Electron transfer in a crystalline cytochrome with four hemes.

Diffusion of electrons over distances on the order of 100 µm has been observed in crystals of a small tetraheme cytochrome (STC) from Shewanella oneidensis [J. Huang et al. J. Am. Chem. Soc. 142, 10459-10467 (2020)]. Electron transfer between hemes in adjacent subunits of the crystal is slower and more strongly dependent on temperature than had been expected based on semiclassical electron-transfer theory. We here explore explanations for these findings by molecular-dynamics simulations of crystalline and monomeric STC. New procedures are developed for including time-dependent quantum mechanical energy differences in the gap between the energies of the reactant and product states and for evaluating fluctuations of the electronic-interaction matrix element that couples the two hemes. Rate constants for electron transfer are calculated from the time- and temperature-dependent energy gaps, coupling factors, and Franck-Condon-weighted densities of states using an expression with no freely adjustable parameters. Back reactions are considered, as are the effects of various protonation states of the carboxyl groups on the heme side chains. Interactions with water are found to dominate the fluctuations of the energy gap between the reactant and product states. The calculated rate constant for electron transfer from heme IV to heme Ib in a neighboring subunit at 300 K agrees well with the measured value. However, the calculated activation energy of the reaction in the crystal is considerably smaller than observed. We suggest two possible explanations for this discrepancy. The calculated rate constant for transfer from heme I to II within the same subunit of the crystal is about one-third that for monomeric STC in solution.

Chloroplast phosphatases LPPγ and LPPε1 facilitate conversion of extraplastidic phospholipids to galactolipids.

Galactolipids comprise the majority of chloroplast membranes in plants, and their biosynthesis requires dephosphorylation of phosphatidic acid at the chloroplast envelope membranes. In Arabidopsis (Arabidopsis thaliana), the lipid phosphate phosphatases LPPγ, LPPε1, and LPPε2 have been previously implicated in chloroplast lipid assembly, with LPPγ being essential, as null mutants were reported to exhibit embryo lethality. Here, we show that lppγ mutants are in fact viable and that LPPγ, LPPε1, and LPPε2 do not appear to have central roles in the plastid pathway of membrane lipid biosynthesis. Redundant LPPγ and LPPε1 activity at the outer envelope membrane is important for plant development, and the respective lppγ lppε1 double mutant exhibits reduced flux through the ER pathway of galactolipid synthesis. While LPPε2 is imported and associated with interior chloroplast membranes, its role remains elusive and does not include basal nor phosphate limitation-induced biosynthesis of glycolipids. The specific physiological roles of LPPγ, LPPε1, and LPPε2 are yet to be uncovered, as does the identity of the phosphatidic acid phosphatase required for plastid galactolipid biosynthesis.

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.

Redox regulation in chloroplast thylakoid lumen: The pmf changes everything, again.

Photosynthesis is the foundation of life on Earth. However, if not well regulated, it can also generate excessive reactive oxygen species (ROS), which can cause photodamage. Regulation of photosynthesis is highly dynamic, responding to both environmental and metabolic cues, and occurs at many levels, from light capture to energy storage and metabolic processes. One general mechanism of regulation involves the reversible oxidation and reduction of protein thiol groups, which can affect the activity of enzymes and the stability of proteins. Such redox regulation has been well studied in stromal enzymes, but more recently, evidence has emerged of redox control of thylakoid lumenal enzymes. This review/hypothesis paper summarizes the latest research and discusses several open questions and challenges to achieving effective redox control in the lumen, focusing on the distinct environments and regulatory components of the thylakoid lumen, including the need to transport electrons across the thylakoid membrane, the effects of pH changes by the proton motive force (pmf) in the stromal and lumenal compartments, and the observed differences in redox states. These constraints suggest that activated oxygen species are likely to be major regulatory contributors to lumenal thiol redox regulation, with key components and processes yet to be discovered.

Identification of quantitative trait loci for drought tolerance in Bukoba/Kijivu Andean mapping population of common bean.

