Proton Gradient Regulation5-Like1-Mediated Cyclic Electron Flow Is Crucial for Acclimation to Anoxia and Complementary to Nonphotochemical Quenching in Stress Adaptation

To investigate the functional importance of Proton Gradient Regulation5-Like1 (PGRL1) for photosynthetic performances in the moss Physcomitrella patens, we generated a pgrl1 knockout mutant. Functional analysis revealed diminished nonphotochemical quenching (NPQ) as well as decreased capacity for cyclic electron flow (CEF) in pgrl1. Under anoxia, where CEF is induced, quantitative proteomics evidenced severe down-regulation of photosystems but up-regulation of the chloroplast NADH dehydrogenase complex, plastocyanin, and Ca²⁺ sensors in the mutant, indicating that the absence of PGRL1 triggered a mechanism compensatory for diminished CEF. On the other hand, proteins required for NPQ, such as light-harvesting complex stress-related protein1 (LHCSR1), violaxanthin de-epoxidase, and PSII subunit S, remained stable. To further investigate the interrelation between CEF and NPQ, we generated a pgrl1 npq4 double mutant in the green alga Chlamydomonas reinhardtii lacking both PGRL1 and LHCSR3 expression. Phenotypic comparative analyses of this double mutant, together with the single knockout strains and with the P. patens pgrl1, demonstrated that PGRL1 is crucial for acclimation to high light and anoxia in both organisms. Moreover, the data generated for the C. reinhardtii double mutant clearly showed a complementary role of PGRL1 and LHCSR3 in managing high light stress response. We conclude that both proteins are needed for photoprotection and for survival under low oxygen, underpinning a tight link between CEF and NPQ in oxygenic photosynthesis. Given the complementarity of the energy-dependent component of NPQ (qE) and PGRL1-mediated CEF, we suggest that PGRL1 is a capacitor linked to the evolution of the PSII subunit S-dependent qE in terrestrial plants.The conversion of solar energy into chemical energy and building material by oxygenic photosynthesis, as performed by plants, green algae, and cyanobacteria, supports much of the life on our planet. The production of oxygen and the assimilation of carbon dioxide into organic matter determines, to a large extent, the composition of our atmosphere. Plant photosynthesis is achieved thanks to a series of reactions that occur mainly in the chloroplast, resulting in light-dependent water oxidation, NADP⁺ reduction, and ATP formation (Whatley et al., 1963). Two separate photosystems (PSI and PSII) and an ATP synthase (ATPase) embedded in the thylakoid membrane catalyze these reactions. The ATPase produces ATP at the expense of the proton motive force that is generated by the light reactions (Mitchell, 1961). The cytochrome (cyt) b₆f complex assures the link between the two photosystems by transferring electrons from the membrane-bound plastoquinone to a soluble carrier, plastocyanin, or cyt c₆ and functions in the pumping of protons. NADPH and ATP that are produced by linear electron flow from PSII to PSI are fueled into the Calvin Benson Bassham cycle (Bassham et al., 1950) to fix CO₂. In parallel, cyclic electron flow (CEF) between the cyt b₆f complex and PSI may occur, which would solely lead to the production of ATP. CEF around PSI has been first recognized by Arnon (1959) and is involved in the reequilibration of the ATP poise and prevention of overreduction of the PSI acceptor side (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). In microalgae and vascular plants, CEF operates via an NAD(P)H dehydrogenase-like complex (NDH)-dependent and/or PROTON GRADIENT REGULATION5 (PGR5)-related pathway (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). The thylakoid protein Proton Gradient Regulation5-Like1 (PGRL1; DalCorso et al., 2008) has been first discovered as a novel component for the PGR5-dependent CEF pathway in Arabidopsis (Arabidopsis thaliana), as its knockout causes a PGR5-like photosynthetic phenotype and is suggested to operate as a ferredoxin-plastoquinone reductase (Hertle et al., 2013). PGRL1 is also important for efficient CEF in the green alga Chlamydomonas reinhardtii, which becomes particularly evident under settings where CEF is induced, such as in acclimation to iron deficiency, high light (HL), or anaerobic growth conditions (Petroutsos et al., 2009; Iwai et al., 2010; Tolleter et al., 2011; Terashima et al., 2012). Remarkably, a CEF protein supercomplex composed of PSI-light-harvesting complex I (LHCI), LHCII, the cyt b₆f complex, ferredoxin-NADPH oxidoreductase, and PGRL1 was isolated from state 2 conditions (Iwai et al., 2010). Under anaerobic conditions, the Ca²⁺ sensor CAS and Anaerobic response1 (ANR1) were shown to interact with PGRL1 in vivo (Terashima et al., 2012) and were found to be associated with the C. reinhardtii CEF supercomplex. Consistently, depletion of CAS and ANR1 by artificial microRNA expression in C. reinhardtii resulted in strong inhibition of CEF under anoxia, which could be partially rescued by an increase in the extracellular Ca²⁺ concentration, inferring that CEF is Ca²⁺ dependent (Terashima et al., 2012). Notably, the regulation of the proton motive force by a two-pore potassium channel in the thylakoid membrane of Arabidopsis (AtTPK3), is also Ca²⁺ dependent (Carraretto et al., 2013), suggesting that Ca²⁺-dependent activation of CEF and the channel may work hand in hand.qE, the energy-dependent component of nonphotochemical quenching (NPQ) that occurs due to thermal dissipation of excess absorbed light energy (Li et al., 2000; Peers et al., 2009), is dependent on rapid luminal acidification upon photosynthetic electron transfer (Wraight and Crofts, 1970; Li et al., 2000). Thus, processes such as CEF that contribute to the pH gradient across the thylakoid membrane are interrelated to NPQ, as an acidified lumen is required for efficient qE (Joliot and Finazzi, 2010). In vascular plants, PSII subunit S (PSBS) is essential for efficient qE (Li et al., 2000), whereas qE induction in the green alga C. reinhardtii is mediated by light-harvesting complex stress-related protein3 (LHCSR3), an ancient light-harvesting protein that is missing in vascular plants (Peers et al., 2009). The moss Physcomitrella patens, which possesses genes encoding for PSBS and LHCSR proteins, utilizes both types of regulatory proteins to operate qE (Alboresi et al., 2010), suggesting that land plants evolved a novel PSBS-dependent qE mechanism before losing the ancestral LHCSR-dependent qE found in algae. This makes mosses a very interesting subject for investigating the interrelation and evolution of the CEF and NPQ molecular effectors.Mosses diverged from vascular plants early after land colonization and are one of the oldest groups of land plants present on earth. This places the moss model system P. patens (Rensing et al., 2008) evolutionarily in the middle between algae and vascular plants and makes it an ideal model organism for the study of the evolution of photosynthetic organisms. Analysis of photosynthesis in P. patens can provide insights into the events leading to adaptation to the harsher physicochemical conditions of the terrestrial environment (Rensing et al., 2008), as evidenced by the presence and functional overlap of LHCSRs and PSBS (Alboresi et al., 2010).To obtain insights into the interrelation and evolution of CEF and NPQ, we knocked out the PGRL1 gene from P. patens and analyzed functional phenotypic consequences. Moreover, we compared these phenotypes with phenotypic analyses of C. reinhardtii pgrl1, npq4, and pgrl1 npq4 single and double mutants lacking PGRL1, LHCSR3, and both PGRL1 and LHCSR3, respectively. The data provided strong evidence that the green cut protein PGRL1 (Karpowicz et al., 2011) is required for acclimation to anoxia both in algae and mosses. Moreover, an involvement of PGRL1 in the evolution of PSBS-dependent qE in terrestrial plants is implied.     

