic electron flow (CEFI) has been proposed to balance the chloroplast energy budget, but the pathway, mechanism, and physiological role remain unclear. We isolated a new class of mutant in Arabidopsis thaliana, hcef for high CEF1, which shows constitutively elevated CEF1. The first of these, hcef1, was mapped to chloroplast fructose-1,6-bisphosphatase. Crossing hcef1 with pgr5, which is deficient in the antimycin A–sensitive pathway for plastoquinone reduction, resulted in a double mutant that maintained the high CEF1 phenotype, implying that the PGR5-dependent pathway is not involved. By contrast, crossing hcef1 with crr2-2, deficient in thylakoid NADPH dehydrogenase (NDH) complex, results in a double mutant that is highly light sensitive and lacks elevated CEF1, suggesting that NDH plays a direct role in catalyzing or regulating CEF1. Additionally, the NdhI component of the NDH complex was highly expressed in hcef1, whereas other photosynthetic complexes, as well as PGR5, decreased. We propose that (1) NDH is specifically upregulated in hcef1, allowing for increased CEF1; (2) the hcef1 mutation imposes an elevated ATP demand that may trigger CEF1; and (3) alternative mechanisms for augmenting ATP cannot compensate for the loss of CEF1 through NDH.

Light- and Metabolism-related Regulation of the Chloroplast ATP Synthase Has Distinct Mechanisms and Functions

Background: Chloroplast ATP synthase activity is regulated by both light and metabolic factors, but the relationship between these regulatory modes is not established. Results: Mutating three highly conserved acidic amino acid residues in the γ subunit alters light- but not metabolism-induced regulation. Conclusion: Metabolism and light regulation operates via distinct mechanisms. Significance: The chloroplast ATP synthase is a key control point for the light and dark reactions of photosynthesis. The chloroplast CF0-CF1-ATP synthase (ATP synthase) is activated in the light and inactivated in the dark by thioredoxin-mediated redox modulation of a disulfide bridge on its γ subunit. The activity of the ATP synthase is also fine-tuned during steady-state photosynthesis in response to metabolic changes, e.g. altering CO2 levels to adjust the thylakoid proton gradient and thus the regulation of light harvesting and electron transfer. The mechanism of this fine-tuning is unknown. We test here the possibility that it also involves redox modulation. We found that modifying the Arabidopsis thaliana γ subunit by mutating three highly conserved acidic amino acids, D211V, E212L, and E226L, resulted in a mutant, termed mothra, in which ATP synthase which lacked light-dark regulation had relatively small effects on maximal activity in vivo. In situ equilibrium redox titrations and thiol redox-sensitive labeling studies showed that the γ subunit disulfide/sulfhydryl couple in the modified ATP synthase has a more reducing redox potential and thus remains predominantly oxidized under physiological conditions, implying that the highly conserved acidic residues in the γ subunit influence thiol redox potential. In contrast to its altered light-dark regulation, mothra retained wild-type fine-tuning of ATP synthase activity in response to changes in ambient CO2 concentrations, indicating that the light-dark- and metabolic-related regulation occur through different mechanisms, possibly via small molecule allosteric effectors or covalent modification.

Potential Involvement of N-Terminal Acetylation in the Quantitative Regulation of the ε Subunit of Chloroplast ATP Synthase under Drought Stress

In plants, modulation of photosynthetic energy conversion in varying environments is often accompanied by adjustment of the abundance of photosynthetic components. In wild watermelon (Citrullus lanatus L.), proteome analysis revealed that the ε subunit of chloroplast ATP synthase occurs as two distinct isoforms with largely-different isoelectric points, although encoded by a single gene. Mass spectrometry (MS) analysis of the ε isoforms indicated that the structural difference between the ε isoforms lies in the presence or absence of an acetyl group at the N-terminus. The protein level of the non-acetylated ε isoform preferentially decreased in drought, whereas the abundance of the acetylated ε isoform was unchanged. Moreover, metalloprotease activity that decomposed the ε subunit was detected in a leaf extract from drought-stressed plants. Furthermore, in vitro assay suggested that the non-acetylated ε subunit was more susceptible to degradation by metalloaminopeptidase. We propose a model in which quantitative regulation of the ε subunit involves N-terminal acetylation and stress-induced proteases.

