Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses.

Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana, Nicotiana tabacum, and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

The thylakoid proton antiporter KEA3 regulates photosynthesis in response to the chloroplast energy status.

Plant photosynthesis contains two functional modules, the light-driven reactions in the thylakoid membrane and the carbon-fixing reactions in the chloroplast stroma. In nature, light availability for photosynthesis often undergoes massive and rapid fluctuations. Efficient and productive use of such variable light supply requires an instant crosstalk and rapid synchronization of both functional modules. Here, we show that this communication involves the stromal exposed C-terminus of the thylakoid K+-exchange antiporter KEA3, which regulates the ΔpH across the thylakoid membrane and therefore pH-dependent photoprotection. By combining in silico, in vitro, and in vivo approaches, we demonstrate that the KEA3 C-terminus senses the energy state of the chloroplast in a pH-dependent manner and regulates transport activity in response. Together our data pinpoint a regulatory feedback loop by which the stromal energy state orchestrates light capture and photoprotection via multi-level regulation of KEA3.

The role of photorespiration in preventing feedback regulation via ATP synthase in Nicotiana tabacum.

Photorespiration consumes substantial amounts of energy in the forms of adenosine triphosphate (ATP) and reductant making the pathway an important component in leaf energetics. Because of this high reductant demand, photorespiration is proposed to act as a photoprotective electron sink. However, photorespiration consumes more ATP relative to reductant than the C3 cycle meaning increased flux disproportionally increases ATP demand relative to reductant. Here we explore how energetic consumption from photorespiration impacts the flexibility of the light reactions in nicotiana tabacum. Specifically, we demonstrate that decreased photosynthetic efficiency (ϕII ) at low photorespiratory flux was related to feedback regulation at the chloroplast ATP synthase. Additionally, decreased ϕII at high photorespiratory flux resulted in the accumulation of photoinhibition at photosystem II centers. These results are contrary to the proposed role of photorespiration as a photoprotective electron sink. Instead, our results suggest a novel role of ATP consumption from photorespiration in maintaining ATP synthase activity, with implications for maintaining energy balance and preventing photodamage that will be critical for plant engineering strategies.

Energetic considerations for engineering novel biochemistries in photosynthetic organisms.

Humans have been harnessing biology to make valuable compounds for generations. From beer and biofuels to pharmaceuticals, biology provides an efficient alternative to industrial processes. With the continuing advancement of molecular tools to genetically modify organisms, biotechnology is poised to solve urgent global problems related to environment, increasing population, and public health. However, the light dependent reactions of photosynthesis are constrained to produce a fixed stoichiometry of ATP and reducing equivalents that may not match the newly introduced synthetic metabolism, leading to inefficiency or damage. While photosynthetic organisms have evolved several ways to modify the ATP/NADPH output from their thylakoid electron transport chain, it is unknown if the native energy balancing mechanisms grant enough flexibility to match the demands of the synthetic metabolism. In this review we discuss the role of photosynthesis in the biotech industry, and the energetic considerations of using photosynthesis to power synthetic biology.

Characterization of mutants deficient in N-terminal phosphorylation of the chloroplast ATP synthase subunit ß.

Understanding the regulation of photosynthetic light harvesting and electron transfer is of great importance to efforts to improve the ability of the electron transport chain to supply downstream metabolism. A central regulator of the electron transport chain is ATP synthase, the molecular motor that harnesses the chemiosmotic potential generated from proton-coupled electron transport to synthesize ATP. ATP synthase is regulated both thermodynamically and post-translationally, with proposed phosphorylation sites on multiple subunits. In this study we focused on two N-terminal serines on the catalytic subunit ß in tobacco (Nicotiana tabacum), previously proposed to be important for dark inactivation of the complex to avoid ATP hydrolysis at night. Here we show that there is no clear role for phosphorylation in the dark inactivation of ATP synthase. Instead, mutation of one of the two phosphorylated serine residues to aspartate to mimic constitutive phosphorylation strongly decreased ATP synthase abundance. We propose that the loss of N-terminal phosphorylation of ATPß may be involved in proper ATP synthase accumulation during complex assembly.

Light acclimation interacts with thylakoid ion transport to govern the dynamics of photosynthesis in Arabidopsis.