The Response of Cyclic Electron Flow around Photosystem I to Changes in Photorespiration and Nitrate Assimilation

Photosynthesis captures light energy to produce ATP and NADPH. These molecules are consumed in the conversion of CO₂ to sugar, photorespiration, and NO₃⁻ assimilation. The production and consumption of ATP and NADPH must be balanced to prevent photoinhibition or photodamage. This balancing may occur via cyclic electron flow around photosystem I (CEF), which increases ATP/NADPH production during photosynthetic electron transport; however, it is not clear under what conditions CEF changes with ATP/NADPH demand. Measurements of chlorophyll fluorescence and dark interval relaxation kinetics were used to determine the contribution of CEF in balancing ATP/NADPH in hydroponically grown Arabidopsis (Arabidopsis thaliana) supplied different forms of nitrogen (nitrate versus ammonium) under changes in atmospheric CO₂ and oxygen. Measurements of CEF were made under low and high light and compared with ATP/NADPH demand estimated from CO₂ gas exchange. Under low light, contributions of CEF did not shift despite an up to 17% change in modeled ATP/NADPH demand. Under high light, CEF increased under photorespiratory conditions (high oxygen and low CO₂), consistent with a primary role in energy balancing. However, nitrogen form had little impact on rates of CEF under high or low light. We conclude that, according to modeled ATP/NADPH demand, CEF responded to energy demand under high light but not low light. These findings suggest that other mechanisms, such as the malate valve and the Mehler reaction, were able to maintain energy balance when electron flow was low but that CEF was required under higher flow.Photosynthesis must balance both the amount of energy harvested by the light reactions and how it is stored to match metabolic demands. Light energy is harvested by the photosynthetic antenna complexes and stored by the electron and proton transfer complexes as ATP and NADPH. It is used primarily to meet the energy demands for assimilating carbon (from CO₂) and nitrogen (from NO₃⁻ and NH₄⁺; Keeling et al., 1976; Edwards and Walker, 1983; Miller et al., 2007). These processes require different ratios of ATP and NADPH, requiring a finely balanced output of energy in these forms. For example, if ATP were to be consumed at a greater rate than NADPH, electron transport would rapidly become limiting by the lack of NADP⁺, decreasing rates of proton translocation and ATP regeneration. Alternatively, if NADPH were consumed faster than ATP, proton translocation through ATP synthase would be reduced due to limiting ADP and the difference in pH between lumen and stroma would increase, restricting plastoquinol oxidation at the cytochrome b₆f complex and initiating nonphotochemical quenching (Kanazawa and Kramer, 2002). The stoichiometric balancing of ATP and NADPH must occur rapidly, because pool sizes of ATP and NADPH are relatively small and fluxes through primary metabolism are large (Noctor and Foyer, 2000; Avenson et al., 2005; Cruz et al., 2005; Amthor, 2010).The balancing of ATP and NADPH supply is further complicated by the rigid nature of linear electron flow (LEF). In LEF, electrons are transferred from water to NADP⁺, oxidizing water to oxygen and reducing NADP⁺ to NADPH. This electron transfer is coupled to proton translocation and generates a proton motive force, which powers the regeneration of ATP. The stoichiometry of ATP/NADPH produced by these reactions is thought to be 1.29 based on the ratio of proton pumping and the requirement for ATP synthase in the thylakoid (Sacksteder et al., 2000; Seelert et al., 2000). However, under ambient CO₂, oxygen, and temperature, the ATP/NADPH required by CO₂ fixation, photorespiration, and NO₃⁻ assimilation is approximately 1.6 (Edwards and Walker, 1983). The ATP/NADPH demand from central metabolism changes significantly from 1.6 if the ratio of CO₂ or oxygen changes, driving different rates of photosynthesis and photorespiration (see “Theory”). Such changes in energy demand require a flexible mechanism to balance ATP/NADPH that responds to environmental conditions.The difference between ATP/NADPH supply from LEF and demand from primary metabolism could be balanced via cyclic electron flow around PSI (CEF; Avenson et al., 2005; Shikanai, 2007; Joliot and Johnson, 2011; Kramer and Evans, 2011). During CEF, electrons from either NADPH or ferredoxin are cycled around PSI into the plastoquinone pool and regenerate ATP without reducing NADP⁺ (Golbeck et al., 2006). Therefore, CEF has been suggested to be important for optimal photosynthesis and plant growth, but its physiological role in energy balancing is not clear (Munekage et al., 2002, 2004; Livingston et al., 2010). For example, there was no shift in CEF in Arabidopsis (Arabidopsis thaliana) measured under low light (less than 300 μmol m⁻² s⁻¹) and different oxygen partial pressures, which would significantly change the ATP/NADPH demand of primary metabolism (Avenson et al., 2005). Similar results were seen under low light in leaves of barley (Hordeum vulgare) and Hedera helix (Genty et al., 1990). While CEF did not shift with energy demand in steady-state photosynthesis under low light, it did increase with photorespiration as expected at high light (Miyake et al., 2004, 2005). These observations could be explained if CEF becomes more important for energy balancing under high irradiances when other mechanisms become saturated.To determine under which conditions CEF responded to ATP/NADPH demand, we used biochemical models of leaf CO₂ fixation to model ATP and NADPH demand under a variety of conditions (see “Theory”). We then used in vivo spectroscopy to measure the relative response of CEF to modeled ATP/NADPH demand from CO₂ fixation and NO₃⁻ assimilation in hydroponically grown Arabidopsis. Our findings indicate that CEF responded to modeled ATP/NADPH demand under high light but not under low light or nitrate availability.     

Thioredoxin m4 Controls Photosynthetic Alternative Electron Pathways in Arabidopsis

In addition to the linear electron flow, a cyclic electron flow (CEF) around photosystem I occurs in chloroplasts. In CEF, electrons flow back from the donor site of photosystem I to the plastoquinone pool via two main routes: one that involves the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (PGR) and one that is dependent of the NADH dehydrogenase-like complex. While the importance of CEF in photosynthesis and photoprotection has been clearly established, little is known about its regulation. We worked on the assumption of a redox regulation and surveyed the putative role of chloroplastic thioredoxins (TRX). Using Arabidopsis (Arabidopsis thaliana) mutants lacking different TRX isoforms, we demonstrated in vivo that TRXm4 specifically plays a role in the down-regulation of the NADH dehydrogenase-like complex-dependent plastoquinone reduction pathway. This result was confirmed in tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. In vitro assays performed with isolated chloroplasts and purified TRXm4 indicated that TRXm4 negatively controls the PGR pathway as well. The physiological significance of this regulation was investigated under steady-state photosynthesis and in the pgr5 mutant background. Lack of TRXm4 reversed the growth phenotype of the pgr5 mutant, but it did not compensate for the impaired photosynthesis and photoinhibition sensitivity. This suggests that the physiological role of TRXm4 occurs in vivo via a mechanism distinct from direct up-regulation of CEF.In plant thylakoids, photosynthesis involves a linear electron flow (LEF) from water to NADP⁺ via PSII, cytochrome b₆/f, PSI, and soluble carriers. LEF produces NADPH and generates a transthylakoidal electrochemical proton gradient that drives the synthesis of ATP. Besides LEF, cyclic electron flow (CEF) can also occur, involving only PSI (for review, see Johnson, 2011; Kramer and Evans, 2011). These additional reactions include two main distinct pathways involving either the Proton Gradient Regulation5 (PGR5)/PGRL1 complex (Munekage et al., 2002; DalCorso et al., 2008) or the NADH dehydrogenase-like complex (NDH; for review, see Battchikova et al., 2011; Ifuku et al., 2011). The functioning of either CEF pathway, which generates a pH gradient ΔpH without any accumulation of NADPH, is thought to achieve the appropriate ATP/NADPH balance required for the biochemical needs of the plant, especially under certain environmental conditions such as low CO₂ (Golding and Johnson, 2003), heat (Clarke and Johnson, 2001), cold (Clarke and Johnson, 2001), drought (Golding and Johnson, 2003; Kohzuma et al., 2009), high light (Munekage et al., 2004), or dark-to-light transitions (Joliot and Joliot, 2005; Fan et al., 2007). CEF-generated ΔpH is also involved in photoprotection owing to the down-regulation of PSII via nonphotochemical quenching (Munekage et al., 2004; Takahashi et al., 2009). Very recently, the role of the PGR5 protein as a regulator of LEF has been established. It has proved to be essential in the protection of PSI from photodamage (Suorsa et al., 2012).The two cyclic pathways are redundant (Munekage et al., 2004), sharing ferredoxin (Fd) as a common stromal electron donor (Yamamoto et al., 2011) and electron carriers from plastoquinone (PQ) to PSI with LEF. Thus, LEF and either of the CEF pathways may be in competition. The molecular events that allow CEF to challenge LEF remain enigmatic, particularly when considering that the conditions that require CEF are also those under which LEF is in excess. Efforts to understand the appropriate functioning of CEF have led to the proposition of several models segregating cyclic and linear pathways at a structural level (for review, see Eberhard et al., 2008; Cardol et al., 2011; Johnson, 2011; Rochaix, 2011). According to the restricted diffusion model, founded on the uneven distribution of the photosynthetic protein complexes in the thylakoids, there is little competition between CEF and LEF, as CEF occurs in stroma lamellae where PSI is concentrated while LEF takes place in the grana stacks. In line with the supercomplex model, whose relevance was demonstrated in the microalga Chlamydomonas reinhardtii, CEF happens within tightly bound supercomplexes containing PSI, with its own light-harvesting complex (LHCI), the PSII light-harvesting complex (LHCII), cytochrome b₆/f, Fd, Fd NADP reductase (FNR), and the integral membrane protein PGRL1 (Iwai et al., 2010). In higher plants, an association between NDH and PSI subunits suggests the formation of such supercomplexes (Peng et al., 2009). The availability of FNR, found either free in the stroma or bound to the thylakoids (Zhang et al., 2001), has also been proposed to modulate partitioning between LEF and CEF (Joliot and Joliot, 2006; Joliot and Johnson, 2011). In addition, more dynamic models that illustrate competitive processes involved in the distribution of electrons between the cyclic and linear flows have been proposed. The competition between cytochrome b₆/f and FNR for electrons from Fd could regulate the segregation between LEF and CEF (Breyton et al., 2006; Yamamoto et al., 2006; Hald et al., 2008). A few years ago, Joliot and Joliot (2006) suggested that the ATP/ADP ratio was one of the parameters that triggered on the transition between LEF and CEF. It was also established that the redox poise of chloroplast stroma contributed to the regulation of the photosynthetic pathway and played an important role in defining the extent of CEF. Breyton et al. (2006) scrutinized this redox regulation and established that the fraction of PSI complexes engaged in CEF could be modulated by changes in the stromal redox state. Overreduction of the NADPH pool was involved in the repartition between LEF and CEF (Joliot and Joliot, 2006). The NADPH/NADP⁺ ratio was proposed as a regulator of PGR-dependent CEF in vivo (Okegawa et al., 2008).All the published data supporting a role for the redox status in the regulation of CEF urged us to investigate a putative role of thioredoxins (TRX) in the regulation of CEF. TRX are ubiquitous disulfide reductases regulating the redox status of target proteins (for review, see Lemaire et al., 2007; Meyer et al., 2009). In chloroplast, TRX mediate the light regulation of numerous enzymes, among which some belong to the Calvin cycle (for review, see Schürmann and Buchanan, 2008; Montrichard et al., 2009; Lindahl et al., 2011). Global proteomic approaches have revealed that well-known photosynthetic complex subunits may be partners of TRX, such as PsbO in PSII, plastocyanin, Rieske Fe-S protein in cytochrome b₆/f, and PsaK and PsaN in PSI (for review, see Montrichard et al., 2009; Lindahl et al., 2011). Furthermore, regarding the regulation of photosynthesis, TRX have also been involved in state transitions (Rintamäki et al., 2000; Buchanan and Balmer, 2005), and their participation in the control of the redox poise of the electron transport chain has also been suggested (Johnson, 2003).In this work, we have investigated the possible role of TRX in the regulation of CEF. Using Arabidopsis (Arabidopsis thaliana) mutants with altered expression of genes encoding different plastid TRX, we have established in vivo the inhibitor activity of TRXm4 on the NDH-dependent pathway for plastoquinone reduction. This result was confirmed in transplastomic tobacco (Nicotiana tabacum) plants overexpressing the TRXm4 orthologous gene. Moreover, in vitro assays performed with isolated chloroplasts indicated that TRXm4 negatively controls the PGR-dependent electron flow as well.     