Thioredoxin-insensitive plastid ATP synthase that performs moonlighting functions

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and acts as a key feedback regulatory component of photosynthesis. Arabidopsis possesses two homologues of the regulatory γ subunit of the ATP synthase, encoded by the ATPC1 and ATPC2 genes. Using a series of mutants, we show that both these subunits can support photosynthetic ATP synthesis in vivo with similar specific activities, but that in wild-type plants, only γ1 is involved in ATP synthesis in photosynthesis. The γ1-containing ATP synthase shows classical light-induced redox regulation, whereas the mutant expressing only γ2-ATP synthase (gamma exchange-revised ATP synthase, gamera) shows equally high ATP synthase activity in the light and dark. In situ redox titrations demonstrate that the regulatory thiol groups on γ2-ATP synthase remain reduced under physiological conditions but can be oxidized by the strong oxidant diamide, implying that the redox potential for the thiol/disulphide transition in γ2 is substantially higher than that for γ1. This regulatory difference may be attributed to alterations in the residues near the redox-active thiols. We propose that γ2-ATP synthase functions to catalyze ATP hydrolysis-driven proton translocation in nonphotosynthetic plastids, maintaining a sufficient transthylakoid proton gradient to drive protein translocation or other processes. Consistent with this interpretation, ATPC2 is predominantly expressed in the root, whereas modifying its expression results in alteration of root hair development. Phylogenetic analysis suggests that γ2 originated from ancient gene duplication, resulting in divergent evolution of functionally distinct ATP synthase complexes in dicots and mosses.

Limitations to photosynthesis by proton motive force-induced photosystem II photodamage

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field. DOI: http://dx.doi.org/10.7554/eLife.16921.001

Chloroplast ATP Synthase Modulation of the Thylakoid Proton Motive Force: Implications for Photosystem I and Photosystem II Photoprotection

In wild type plants, decreasing CO2 lowers the activity of the chloroplast ATP synthase, slowing proton efflux from the thylakoid lumen resulting in buildup of thylakoid proton motive force (pmf). The resulting acidification of the lumen regulates both light harvesting, via the qE mechanism, and photosynthetic electron transfer through the cytochrome b6f complex. Here, we show that the cfq mutant of Arabidopsis, harboring single point mutation in its γ-subunit of the chloroplast ATP synthase, increases the specific activity of the ATP synthase and disables its down-regulation under low CO2. The increased thylakoid proton conductivity (gH+) in cfq results in decreased pmf and lumen acidification, preventing full activation of qE and more rapid electron transfer through the b6f complex, particularly under low CO2 and fluctuating light. These conditions favor the accumulation of electrons on the acceptor side of PSI, and result in severe loss of PSI activity. Comparing the current results with previous work on the pgr5 mutant suggests a general mechanism where increased PSI photodamage in both mutants is caused by loss of pmf, rather than inhibition of CEF per se. Overall, our results support a critical role for ATP synthase regulation in maintaining photosynthetic control of electron transfer to prevent photodamage.

Defects in the Expression of Chloroplast Proteins Leads to H2O2 Accumulation and Activation of Cyclic Electron Flow around Photosystem I