Understanding photosynthesis in natural, dynamic light environments requires knowledge of long-term acclimation, short-term responses, and their mechanistic interactions. To approach the latter, we systematically determined and characterized light-environmental effects on thylakoid ion transport-mediated short-term responses during light fluctuations. For this, Arabidopsis thaliana wild-type and mutants of the Cl- channel VCCN1 and the K+ exchange antiporter KEA3 were grown under eight different light environments and characterized for photosynthesis-associated parameters and factors in steady state and during light fluctuations. For a detailed characterization of selected light conditions, we monitored ion flux dynamics at unprecedented high temporal resolution by a modified spectroscopy approach. Our analyses reveal that daily light intensity sculpts photosynthetic capacity as a main acclimatory driver with positive and negative effects on the function of KEA3 and VCCN1 during high-light phases, respectively. Fluctuations in light intensity boost the accumulation of the photoprotective pigment zeaxanthin (Zx). We show that KEA3 suppresses Zx accumulation during the day, which together with its direct proton transport activity accelerates photosynthetic transition to lower light intensities. In summary, both light-environment factors, intensity and variability, modulate the function of thylakoid ion transport in dynamic photosynthesis with distinct effects on lumen pH, Zx accumulation, photoprotection, and photosynthetic efficiency.

H+ Transport by K+ EXCHANGE ANTIPORTER3 Promotes Photosynthesis and Growth in Chloroplast ATP Synthase Mutants.

The composition of the thylakoid proton motive force (pmf) is regulated by thylakoid ion transport. Passive ion channels in the thylakoid membrane dissipate the membrane potential (Δψ) component to allow for a higher fraction of pmf stored as a proton concentration gradient (ΔpH). K+/H+ antiport across the thylakoid membrane via K+ EXCHANGE ANTIPORTER3 (KEA3) instead reduces the ΔpH fraction of the pmf. Thereby, KEA3 decreases nonphotochemical quenching (NPQ), thus allowing for higher light use efficiency, which is particularly important during transitions from high to low light. Here, we show that in the background of the Arabidopsis (Arabidopsis thaliana) chloroplast (cp)ATP synthase assembly mutant cgl160, with decreased cpATP synthase activity and increased pmf amplitude, KEA3 plays an important role for photosynthesis and plant growth under steady-state conditions. By comparing cgl160 single with cgl160 kea3 double mutants, we demonstrate that in the cgl160 background loss of KEA3 causes a strong growth penalty. This is due to a reduced photosynthetic capacity of cgl160 kea3 mutants, as these plants have a lower lumenal pH than cgl160 mutants, and thus show substantially increased pH-dependent NPQ and decreased electron transport through the cytochrome b 6 f complex. Overexpression of KEA3 in the cgl160 background reduces pH-dependent NPQ and increases photosystem II efficiency. Taken together, our data provide evidence that under conditions where cpATP synthase activity is low, a KEA3-dependent reduction of ΔpH benefits photosynthesis and growth.

The plastid NAD(P)H dehydrogenase-like complex: structure, function and evolutionary dynamics.

The thylakoid NAD(P)H dehydrogenase-like (NDH) complex is a large protein complex that reduces plastoquinone and pumps protons into the lumen generating protonmotive force. In plants, the complex consists of both nuclear and chloroplast-encoded subunits. Despite its perceived importance for stress tolerance and ATP generation, chloroplast-encoded NDH subunits have been lost numerous times during evolution in species occupying seemingly unrelated environmental niches. We have generated a phylogenetic tree that reveals independent losses in multiple phylogenetic lineages, and we use this tree as a reference to discuss possible evolutionary contexts that may have relaxed selective pressure for retention of ndh genes. While we are still yet unable to pinpoint a singular specific lifestyle that negates the need for NDH, we are able to rule out several long-standing explanations. In light of this, we discuss the biochemical changes that would be required for the chloroplast to dispense with NDH functionality with regards to known and proposed NDH-related reactions.

Photosynthesis in Arabidopsis Is Unaffected by the Function of the Vacuolar K+ Channel TPK3.