Structural Basis of Efficient Electron Transport between Photosynthetic Membrane Proteins and Plastocyanin in Spinach Revealed Using Nuclear Magnetic Resonance

In the photosynthetic light reactions of plants and cyanobacteria, plastocyanin (Pc) plays a crucial role as an electron carrier and shuttle protein between two membrane protein complexes: cytochrome b₆f (cyt b₆f) and photosystem I (PSI). The rapid turnover of Pc between cyt b₆f and PSI enables the efficient use of light energy. In the Pc-cyt b₆f and Pc-PSI electron transfer complexes, the electron transfer reactions are accomplished within <10⁻⁴ s. However, the mechanisms enabling the rapid association and dissociation of Pc are still unclear because of the lack of an appropriate method to study huge complexes with short lifetimes. Here, using the transferred cross-saturation method, we investigated the residues of spinach (Spinacia oleracea) Pc in close proximity to spinach PSI and cyt b₆f, in both the thylakoid vesicle–embedded and solubilized states. We demonstrated that the hydrophobic patch residues of Pc are in close proximity to PSI and cyt b₆f, whereas the acidic patch residues of Pc do not form stable salt bridges with either PSI or cyt b₆f, in the electron transfer complexes. The transient characteristics of the interactions on the acidic patch facilitate the rapid association and dissociation of Pc.     

Characterization and Evolution of Tetrameric Photosystem I from the Thermophilic Cyanobacterium Chroococcidiopsis sp TS-821

Photosystem I (PSI) is a reaction center associated with oxygenic photosynthesis. Unlike the monomeric reaction centers in green and purple bacteria, PSI forms trimeric complexes in most cyanobacteria with a 3-fold rotational symmetry that is primarily stabilized via adjacent PsaL subunits; however, in plants/algae, PSI is monomeric. In this study, we discovered a tetrameric form of PSI in the thermophilic cyanobacterium Chroococcidiopsis sp TS-821 (TS-821). In TS-821, PSI forms tetrameric and dimeric species. We investigated these species by Blue Native PAGE, Suc density gradient centrifugation, 77K fluorescence, circular dichroism, and single-particle analysis. Transmission electron microscopy analysis of native membranes confirms the presence of the tetrameric PSI structure prior to detergent solubilization. To investigate why TS-821 forms tetramers instead of trimers, we cloned and analyzed its psaL gene. Interestingly, this gene product contains a short insert between the second and third predicted transmembrane helices. Phylogenetic analysis based on PsaL protein sequences shows that TS-821 is closely related to heterocyst-forming cyanobacteria, some of which also have a tetrameric form of PSI. These results are discussed in light of chloroplast evolution, and we propose that PSI evolved stepwise from a trimeric form to tetrameric oligomer en route to becoming monomeric in plants/algae.     

Interactions of C4 Subtype Metabolic Activities and Transport in Maize Are Revealed through the Characterization of DCT2 Mutants

C₄ photosynthesis in grasses requires the coordinated movement of metabolites through two specialized leaf cell types, mesophyll (M) and bundle sheath (BS), to concentrate CO₂ around Rubisco. Despite the importance of transporters in this process, few have been identified or rigorously characterized. In maize (Zea mays), DCT2 has been proposed to function as a plastid-localized malate transporter and is preferentially expressed in BS cells. Here, we characterized the role of DCT2 in maize leaves using Activator-tagged mutant alleles. Our results indicate that DCT2 enables the transport of malate into the BS chloroplast. Isotopic labeling experiments show that the loss of DCT2 results in markedly different metabolic network operation and dramatically reduced biomass production. In the absence of a functioning malate shuttle, dct2 lines survive through the enhanced use of the phosphoenolpyruvate carboxykinase carbon shuttle pathway that in wild-type maize accounts for ∼25% of the photosynthetic activity. The results emphasize the importance of malate transport during C₄ photosynthesis, define the role of a primary malate transporter in BS cells, and support a model for carbon exchange between BS and M cells in maize.     