We describe a new member of the class of mutants in Arabidopsis exhibiting high rates of cyclic electron flow around photosystem I (CEF), a light-driven process that produces ATP but not NADPH. High cyclic electron flow 2 (hcef2) shows strongly increased CEF activity through the NADPH dehydrogenase complex (NDH), accompanied by increases in thylakoid proton motive force (pmf), activation of the photoprotective qE response, and the accumulation of H2O2. Surprisingly, hcef2 was mapped to a non-sense mutation in the TADA1 (tRNA adenosine deaminase arginine) locus, coding for a plastid targeted tRNA editing enzyme required for efficient codon recognition. Comparison of protein content from representative thylakoid complexes, the cytochrome bf complex, and the ATP synthase, suggests that inefficient translation of hcef2 leads to compromised complex assembly or stability leading to alterations in stoichiometries of major thylakoid complexes as well as their constituent subunits. Altered subunit stoichiometries for photosystem I, ratios and properties of cytochrome bf hemes, and the decay kinetics of the flash-induced thylakoid electric field suggest that these defect lead to accumulation of H2O2 in hcef2, which we have previously shown leads to activation of NDH-related CEF. We observed similar increases in CEF, as well as increases in H2O2 accumulation, in other translation defective mutants. This suggests that loss of coordination in plastid protein levels lead to imbalances in photosynthetic energy balance that leads to an increase in CEF. These results taken together with a large body of previous observations, support a general model in which processes that lead to imbalances in chloroplast energetics result in the production of H2O2, which in turn activates CEF. This activation could be from either H2O2 acting as a redox signal, or by a secondary effect from H2O2 inducing a deficit in ATP.

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead, ATP synthase redox regulation may be impacting a number of cellular processes such as (1) the accumulation of chloroplast proteins and/or ions or (2) the responses of photosynthesis to rapid changes in light intensity. A model highlighting the complex interplay between ATP synthase regulation and pmf in maintaining various chloroplast functions in the dark is presented. Significance Statement: We uncover an unexpected role for thioredoxin modulation of the chloroplast ATP synthase in regulating the dark-stability of the photosynthetic apparatus, most likely by controlling thylakoid membrane transport of proteins and ions.

Preferential Decay of the CF1-ε Subunit Induces Thylakoid Uncoupling in Wild Watermelon Under Drought Stress

Photosynthetic energy conversion in plants involves the formation of a proton gradient across thylakoid membranes, but the mechanisms for balancing the membrane potential have been poorly elucidated. We found that drought stress induces selective decomposition of the e subunit in the CFoCF1 ATP synthase. Thylakoid membranes from stressed leaves showed reduced efficiencies for proton gradient formation and energy coupling, but addition of the recombinant e subunit significantly suppressed their “leaky” property. We conclude that the selective decomposition of the e subunit induces partial uncoupling of thylakoid membranes under drought, and hence contributes to the avoidance of over-acidification in the thylakoid lumen under excess light conditions.

Chapter 23 Mechanisms of Drought and High Light Stress Tolerance Studied in a Xerophyte, Citrullus lanatus (Wild Watermelon)

The majority of higher plants are unable to survive extreme drought in the presence of strong solar radiation. However, a small group of vascular plants termed ‘‘xerophytes’’ have evolved drought and high light stress tolerance, and successfully thrives in the arid areas. This chapter will focus on the physiological, biochemical and molecular responses of wild watermelon (Citrullus lanatus), a xerophyte which is indigenous to the Kalahari Desert despite carrying out C3-type photosynthesis. The electrochromic shift of carotenoids in the thylakoid membranes was analyzed in vivo, which revealed that the proton efflux through chloroplast ATP synthase was strongly suppressed under drought and high light stresses. In addition, cyclic electron flow around photosystem I was significantly activated under the stress, suggesting the functional relevance of these processes to the build-up of large ΔpH across thylakoid membranes, for sustaining high qE quenching under excess light conditions. Biochemical analyses showed that key components for ROS metabolism, such as chloroplastic ascorbate peroxidase and monodehydroascorbate reductase, were markedly fortified in this plant. Moreover, unique responses of wild watermelon under the stress were described like metabolism and function of citrulline, a novel compatible solute with potent activity for scavenging hydroxyl radicals. Furthermore, characteristic gene expression patterns were observed in this plant under drought, which are exemplified by the induction of cytochrome b 561, a trans-plasma membrane protein for transferring reducing equivalents from cytosol to the apoplasts. Interestingly, unprecedentedly high activity of ascorbate oxidase was observed in the leaf apoplasts, suggesting the electron flux from cytosol to this terminal oxidase may be activated under drought. Taken together, these findings offer intriguing implications on how terrestrial plants can achieve effective adaptation to the harsh environmental conditions.