Photosynthesis is limited by the slow relaxation of nonphotochemical quenching, which primarily dissipates excess absorbed light energy as heat. Because the heat dissipation process is proportional to light-driven thylakoid lumen acidification, manipulating thylakoid ion and proton flux via transport proteins could improve photosynthesis. However, an important aspect of the current understanding of the thylakoid ion transportome is inaccurate. Using fluorescent protein fusions, we show that the Arabidopsis (Arabidopsis thaliana) two-pore K+ channel TPK3, which had been reported to mediate thylakoid K+ flux, localizes to the tonoplast, not the thylakoid. The localization of TPK3 outside of the thylakoids is further supported by the absence of TPK3 in isolated thylakoids as well as the inability of isolated chloroplasts to import TPK3 protein. In line with the subcellular localization of TPK3 in the vacuole, we observed that photosynthesis in the Arabidopsis null mutant tpk3-1, which carries a transfer DNA insertion in the first exon, remains unaffected. To gain a comprehensive understanding of how thylakoid ion flux impacts photosynthetic efficiency under dynamic growth light regimes, we performed long-term photosynthesis imaging of established and newly isolated transthylakoid K+- and Cl--flux mutants. Our results underpin the importance of the thylakoid ion transport proteins potassium cation efflux antiporter KEA3 and voltage-dependent chloride channel VCCN1 and suggest that the activity of yet unknown K+ channel(s), but not TPK3, is critical for optimal photosynthesis in dynamic light environments.

A Cytosolic Bypass and G6P Shunt in Plants Lacking Peroxisomal Hydroxypyruvate Reductase.

The oxygenation of ribulose 1,5-bisphosphate by Rubisco is the first step in photorespiration and reduces the efficiency of photosynthesis in C3 plants. Our recent data indicate that mutants in photorespiration have increased rates of photosynthetic cyclic electron flow around photosystem I. We investigated mutant lines lacking peroxisomal hydroxypyruvate reductase to determine if there are connections between 2-phosphoglycolate accumulation and cyclic electron flow in Arabidopsis (Arabidopsis thaliana). We found that 2-phosphoglycolate is a competitive inhibitor of triose phosphate isomerase, an enzyme in the Calvin-Benson cycle that converts glyceraldehyde 3-phosphate to dihydroxyacetone phosphate. This block in metabolism could be overcome if glyceraldehyde 3-phosphate is exported to the cytosol, where cytosolic triose phosphate isomerase could convert it to dihydroxyacetone phosphate. We found evidence that carbon is reimported as glucose-6-phosphate, forming a cytosolic bypass around the block of stromal triose phosphate isomerase. However, this also stimulates a glucose-6-phosphate shunt, which consumes ATP, which can be compensated by higher rates of cyclic electron flow.

Photometric screens identified Arabidopsis peroxisome proteins that impact photosynthesis under dynamic light conditions.

Plant peroxisomes function collaboratively with other subcellular organelles, such as chloroplasts and mitochondria, in several metabolic processes. To comprehensively investigate the impact of peroxisomal function on photosynthesis, especially under conditions that are more relevant to natural environments, a systematic screen of over 150 Arabidopsis mutants of genes encoding peroxisomal proteins was conducted using the automated Dynamic Environment Photosynthesis Imager (DEPI). Dynamic and high-light (HL) conditions triggered significant photosynthetic defects in a subset of the mutants, including those of photorespiration (PR) and other peroxisomal processes, some of which may also be related to PR. Further analysis of the PR mutants revealed activation of cyclic electron flow (CEF) around photosystem I and higher accumulation of hydrogen peroxide (H2 O2 ) under HL conditions. We hypothesize that impaired PR disturbs the balance of ATP and NADPH, leading to the accumulation of H2 O2 that activates CEF to produce ATP to compensate for the imbalance of reducing equivalents. The identification of peroxisomal mutants involved in PR and other peroxisomal functions in the photometric screen will enable further investigation of regulatory links between photosynthesis and PR and interorganellar interaction at the mechanistic level.

Transcriptome and phenotyping analyses support a role for chloroplast sigma factor 2 in red-light-dependent regulation of growth, stress, and photosynthesis.

Sigma factor (SIG) proteins contribute to promoter specificity of the plastid-encoded RNA polymerase during chloroplast genome transcription. All six members of the SIG family, that is, SIG1-SIG6, are nuclear-encoded proteins targeted to chloroplasts. Sigma factor 2 (SIG2) is a phytochrome-regulated protein important for stoichiometric control of the expression of plastid- and nuclear-encoded genes that impact plastid development and plant growth and development. Among SIG factors, SIG2 is required not only for transcription of chloroplast genes (i.e., anterograde signaling), but also impacts nuclear-encoded, photosynthesis-related, and light signaling-related genes (i.e., retrograde signaling) in response to plastid functional status. Although SIG2 is involved in photomorphogenesis in Arabidopsis, the molecular bases for its role in light signaling that impacts photomorphogenesis and aspects of photosynthesis have only recently begun to be investigated. Previously, we reported that SIG2 is necessary for phytochrome-mediated photomorphogenesis specifically under red (R) and far-red light, thereby suggesting a link between phytochromes and nuclear-encoded SIG2 in light signaling. To explore transcriptional roles of SIG2 in R-dependent growth and development, we performed RNA sequencing analysis to compare gene expression in sig2-2 mutant and Col-0 wild-type seedlings at two developmental stages (1- and 7-day). We identified a subset of misregulated genes involved in growth, hormonal cross talk, stress responses, and photosynthesis. To investigate the functional relevance of these gene expression analyses, we performed several comparative phenotyping tests. In these analyses, strong sig2 mutants showed insensitivity to bioactive GA 3, high intracellular levels of hydrogen peroxide (H2O2) indicative of a stress response, and specific defects in photosynthesis, including elevated levels of cyclic electron flow (CEF) and nonphotochemical quenching (NPQ). We demonstrated that SIG2 regulates a broader range of physiological responses at the molecular level than previously reported, with specific roles in red-light-mediated photomorphogenesis.