The Type II NADPH Dehydrogenase Facilitates Cyclic Electron Flow,Energy-Dependent Quenching,and Chlororespiratory Metabolism during Acclimation of Chlamydomonas reinhardtii to Nitrogen Deprivation

When photosynthetic organisms are deprived of nitrogen (N), the capacity to grow and assimilate carbon becomes limited, causing a decrease in the productive use of absorbed light energy and likely a rise in the cellular reduction state. Although there is a scarcity of N in many terrestrial and aquatic environments, a mechanistic understanding of how photosynthesis adjusts to low-N conditions and the enzymes/activities integral to these adjustments have not been described. In this work, we use biochemical and biophysical analyses of photoautotrophically grown wild-type and mutant strains of Chlamydomonas reinhardtii to determine the integration of electron transport pathways critical for maintaining active photosynthetic complexes even after exposure of cells to N deprivation for 3 d. Key to acclimation is the type II NADPH dehydrogenase, NDA2, which drives cyclic electron flow (CEF), chlororespiration, and the generation of an H⁺ gradient across the thylakoid membranes. N deprivation elicited a doubling of the rate of NDA2-dependent CEF, with little contribution from PGR5/PGRL1-dependent CEF. The H⁺ gradient generated by CEF is essential to sustain nonphotochemical quenching, while an increase in the level of reduced plastoquinone would promote a state transition; both are necessary to down-regulate photosystem II activity. Moreover, stimulation of NDA2-dependent chlororespiration affords additional relief from the elevated reduction state associated with N deprivation through plastid terminal oxidase-dependent water synthesis. Overall, rerouting electrons through the NDA2 catalytic hub in response to photoautotrophic N deprivation sustains cell viability while promoting the dissipation of excess excitation energy through quenching and chlororespiratory processes.Oxygenic photosynthesis involves the conversion of light energy into chemical bond energy by plants, green algae, and cyanobacteria and the use of that energy to fix CO₂. The photosynthetic electron transport system, located in thylakoid membranes, involves several major protein complexes: PSII (water-plastoquinone oxidoreductase), cytochrome b₆f (cyt b6f; plastoquinone-plastocyanin oxidoreductase), PSI (plastocyanin-ferredoxin oxidoreductase), and the ATP synthase (CF_oCF₁). Light energy absorbed by the photosynthetic apparatus is used to establish both linear electron flow (LEF) and cyclic electron flow (CEF), which drive the production of ATP and NADPH, the chemical products of the light reactions needed for CO₂ fixation in the Calvin-Benson-Bassham (CBB) cycle.With the absorption of light energy by pigment-protein complexes associated with PSII, energy is funneled into unique chlorophyll (Chl) molecules located in the PSII reaction center (RC), where it can elicit a charge separation that generates a large enough oxidizing potential to extract electrons from water. In LEF, electrons from PSII RCs are transferred sequentially along a set of electron carriers, initially reducing the plastoquinone (PQ) pool, then the cyt b₆f complex, and subsequently the lumenal electron carrier plastocyanin (PC). Light energy absorbed by PSI excites a special pair of Chl molecules (P700), causing a charge separation that generates the most negative redox potential in nature (Nelson and Yocum, 2006). The energized electron, which is replaced by electrons from PC, is sequentially transferred to ferredoxin and ferredoxin NADP⁺ reductase, generating reductant in the form of NADPH.Electron transport from water to NADPH in LEF is accompanied by the transport of H⁺ into the thylakoid lumen. For each water molecule oxidized, two H⁺ are released in the thylakoid lumen. In addition, H⁺ are moved into the lumen by the transfer of electrons through cyt b₆f (Q cycle). H⁺ accumulation in the thylakoid lumen dramatically alters the lumenal pH, and the transmembrane H⁺ gradient (ΔpH) together with the transmembrane ion gradient constitute the proton motive force (pmf), which drives ATP formation by ATP synthase (Mitchell, 1961, 1966, 2011). This pmf also promotes other cellular processes, including the dissipation of excess absorbed excitation energy as heat in a photoprotective process (see below; Li et al., 2009; Erickson et al., 2015). The NADPH and ATP molecules generated by LEF and CEF fuel the synthesis of reduced carbon backbones (in the CBB cycle) used in the production of many cellular metabolites and fixed carbon storage polymers.A basic role for CEF is to increase the ATP-NADPH ratio, which can satisfy the energy requirements of the cell and augment the synthesis of ATP by LEF, which is required to sustain CO₂ fixation by the CBB cycle (Allen, 2003; Kramer et al., 2004; Iwai et al., 2010; Alric, 2014). There are two distinct CEF pathways identified in plants and algae. In both pathways, electrons flow from the PQ pool through cyt b₆f to reduce the oxidized form of P700 (P700⁺). In one CEF pathway, electrons are transferred back to the PQ pool prior to the formation of NADPH. This route involves the proteins PGR5 and PGRL1 (DalCorso et al., 2008; Tolleter et al., 2011; Hertle et al., 2013) and is termed PGR5/L1-dependent CEF. A second route for CEF includes an NADPH dehydrogenase that oxidizes NADPH (product of LEF) to NADP⁺, simultaneously reducing the PQ (Allen, 2003; Kramer et al., 2004; Rumeau et al., 2007). The reduced PQ pool is then oxidized by cyt b₆f, causing H⁺ translocation into the thylakoid lumen, followed by the transfer of electrons to P700⁺ via PC. In the green alga Chlamydomonas reinhardtii, this second route for CEF involves a type II NADPH dehydrogenase (NDA2; Jans et al., 2008; Desplats et al., 2009).Oxygenic photosynthetic organisms have inhabited the planet for approximately 3 billion years and have developed numerous strategies to acclimate to environmental fluctuations. These acclimation processes confer flexibility to the photosynthetic machinery, allowing it to adjust to changes in conditions that impact the metabolic/energetic state of the organism and, most importantly, the formation of reactive oxygen species that may damage the photosynthetic apparatus and other cellular components (Li et al., 2009). Several ways in which the photosynthetic apparatus adjusts to environmental fluctuations have been established. A well-studied acclimation process, nonphotochemical quenching (NPQ), reduces the excitation pressure on PSII when oxidized downstream electron acceptors are not available (Eberhard et al., 2008; Li et al., 2009; Erickson et al., 2015). Several processes constitute NPQ, as follows. (1) qT, which involves the physical movement of light-harvesting complexes (LHCs) from one photosystem to another (this is also designated state transitions; Rochaix, 2014). (2) qE, which involves thermal dissipation of the excitation energy. This energy-dependent process requires an elevated ΔpH and involves an LHC-like protein, LHCSR3 (in C. reinhardtii) or PSBS (in plants), as well as the accumulation of specific xanthophylls (mainly lutein in C. reinhardtii and zeaxanthin in plants; Niyogi et al., 1997b; Li et al., 2000, 2004; Peers et al., 2009). (3) qZ, which is energy independent and involves the accumulation of zeaxanthin (Dall’Osto et al., 2005; Nilkens et al., 2010). (4) qI, which promotes quenching following physical damage to PSII core subunits (Aro et al., 1993). Additional mechanisms that can impact LEF and CEF are the synthesis and degradation of pigment molecules, changes in levels of RC and antenna complexes, and the control of electron distribution between LEF and CEF as the energetic demands of the cell change (Allen, 2003; Kramer et al., 2004). In addition, electrons can be consumed by mitochondrial and chlororespiratory activities (Bennoun, 1982; Peltier and Cournac, 2002; Johnson et al., 2014; Bailleul et al., 2015). The latter mainly involves the plastid terminal oxidase PTOX2, which catalyzes the oxidation of the PQ pool and the reduction of oxygen and H⁺ to form water molecules (Houille-Vernes et al., 2011; Nawrocki et al., 2015).Photosynthetic processes also must be modulated as organisms experience changes in the levels of available nutrients (Grossman and Takahashi, 2001). The macronutrient nitrogen (N), which represents 3% to 5% of the dry weight of photosynthetic organisms, is required to synthesize many biological molecules (e.g. amino acids, nucleic acids, and various metabolites) and also participates in posttranslational modifications of proteins (e.g. S-nitrosylation; Romero-Puertas et al., 2013). Importantly, N is highly abundant in chloroplasts in the form of DNA, ribosomes, Chl, and polypeptides (e.g. Rubisco and LHCs; Evans, 1989; Raven, 2013). Furthermore, there is a strong integration between N and carbon assimilation. During N limitation under photoautotrophic conditions, the inability of the organism to synthesize amino acids and other N-containing molecules necessary for cell growth and division can feed back to inhibit both carbon fixation by the CBB cycle and electron transport processes and also can negatively impact the expression of genes encoding key CBB cycle enzymes (Terashima and Evans, 1988; Huppe and Turpin, 1994; Nunes-Nesi et al., 2010).C. reinhardtii is a well-established model organism in which to study photosynthesis and acclimation processes, including acclimation to nutrient limitation (Wykoff et al., 1998; Grossman and Takahashi, 2001; Moseley et al., 2006; Grossman et al., 2009; Terauchi et al., 2010; Aksoy et al., 2013). This unicellular alga grows rapidly as a photoheterotroph (on fixed carbon in the light) or as a heterotroph (on fixed carbon in the dark), has completely sequenced nuclear, chloroplast, and mitochondrial genomes, can be used for classical genetic analyses, and is haploid, which makes some aspects of molecular manipulation (e.g. the generation of knockout mutants) easier (Merchant et al., 2007; Blaby et al., 2014). In the past few years, there have been many studies on the ways in which C. reinhardtii responds to N deprivation (Bulté and Wollman, 1992; Blaby et al., 2013; Goodenough et al., 2014; Schmollinger et al., 2014; Wei et al., 2015; Juergens et al., 2015). Cells deprived of N under photoheterotrophic conditions (i.e. acetate as an external carbon source) minimize the use of N (referred to as N sparing) and induce mechanisms associated with scavenging N from both external and internal pools, all of which eventually lead to proteome modifications and an elevated carbon-N ratio (Schmollinger et al., 2014). Acclimation under photoheterotrophic conditions also causes dramatic modifications of cellular metabolism and energetics: photosynthesis is down-regulated at multiple levels, with a portion of its N content recycled (mainly Chl and polypeptides of the photosynthetic apparatus), while there is enhanced accumulation of mitochondrial complexes leading to increased respiratory activity (Schmollinger et al., 2014; Juergens et al., 2015). Additionally, while fixed carbon cannot be used for growth in the absence of N, it may be stored as starch and triacylglycerol (Work et al., 2010; Siaut et al., 2011; Davey et al., 2014; Goodenough et al., 2014).In contrast to the acclimation of photoheterotrophically grown C. reinhardtii to N deprivation, little is known about how the photosynthetic machinery in this alga adjusts in response to N deprivation under photoautotrophic conditions, when the cells absolutely require photosynthetic energy generation for maintenance. Specifically, we sought to understand how photosynthesis adjusts to metabolic restrictions that slow down the CBB cycle, which in turn could cause the accumulation of photoreductant, particularly NADPH, as the demand for electrons declines (Peltier and Schmidt, 1991; Rumeau et al., 2007). Based on analyses of mutants and the use of spectroscopic and fluorescence measurements, we established a critical role for NDA2 in the acclimation of C. reinhardtii to N deprivation under photoautotrophic conditions, including (1) an augmented capacity for alternative routes of electron utilization (which decrease the NADPH-NADP⁺ ratio) based on increased NDA2-dependent CEF and chlororespiration, and (2) elevated qE, which relies on the H⁺ gradient generated by NDA2-dependent CEF.     