KEY MESSAGE: Quantitative Trait Loci "hotspots" for drought tolerance were identified on chromosomes Pv06, Pv07 and Pv10 of common bean. Drought is a major production constraint of common bean (Phaseolus vulgaris L.) worldwide. The objective of this study was to identify the Quantitative Trait Loci (QTL) for drought tolerance in an Andean population of Recombinant Inbred Lines (RILs). A total of 155 F5:7 RILs derived from a cross between Kijivu (drought tolerant) and Bukoba (drought susceptible) were evaluated for drought tolerance in field and pot experiments. Four field experiments were conducted at three locations in Zambia in 2020 and 2021. All field trials were conducted in the dry season under irrigation. The 155 RILs were genotyped with 11,292 SNPs, and composite interval mapping was conducted to identify QTL for drought tolerance. Seed yield for Kijivu under drought stress was consistently higher than for Bukoba across all four field trials. A total of 60 QTL were identified for morphological, agronomic, and physiological traits under drought stress and non-stress conditions. However, the majority of these QTL were specific to drought stress. QTL "hotspots" for drought tolerance were identified on chromosomes Pv06, Pv07, and Pv10. Extensive co-localizations for agronomic and morpho-physiological traits under drought stress were observed at the three drought-tolerance QTL hotspots. Additionally, these three QTL hotspots overlapped with previously identified QTL for drought tolerance, while several others identified QTL are novel. The three identified QTL hotspots could be used in future marker-assisted selection for drought tolerance in common bean.

Long-Range Electron Transport Rates Depend on Wire Dimensions in Cytochrome Nanowires.

The ability to redirect electron transport to new reactions in living systems opens possibilities to store energy, generate new products, or probe physiological processes. Recent work by Huang et al. showed that 3D crystals of small tetraheme cytochromes (STC) can transport electrons over nanoscopic to mesoscopic distances by an electron hopping mechanism, making them promising materials for nanowires. However, fluctuations at room temperature may distort the nanostructure, hindering efficient electron transport. Classical molecular dynamics simulations of these fluctuations at the nano- and mesoscopic scales allowed us to develop a graph network representation to estimate maximum electron flow that can be driven through STC wires. In longer nanowires, transient structural fluctuations at protein-protein interfaces tended to obstruct efficient electron transfer, but these blockages are ameliorated in thicker crystals where alternative electron transfer pathways become more efficient. The model implies that more flexible proteinprotein interfaces limit the required minimum diameter to carry currents commensurate with conventional electronics.

Plants cope with fluctuating light by frequency-dependent nonphotochemical quenching and cyclic electron transport.

In natural environments, plants are exposed to rapidly changing light. Maintaining photosynthetic efficiency while avoiding photodamage requires equally rapid regulation of photoprotective mechanisms. We asked what the operation frequency range of regulation is in which plants can efficiently respond to varying light. Chlorophyll fluorescence, P700, plastocyanin, and ferredoxin responses of wild-types Arabidopsis thaliana were measured in oscillating light of various frequencies. We also investigated the npq1 mutant lacking violaxanthin de-epoxidase, the npq4 mutant lacking PsbS protein, and the mutants crr2-2, and pgrl1ab impaired in different pathways of the cyclic electron transport. The fastest was the PsbS-regulation responding to oscillation periods longer than 10 s. Processes involving violaxanthin de-epoxidase dampened changes in chlorophyll fluorescence in oscillation periods of 2 min or longer. Knocking out the PGR5/PGRL1 pathway strongly reduced variations of all monitored parameters, probably due to congestion in the electron transport. Incapacitating the NDH-like pathway only slightly changed the photosynthetic dynamics. Our observations are consistent with the hypothesis that nonphotochemical quenching in slow light oscillations involves violaxanthin de-epoxidase to produce, presumably, a largely stationary level of zeaxanthin. We interpret the observed dynamics of photosystem I components as being formed in slow light oscillations partially by thylakoid remodeling that modulates the redox rates.

Arabidopsis stromal carbonic anhydrases exhibit non-overlapping roles in photosynthetic efficiency and development.