The higher plant plastid NAD(P)H dehydrogenase-like complex (NDH) is a high efficiency proton pump that increases ATP production by cyclic electron flow.

Cyclic electron flow around photosystem I (CEF) is critical for balancing the photosynthetic energy budget of the chloroplast by generating ATP without net production of NADPH. We demonstrate that the chloroplast NADPH dehydrogenase complex, a homolog to respiratory Complex I, pumps approximately two protons from the chloroplast stroma to the lumen per electron transferred from ferredoxin to plastoquinone, effectively increasing the efficiency of ATP production via CEF by 2-fold compared with CEF pathways involving non-proton-pumping plastoquinone reductases. By virtue of this proton-pumping stoichiometry, we hypothesize that NADPH dehydrogenase not only efficiently contributes to ATP production but operates near thermodynamic reversibility, with potentially important consequences for remediating mismatches in the thylakoid energy budget.

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.

The regulation of the chloroplast proton motive force plays a key role for photosynthesis in fluctuating light.

Plants use sunlight as their primary energy source. During photosynthesis, absorbed light energy generates reducing power by driving electron transfer reactions. These are coupled to the transfer of protons into the thylakoid lumen, generating a proton motive force (pmf) required for ATP synthesis. Sudden alterations in light availability have to be met by regulatory mechanisms to avoid the over-accumulation of reactive intermediates and maximize energy efficiency. Here, the acidification of the lumen, as an intermediate product of photosynthesis, plays an important role by regulating photosynthesis in response to excitation energy levels. Recent findings reveal pmf regulation and the modulation of its composition as key determinants for efficient photosynthesis, plant growth, and survival in fluctuating light environments.

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.

Redox regulation of the antimycin A sensitive pathway of cyclic electron flow around photosystem I in higher plant thylakoids.

The chloroplast must regulate supply of reducing equivalents and ATP to meet rapid changes in downstream metabolic demands. Cyclic electron flow around photosystem I (CEF) is proposed to balance the ATP/NADPH budget by using reducing equivalents to drive plastoquinone reduction, leading to the generation of proton motive force and subsequent ATP synthesis. While high rates of CEF have been observed in vivo, isolated thylakoids show only very slow rates, suggesting that the activity of a key complex is lost or down-regulated upon isolation. We show that isolation of thylakoids while in the continuous presence of reduced thiol reductant dithiothreitol (DTT), but not oxidized DTT, maintains high CEF activity through an antimycin A sensitive ferredoxin:quinone reductase (FQR). Maintaining low concentrations (~2 mM) of reduced DTT while modulating the concentration of oxidized DTT leads to reversible activation/inactivation of CEF with an apparent midpoint potential of -306 mV (±10 mV) and n=2, consistent with redox modulation of a thiol/disulfide couple and thioredoxin-mediated regulation of the plastoquinone reductase involved in the antimycin A-sensitive pathway, possibly at the level of the PGRL1 protein. Based on proposed differences in regulatory modes, we propose that the FQR and NADPH:plastoquinone oxidoreductase (NDH) pathways for CEF are activated under different conditions and fulfill different roles in chloroplast energy balance.

Activation of cyclic electron flow by hydrogen peroxide in vivo.

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H2O2. We thus tested the hypothesis that CEF can be activated by H2O2 in vivo. CEF was strongly increased by H2O2 both by infiltration or in situ production by chloroplast-localized glycolate oxidase, implying that H2O2 can activate CEF either directly by redox modulation of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H2O2, suggesting activation of previously expressed CEF-related machinery. H2O2-dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H2O2. We propose that H2O2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants.

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.