Activation of the Stt7/STN7 Kinase through Dynamic Interactions with the Cytochrome b6f Complex

Photosynthetic organisms have the ability to adapt to changes in light quality by readjusting the cross sections of the light-harvesting systems of photosystem II (PSII) and photosystem I (PSI). This process, called state transitions, maintains the redox poise of the photosynthetic electron transfer chain and ensures a high photosynthetic yield when light is limiting. It is mediated by the Stt7/STN7 protein kinase, which is activated through the cytochrome b₆f complex upon reduction of the plastoquinone pool. Its probable major substrate, the light-harvesting complex of PSII, once phosphorylated, dissociates from PSII and docks to PSI, thereby restoring the balance of absorbed light excitation energy between the two photosystems. Although the kinase is known to be inactivated under high-light intensities, the molecular mechanisms governing its regulation remain unknown. In this study we monitored the redox state of a conserved and essential Cys pair of the Stt7/STN7 kinase and show that it forms a disulfide bridge. We could not detect any change in the redox state of these Cys during state transitions and high-light treatment. It is only after prolonged anaerobiosis that this disulfide bridge is reduced. It is likely to be mainly intramolecular, although kinase activation may involve a transient covalently linked kinase dimer with two intermolecular disulfide bonds. Using the yeast two-hybrid system, we have mapped one interaction site of the kinase on the Rieske protein of the cytochrome b₆f complex.Photosynthetic organisms are subjected to constant changes in light quality and quantity and need to adapt to these changes in order to optimize, on the one hand, their photosynthetic yield, and to minimize photo-oxidative damage on the other. The photosynthetic electron transfer chain consists of photosystem II (PSII), the plastoquinone (PQ) pool, the cytochrome b₆f complex (Cyt b₆f), plastocyanin, and photosystem I (PSI). All of these complexes and components are integrated or closely associated with the thylakoid membrane. The two antenna systems of PSII and PSI capture and direct the light excitation energy to the corresponding reaction centers in which a chlorophyll dimer is oxidized and charge separation occurs across the thylakoid membrane. These processes lead to the onset of electron flow from water on the donor side of PSII to ferredoxin on the acceptor side of PSI coupled with proton translocation across the thylakoid membrane. In order to sustain optimal electron flow along this electron transfer chain, the redox poise needs to be maintained under changing environmental conditions. Several mechanisms have evolved for the maintenance of this redox balance. In the case of over-reduction of the acceptor side of PSI, excess electrons can reduce molecular oxygen through the Mehler reaction to superoxide, which is then converted to hydrogen peroxide by a plastid superoxide dismutase and ultimately to water by a peroxidase (Asada, 2000). Over-reduction of the PQ pool can be alleviated by PTOX, the plastid terminal oxidase responsible for oxidizing PQH₂ to form hydrogen peroxide, which is subsequently converted to water (Carol et al., 1999; Cournac et al., 2000; Wu et al., 1999).In addition to these electron sinks that prevent the over-reduction of the electron transfer chain, the photosynthetic apparatus is able to maintain the redox poise of the PQ pool by readjusting the relative cross sections of the light harvesting systems of PSII and PSI upon unequal excitation of the two photosystems. This readjustment can occur both in the short term through state transitions and in the long term by changing the stoichiometry between PSII and PSI (Bonaventura and Myers, 1969; Murata, 1969; Pfannschmidt, 2003). State transitions occur because of perturbations of the redox state of the PQ pool due to unequal excitation of PSII and PSI, limitations in electron acceptors downstream of PSI, and/or in CO₂ availability. Excess excitation of PSII relative to PSI leads to reduction of the PQ pool and thus favors the docking of PQH₂ to the Qo site of the Cyt b₆f complex. This process activates the Stt7/STN7 protein kinase (Vener et al., 1997; Zito et al., 1999), which is closely associated with this complex and leads to the phosphorylation of some LHCII proteins and to their detachment from PSII and binding to PSI (Depège et al., 2003; Lemeille et al., 2009). Although both Lhcb1 and Lhcb2 are phosphorylated, only the phosphorylated form of Lhcb2 is associated with PSI whereas phosphorylated Lhcb1 is excluded from this complex (Longoni et al., 2015). This state corresponds to state 2. In this way the change in the relative antenna sizes of the two photosystems restores the redox poise of the PQ pool. The process is reversible as over-excitation of PSI relative to PSII leads to the oxidation of the PQ pool and to the inactivation of the kinase. Under these conditions, phosphorylated LHCII associated with PSI is dephosphorylated by the PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010) and returns to PSII (state 1). It should be noted, however, that a strict causal link between LHCII phosphorylation and its migration from PSII to PSI has been questioned recently by the finding that some phosphorylated LHCII remains associated with PSII supercomplexes and that LHCII serves as antenna for both photosystems under most natural light conditions (Drop et al., 2014; Wientjes et al., 2013).State transitions are important at low light but do not occur under high light because the LHCII kinase is inactivated under these conditions (Schuster et al., 1986). It was proposed that inactivation of the kinase is mediated by the ferredoxin-thioredoxin system and that a disulfide bond in the kinase rather than in the substrate may be the target site of thioredoxin (Rintamäki et al., 1997, 2000). Analysis of the Stt7/STN7 protein sequences indeed reveals the presence of two conserved Cys residues close to the N-terminal end of this kinase, which are conserved in all species examined and both are essential for kinase activity although they are located outside of the kinase catalytic domain (Fig. 1) (Depège et al., 2003; Lemeille et al., 2009). Based on protease protection studies, this model of the Stt7/STN7 kinase proposes that the N-terminal end of the kinase is on the lumen side of the thylakoid membrane separated from the catalytic domain on the stromal side by an unusual transmembrane domain containing several Pro residues (Lemeille et al., 2009). This configuration of the kinase allows its catalytic domain to act on the substrate sites of the LHCII proteins, which are exposed to the stroma. Although in this model the conserved Cys residues in the lumen are on the opposite side from the stromal thioredoxins, it is possible that thiol-reducing equivalents are transferred across the thylakoid membrane through the CcdA and Hcf164 proteins, which have been shown to operate in this way during heme and Cyt b₆f assembly (Lennartz et al., 2001; Page et al., 2004) or through the LTO1 protein (Du et al., 2015; Karamoko et al., 2011).Figure 1.Conserved Cys in the Stt7/STN7 kinase. Alignment of the sequences of the Stt7/STN protein kinase from Selaginella moelendorffii (Sm), Physcomitrella patens (Pp), Oryza sativa (Os), Populus trichocarpa (Pt), Arabidopsis thaliana (At), Chlamydomonas reinhardtii ...Here we have examined the redox state of the Stt7/STN7 kinase during state transitions and after illumination with high light to test the proposed model. We find that the Stt7/STN7 kinase contains a disulfide bridge that appears to be intramolecular and maintained not only during state transitions but also in high light when the kinase is inactive. Although these results suggest at first sight that the disulfide bridge of Stt7/STN7 is maintained during its activation and inactivation, we propose that a transient opening of this bridge occurs during the activation process followed by the formation of an intermolecular disulfide bridge and the appearance of a short-lived, covalently linked kinase dimer.     

Nanodomains of Cytochrome b6f and Photosystem II Complexes in Spinach Grana Thylakoid Membranes

The cytochrome b₆f (cytb₆f) complex plays a central role in photosynthesis, coupling electron transport between photosystem II (PSII) and photosystem I to the generation of a transmembrane proton gradient used for the biosynthesis of ATP. Photosynthesis relies on rapid shuttling of electrons by plastoquinone (PQ) molecules between PSII and cytb₆f complexes in the lipid phase of the thylakoid membrane. Thus, the relative membrane location of these complexes is crucial, yet remains unknown. Here, we exploit the selective binding of the electron transfer protein plastocyanin (Pc) to the lumenal membrane surface of the cytb₆f complex using a Pc-functionalized atomic force microscope (AFM) probe to identify the position of cytb₆f complexes in grana thylakoid membranes from spinach (Spinacia oleracea). This affinity-mapping AFM method directly correlates membrane surface topography with Pc-cytb₆f interactions, allowing us to construct a map of the grana thylakoid membrane that reveals nanodomains of colocalized PSII and cytb₆f complexes. We suggest that the close proximity between PSII and cytb₆f complexes integrates solar energy conversion and electron transfer by fostering short-range diffusion of PQ in the protein-crowded thylakoid membrane, thereby optimizing photosynthetic efficiency.     

Systemic Induction of Photosynthesis via Illumination of the Shoot Apex Is Mediated Sequentially by Phytochrome B,Auxin and Hydrogen Peroxide in Tomato

Systemic signaling of upper leaves promotes the induction of photosynthesis in lower leaves, allowing more efficient use of light flecks. However, the nature of the systemic signals has remained elusive. Here, we show that preillumination of the tomato (Solanum lycopersicum) shoot apex alone can accelerate photosynthetic induction in distal leaves and that this process is light quality dependent, where red light promotes and far-red light delays photosynthetic induction. Grafting the wild-type rootstock with a phytochome B (phyB) mutant scion compromised light-induced photosynthetic induction as well as auxin biosynthesis in the shoot apex, auxin signaling, and RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1)-dependent hydrogen peroxide (H₂O₂) production in the systemic leaves. Light-induced systemic H₂O₂ production in the leaves of the rootstock also was absent in plants grafted with an auxin-resistant diageotropica (dgt) mutant scion. Cyclic electron flow around photosystem I and associated ATP production were increased in the systemic leaves by exposure of the apex to red light. This enhancement was compromised in the systemic leaves of the wild-type rootstock with phyB and dgt mutant scions and also in RBOH1-RNA interference leaves with the wild type as scion. Silencing of ORANGE RIPENING, which encodes NAD(P)H dehydrogenase, compromised the systemic induction of photosynthesis. Taken together, these results demonstrate that exposure to red light triggers phyB-mediated auxin synthesis in the apex, leading to H₂O₂ generation in systemic leaves. Enhanced H₂O₂ levels in turn activate cyclic electron flow and ATP production, leading to a faster induction of photosynthetic CO₂ assimilation in the systemic leaves, allowing plants better adaptation to the changing light environment.As a consequence of their sessile lifestyle, plants have evolved a high capacity for the regulation of physiology, growth, and development that facilitates survival in a constantly changing environment. Environmental stimuli perceived within an organ not only influence morphogenetic and physiological changes within that organ but also generate systemic effects in other organs that are remote from the site of signal perception. This crucial phenomenon is called systemic signaling or systemic regulation. Systemic signaling prepares other tissues of a plant for future challenges that may initially only be sensed by a few local tissues or cells. Several types of systemic responses are known. These include systemic acquired resistance, which is typically activated by pathogens such as viruses, bacteria, and fungi (Fu and Dong, 2013), induced systemic resistance, which is triggered by beneficial soil microorganisms or others (Pieterse and Dicke, 2007), and systemic acquired acclimation, which is initiated by abiotic stresses such as high light, UV radiation, heat, cold, and salinity (Mittler and Blumwald, 2015).The light utilization efficiency of photosynthesis is important for the survival of understory plants and plants growing in canopies. In particular, the efficient use of the energy contained in light (sun) flecks is important because light flecks contribute up to 60% to 80% of photosynthetically active radiation received by understory plants (Pearcy and Seemann, 1990; Leakey et al., 2003, 2005). Earlier studies have shown the existence of systemic regulation of stomatal development and of photosynthesis in developing leaves in response to environmental signals perceived by mature leaves, such as changing irradiance and atmospheric CO₂ conditions (Lake et al., 2002; Coupe et al., 2006; Araya et al., 2008). Phytochome B (phyB) is important in the transmission of the systemic signals that modulate stomatal development in young leaves of Arabidopsis (Arabidopsis thaliana; Casson and Hetherington, 2014). In tomato (Solanum lycopersicum), there are two forms of phyB, phyB1 and phyB2, that work together to mediate red (R) light-induced responses, such as hypocotyl elongation and greening in seedlings (Hauser et al., 1995; Weller et al., 2000).Photosynthesis is completely switched off in the dark, specifically to prevent futile cycling of metabolites through the reductive and oxidative pentose phosphate pathways. Hence, leaves need time to reactivate the enzymes of carbon assimilation after a period of darkness. The time taken to reach maximum net rates of photosynthesis upon illumination is called photosynthetic induction (Walker, 1973). Systemic signaling also has been observed for the regulation of photosynthesis in relation to leaf ontology in understory plants (Montgomery and Givnish, 2008). The uppermost leaves, which are generally the first to receive sunlight, display faster photosynthetic induction times than understory leaves (Bai et al., 2008). Photosynthetic induction in understory leaves is enhanced by the preillumination of upper leaves but not lower leaves, suggesting a directional signal transfer (Hou et al., 2015). While this process allows plants to use the light energy in sun flecks more efficiently, the nature of the systemic signals and their transmission pathways remain largely unresolved. Although systemic signaling between different leaf ranks has been suggested to occur through the xylem (Thorpe et al., 2007) and also via electrical signals (Zimmermann et al., 2009), it is likely that systemic signals also pass through the phloem (Turgeon and Wolf, 2009; Hou et al., 2015). In addition, the phytohormone auxin is produced in the shoot apex and redistributed throughout the shoot by rapid nonpolar phloem transport (Ljung et al., 2001). Changes in the light environment can dramatically alter auxin homeostasis, which is regulated in a light quality- and photoreceptor-dependent manner (Halliday et al., 2009).The photosynthetic electron transport chain exhibits enormous flexibility in the relative rates of NADPH and ATP production in order to accommodate the varying requirements of metabolism (Foyer et al., 2012). Noncyclic, pseudocyclic, and cyclic electron flow (CEF) pathways operate in the photosynthetic electron transport chain to drive the proton gradient across the thylakoid membrane (Allen, 2003). Photosynthetic induction is not only associated with the activation of the light- and thiol-dependent activation of carbon assimilation enzymes but also dependent on a high rate of CEF to drive ATP synthesis (Foyer et al., 1992). Considerable overreduction of the electron transport acceptors occurs during the photosynthetic induction period, and this continues until carbon assimilation can be activated. CEF around PSI, an essential component of photosynthesis, drives the proton gradient in a situation when NADP reduction has reached its highest capacity and this essential electron acceptor is no longer available (Yamori et al., 2015; Yamori and Shikanai, 2016). CEF is particularly sensitive to the reduction-oxidation (redox) status of the chloroplast, which in turn is responsive to cellular redox homeostasis. Oxidants such as hydrogen peroxide (H₂O₂), which are produced by pseudocyclic electron flow in the chloroplasts, play a crucial role in the activation of CEF through modulation of the activity of the NADPH-plastoquinone reductase complex (Strand et al., 2015). Hormone-mediated generation of H₂O₂ also can stimulate CO₂ assimilation (Jiang et al., 2012).Auxins such as indole-3-acetic acid (IAA) generate H₂O₂ (Ivanchenko et al., 2013; Peer et al., 2013) and can regulate CO₂ assimilation (Bidwell and Turner, 1966; Hayat et al., 2009; Peng et al., 2013). Therefore, we used tomato plants to test the hypothesis that the systemic signaling that regulates photosynthetic induction in understory leaves arises from light-induced changes in auxin and H₂O₂ homeostasis involving the modulation of CEF in systemic leaves. We present evidence showing that R light perceived in the shoot apex by a phyB-dependent pathway alters IAA signaling in a systemic manner. IAA signals from the apex, perceived in distal leaves, trigger systemic H₂O₂ production that accelerates photosynthetic induction by increasing CEF-dependent ATP production in the systemic leaves. These findings provide new insights into the elaborate plant regulatory network that allows light adaptation in different organs.     

FLAVODIIRON2 and FLAVODIIRON4 Proteins Mediate an Oxygen-Dependent Alternative Electron Flow in Synechocystis sp. PCC 6803 under CO2-Limited Conditions

This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO₂ limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO₂-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO₂ are not available.In photosynthesis, photon energy absorbed by PSI and PSII in thylakoid membranes oxidizes the reaction center chlorophylls (Chls), P700 in PSI and P680 in PSII, and drives the photosynthetic electron transport (PET) system. In PSII, water is oxidized to oxygen as the oxidized P680 accepts electrons from water. These electrons then reduce the cytochrome b₆/f complex through plastoquinone (PQ) in the thylakoid membranes. Photooxidized P700 in PSI accepts electrons from the reduced cytochrome b₆/f complex through plastocyanin or cytochrome c₆. Electrons released in the photooxidation of P700 are used to produce NADPH through ferredoxin and ferredoxin NADP⁺ reductase. Thus, electrons flow from water to NADPH in the so-called photosynthetic linear electron flow (LEF). Importantly, LEF induces a proton gradient across the thylakoid membranes, which provides the driving force for ATP production by ATP synthases in the thylakoid membranes. NADPH and ATP serve as chemical energy donors in the photosynthetic carbon reduction cycle (Calvin cycle).It recently has been proposed that, in cyanobacteria, the photorespiratory carbon oxidation cycle (photorespiration) functions simultaneously with the Calvin cycle to recover carbon for the regeneration of ribulose-1,5-bisphosphate, one of the substrates of Rubisco (Hagemann et al., 2013). Rubisco catalyzes the primary reactions of carbon reduction as well as oxidation cycles. However, the presence of a specific carbon concentration mechanism (CCM) in cyanobacteria had been thought to prevent the operation of photorespiration. CCM maintains a high concentration of CO₂ around Rubisco so that the oxygenase activity of Rubisco is suppressed (Badger and Price, 1992). However, recent studies on mutants deficient in photorespiration enzymes have shown that photorespiration functions, particularly under CO₂-limited conditions, in cyanobacteria as it does in higher plants (Eisenhut et al., 2006, 2008).Decreased consumption of NADPH under CO₂-limited or high-light conditions causes electrons to accumulate in the PET system. As a result, the photooxidation and photoreduction cycles of the reaction center Chls in PSI and PSII become uncoupled from the production of NADPH, inducing alternative electron flow (AEF) pathways (Mullineaux, 2014). In cyanobacteria, several AEFs that differ from those in higher plants are proposed to function as electron sinks (Mullineaux, 2014). Electrons accumulated in the PET system flow to oxygen through FLAVODIIRON1 (FLV1) and FLV3 proteins in PSI and the terminal oxidase, cytochrome c oxidase complex, and cytochrome bd-quinol oxidase (Pils and Schmetterer, 2001; Berry et al., 2002; Helman et al., 2003; Nomura et al., 2006; Lea-Smith et al., 2013). Cyanobacterial FLV comprises a diiron center, a flavodoxin domain with an FMN-binding site, and a flavin reductase domain (Vicente et al., 2002). In Synechocystis sp. PCC 6803, Helman et al. (2003) identified four genes encoding FLV1 to FLV4 and showed that FLV1 and FLV3 were essential for the photoreduction of oxygen by PSI. FLV1 and FLV3 were proposed to function as a heterodimer (Allahverdiyeva et al., 2013). FLV2/4 have been proposed to function in energy dissipation associated with PSII (Zhang et al., 2012). In addition, hydrogenases convert H⁺ to H₂ with NADPH as an electron donor (Appel et al., 2000). Furthermore, Flores et al. (2005) suggested that the nitrate assimilation pathway functions in AEF when the cells live in medium containing nitrate.To elucidate the physiological functions of these AEFs, evaluation of the presence and capacity of each AEF pathway is required. Therefore, in vivo analyses of electron fluxes are essential. We had found that an electron flow uncoupled from photosynthetic oxygen evolution functioned under suppressed (CO₂-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803 but not in Synechococcus elongatus PCC 7942 (Hayashi et al., 2014), indicating that an AEF operated in Synechocystis sp. PCC 6803. This AEF was induced in high-[CO₂]-grown Synechocystis sp. PCC 6803 during the transition from CO₂-saturated photosynthesis to CO₂-limited photosynthesis (Hayashi et al., 2014). In contrast, in Synechocystis sp. PCC 6803 grown at ambient CO₂ concentration, AEF was detected immediately following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014), suggesting that AEF was already induced under ambient atmospheric conditions.The expression of the AEF activity observed under CO₂-limited photosynthesis required the presence of oxygen in Synechocystis sp. PCC 6803 (Hayashi et al., 2014). In Synechocystis sp. PCC 6803, FLV1/3 were proposed to catalyze the photoreduction of oxygen (Helman et al., 2003). However, Hayashi et al. (2014) found no evidence that FLV1/3 operated under CO₂-limited photosynthesis: a mutant Synechocystis sp. PCC 6803 deficient in FLV1/3 maintained almost constant electron flux under CO₂-limited photosynthesis after the transition from CO₂-saturated conditions. Thus, the postulated photoreduction of oxygen by FLV1/3 was not responsible for the electron flux observed under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803.In this study, we aimed to elucidate the molecular mechanism of the oxygen-dependent AEF functioning under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. The possibility that FLV2 and FLV4 catalyze the photoreduction of oxygen under CO₂-limited photosynthesis could not be excluded, given that AEF in high-[CO₂]-grown Synechocystis sp. PCC 6803 was induced following the transition to CO₂-limited photosynthesis (Hayashi et al., 2014). Both FLV2 and FLV4 are predicted to possess oxidoreductase motifs, similar to FLV1 and FLV3 (Helman et al., 2003; Zhang et al., 2012). Furthermore, the expression of two FLV genes (flv2 and flv4) was enhanced under low-[CO₂] conditions (Zhang et al., 2009). Zhang et al. (2012) proposed that FLV2 and FLV4 did not donate electrons to oxygen on the basis of the finding that the Synechocystis sp. PCC 6803 mutants deficient in FLV1/3 showed no light-dependent oxygen uptake (Helman et al., 2003). However, Helman et al. (2003) cultivated Synechocystis sp. PCC 6803 strains deficient in FLV1 and FLV3 proteins under high-[CO₂] conditions, and we cannot exclude the possibility that the FLV2 and FLV4 proteins were not produced in the studied cells. Taken together, it seems plausible that FLV2 and FLV4 mediate oxygen-dependent AEF following the transition to CO₂-limited photosynthesis. To evaluate this possibility, we constructed Synechocystis sp. PCC 6803 mutants deficient in flv2 and flv4 and measured their oxygen evolution and Chl fluorescence simultaneously. The mutants showed suppressed LEF after transition to CO₂-limited photosynthesis, similar to S. elongatus PCC 7942. We also tested the possibility that photorespiration functions as an electron sink under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. A recent study revealed photorespiratory oxygen uptake in a flv1/3 mutant under CO₂-depleted conditions (Allahverdiyeva et al., 2011). In this study, we found that the quantum yield of photosystem II [Y(II)] of mutants deficient in genes encoding proteins that function in photorespiration was similar to that of wild-type Synechocystis sp. PCC 6803. Thus, FLV2 and FLV4 appear to function in the oxygen-dependent AEF under CO₂-limited photosynthesis in Synechocystis sp. PCC 6803. This inference is further supported by the lack of FLV2 and FLV4 homologs in the genome of S. elongatus PCC 7942 (Bersanini et al., 2014). In addition, we found oxygen-reducing activities of recombinant glutathione S-transferase (GST)-FLV4 fusion protein, similar to those of recombinant FLV3 protein (Vicente et al., 2002). In light of these results, we discuss the molecular mechanism of the oxygen-dependent AEF under CO₂-limited photosynthesis and the physiological function of FLV proteins in Synechocystis sp. PCC 6803.     

The Operation of Two Decarboxylases,Transamination, and Partitioning of C4 Metabolic Processes between Mesophyll and Bundle Sheath Cells Allows Light Capture To Be Balanced for the Maize C4 Pathway

The C₄ photosynthesis carbon-concentrating mechanism in maize (Zea mays) has two CO₂ delivery pathways to the bundle sheath (BS; via malate or aspartate), and rates of phosphoglyceric acid reduction, starch synthesis, and phosphoenolpyruvate regeneration also vary between BS and mesophyll (M) cells. The theoretical partitioning of ATP supply between M and BS cells was derived for these metabolic activities from simulated profiles of light penetration across a leaf, with a potential 3-fold difference in the fraction of ATP produced in the BS relative to M (from 0.29 to 0.96). A steady-state metabolic model was tested using varying light quality to differentially stimulate M or BS photosystems. CO₂ uptake, ATP production rate (J_ATP; derived with a low oxygen/chlorophyll fluorescence method), and carbon isotope discrimination were measured on plants under a low light intensity, which is considered to affect C₄ operating efficiency. The light quality treatments did not change the empirical ATP cost of gross CO₂ assimilation (J_ATP/GA). Using the metabolic model, measured J_ATP/GA was compared with the predicted ATP demand as metabolic functions were varied between M and BS. Transamination and the two decarboxylase systems (NADP-malic enzyme and phosphoenolpyruvate carboxykinase) were critical for matching ATP and reduced NADP demand in BS and M when light capture was varied under contrasting light qualities.Interest in the C₄ pathway has been increased by the potential for enhancing crop productivity and maintaining yield stability in the face of global warming and population pressure (Friso et al., 2010; Zhu et al., 2010; Covshoff and Hibberd, 2012). Maize (Zea mays), a C₄ plant of the NADP-malic enzyme (ME) subtype, is a leading grain production cereal (www.fao.org). C₄ photosynthesis is a shared activity between mesophyll (M; abbreviations are listed in BS) cells, coupled to allow the operation of a biochemical carbon-concentrating mechanism (CCM). The CCM effectively minimizes photorespiration by increasing the CO₂ concentration in the bundle sheath (C_BS), where Rubisco is exclusively expressed. Since BS and M are connected by plasmodesmata, some CO₂ retrodiffuses. The refixation of that escaping CO₂ by the CCM increases the activity of the CCM and the total ATP demand (ATP_BS + ATP_M) for gross CO₂ assimilation (GA; [ATP_BS + ATP_M]/GA), from a theoretical minimum of five ATPs (Furbank et al., 1990). Leakiness (Φ), the amount of CO₂ retrodiffusing relative to phosphoenolpyruvate (PEP) carboxylation rate, is therefore a proxy for the coordination between the CCM and assimilatory activity (Henderson et al., 1992; Tazoe et al., 2008; Kromdijk et al., 2010; Ubierna et al., 2011; Bellasio and Griffiths, 2013).

Table I.

Variables and acronyms described in the text

Temperature Responses of C4 Photosynthesis: Biochemical Analysis of Rubisco,Phosphoenolpyruvate Carboxylase,and Carbonic Anhydrase in Setaria viridis

The photosynthetic assimilation of CO₂ in C₄ plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C₄ biochemical models of leaf CO₂ exchange in response to changes in CO₂ availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C₄ plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO₃⁻, and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10°C to 40°C using crude leaf extracts from the C₄ plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO₃⁻ limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C₃ species and the C₄ plant S. viridis.Biochemical models of photosynthesis are often used to predict the effect of environmental conditions on net rates of leaf CO₂ assimilation (Farquhar et al., 1980; von Caemmerer, 2000, 2013; Walker et al., 2013). With climate change, there is increased interest in modeling and understanding the effects of changes in temperature and CO₂ concentration on photosynthesis. The biochemical models of photosynthesis are primarily driven by the kinetic properties of the enzyme Rubisco, the primary carboxylating enzyme of the C₃ photosynthetic pathway, catalyzing the reaction of ribulose-1,5-bisphosphate (RuBP) with either CO₂ or oxygen. However, the CO₂-concentrating mechanism in C₄ photosynthesis utilizes carbonic anhydrase (CA) to help maintain the chemical equilibrium of CO₂ with HCO₃⁻ and phosphoenolpyruvate carboxylase (PEPc) to catalyze the carboxylation of phosphoenolpyruvate (PEP) with HCO₃⁻. These reactions ultimately provide the elevated levels of CO₂ to the compartmentalized Rubisco (Edwards and Walker, 1983). In C₄ plants, it has been demonstrated that PEPc, Rubisco, and CA can limit rates of CO₂ assimilation and influence the efficiency of the CO₂-concentrating mechanism (von Caemmerer, 2000; von Caemmerer et al., 2004; Studer et al., 2014). Therefore, accurate modeling of leaf photosynthesis in C₄ plants in response to future climatic conditions will require temperature parameterizations of Rubisco, PEPc, and CA kinetics from C₄ species.Modeling C₄ photosynthesis relies on the parameterization of both PEPc and Rubisco kinetics, making it more complex than for C₃ photosynthesis (Berry and Farquhar, 1978; von Caemmerer, 2000). However, the activity of CA is not included in these models, as it is assumed to be nonlimiting under most conditions (Berry and Farquhar, 1978; von Caemmerer, 2000). This assumption is implemented by modeling PEPc kinetics as a function of CO₂ partial pressure (pCO₂) and not HCO₃⁻ concentration, assuming CO₂ and HCO₃⁻ are in chemical equilibrium. However, there are questions regarding the amount of CA activity needed to sustain rates of C₄ photosynthesis and if CO₂ and HCO₃⁻ are in equilibrium (von Caemmerer et al., 2004; Studer et al., 2014).The most common steady-state biochemical models of photosynthesis are derived from the Michaelis-Menten models of enzyme activity (von Caemmerer, 2000), which are driven by the V_max and the K_m. Both of these parameters need to be further described by their temperature responses to be used to model photosynthesis in response to temperature. However, the temperature response of plant CA activity has not been completed above 17°C, and there is no known measured temperature response of K_m HCO₃⁻ for PEPc (K_P). Alternatively, Rubisco has been well studied, and there are consistent differences in kinetic values between C₃ and C₄ species at 25°C (von Caemmerer and Quick, 2000; Kubien et al., 2008), but the temperature responses, including both carboxylation and oxygenation reactions, have only been performed in C₃ species (Badger and Collatz, 1977; Jordan and Ogren, 1984; Bernacchi et al., 2001, 2002; Walker et al., 2013).Here, we present the temperature dependency of Rubisco carboxylation and oxygenation reactions, PEPc kinetics for HCO₃⁻, and CA hydration from 10°C to 40°C from the C₄ species Setaria viridis (succession no., A-010) measured using membrane inlet mass spectrometry. Generally, the 25°C values of the Rubisco parameters were similar to previous measurements of C₄ species. The temperature response of the maximum rate of Rubisco carboxylation (V_cmax) was high compared with most previous measurements from both C₃ and C₄ species, and the temperature response of the K_m for oxygenation (K_O) was low compared with most previously measured species. Taken together, the modeled temperature responses of Rubisco activity in S. viridis were similar to the previously reported temperature responses of some C₃ species. Additionally, the temperature response of the maximum rate of PEPc carboxylation (V_pmax) was similar to previous measurements. However, the temperature response of K_P was lower than what has been predicted (Chen et al., 1994). For CA, deactivation of the hydration activity was observed above 25°C. Additionally, models of CA and PEPc show that CA activity limits HCO₃⁻ availability to PEPc above 15°C, suggesting that CA limits PEP carboxylation rates in S. viridis when compared with the assumption that CO₂ and HCO₃⁻ are in full chemical equilibrium.     

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.