Carbonic anhydrases (CAs) are ubiquitous enzymes that accelerate the reversible conversion of CO2 to HCO3 - . The Arabidopsis genome encodes members of the α-, ß- and γ-CA families, and it has been hypothesized that ßCA activity has a role in photosynthesis. In this work, we tested this hypothesis by characterizing the two plastidial ßCAs, ßCA1 and ßCA5, in physiological conditions of growth. We conclusively established that both proteins are localized in the chloroplast stroma and that the loss of ßCA5 induced the expression of ßCA1, supporting the existence of regulatory mechanisms to control the expression of stromal ßCAs. We also established that ßCA1 and ßCA5 have markedly different enzymatic kinetics and physiological relevance. Specifically, we found that ßCA5 had a first-order rate constant ~10-fold lower than ßCA1, and that the loss of ßCA5 is detrimental to growth and could be rescued by high CO2 . Furthermore, we established that, while a ßCA1 mutation showed near wild-type growth and no significant impact on photosynthetic efficiency, the loss of ßCA5 markedly disrupted photosynthetic efficiency and light-harvesting capacity at ambient CO2 . Therefore, we conclude that in physiological autotrophic growth, the loss of the more highly expressed ßCA1 does not compensate for the loss of a less active ßCA5, which in turn is involved in growth and photosynthesis at ambient CO2 levels. These results lend support to the hypothesis that, in Arabidopsis,ßCAs have non-overlapping roles in photosynthesis and identify a critical activity of stromal ßCA5 and a dispensable role for ßCA1.

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.

The time course of acclimation to the stress of triose phosphate use limitation.

Triose phosphate utilisation (TPU) limits the maximum rate at which plants can photosynthesise. However, TPU is almost never found to be limiting photosynthesis under ambient conditions for plants. This, along with previous results showing adaptability of TPU at low temperature, suggest that TPU capacity is regulated to be just above the photosynthetic rate achievable under the prevailing conditions. A set of experiments were performed to study the adaptability of TPU capacity when plants are acclimated to elevated CO2 concentrations. Plants held at 1500 ppm CO2 were initially TPU limited. After 30 h they no longer exhibited TPU limitations but they did not elevate their TPU capacity. Instead, the maximum rates of carboxylation and electron transport declined. A timecourse of regulatory responses was established. A step increase of CO2 first caused PSI to be oxidised but after 40 s both PSI and PSII had excess electrons as a result of acceptor-side limitations. Electron flow to PSI slowed and the proton motive force increased. Eventually, non-photochemical quenching reduced electron flow sufficiently to balance the TPU limitation. Over several minutes rubisco deactivated contributing to regulation of metabolism to overcome the TPU limitation.

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.

The Arabidopsis T-DNA mutant SALK_008491 carries a 14-kb deletion on chromosome 3 that provides rare insights into the plant response to dynamic light stress.

In nature, plants experience rapid changes in light intensity and quality throughout the day. To maximize growth, they have established molecular mechanisms to optimize photosynthetic output while protecting components of the light-dependent reaction and CO2 fixation pathways. Plant phenotyping of mutant collections has become a powerful tool to unveil the genetic loci involved in environmental acclimation. Here, we describe the phenotyping of the transfer-DNA (T-DNA) insertion mutant line SALK_008491, previously known as nhd1-1. Growth in a fluctuating light regime caused a loss in growth rate accompanied by a spike in photosystem (PS) II damage and increased non-photochemical quenching (NPQ). Interestingly, an independent nhd1 null allele did not recapitulate the NPQ phenotype. Through bulk sequencing of a backcrossed segregating F2 pool, we identified an ~14-kb large deletion on chromosome 3 (Chr3) in SALK_008491 affecting five genes upstream of NHD1. Besides NHD1, which encodes for a putative plastid Na+/H+ antiporter, the stromal NAD-dependent D-3-phosphoglycerate dehydrogenase 3 (PGDH3) locus was eradicated. Although some changes in the SALK_008491 mutant's photosynthesis can be assigned to the loss of PGDH3, our follow-up studies employing respective single mutants and complementation with overlapping transformation-competent artificial chromosome (TAC) vectors reveal that the exacerbated fluctuating light sensitivity in SALK_008491 mutants result from the simultaneous loss of PGDH3 and NHD1. Altogether, the data obtained from this large deletion-carrying mutant provide new and unintuitive insights into the molecular mechanisms that function to protect the photosynthetic machinery. Moreover, our study renews calls for caution when setting up reverse genetic studies using T-DNA lines. Although second-site insertions, indels, and SNPs have been reported before, large deletion surrounding the insertion site causes yet another problem. Nevertheless, as shown through this research, such unpredictable genetic events following T-DNA mutagenesis can provide unintuitive insights that allow for understanding complex phenomena such as the plant acclimation to dynamic high light stress.

Genetically-determined variations in photosynthesis indicate roles for specific fatty acid species in chilling responses.

Using a population of recombinant inbred lines (RILs) cowpea (Vigna unguiculata. L. Walp), we tested for co-linkages between lipid contents and chilling responses of photosynthesis. Under low-temperature conditions (19°C/13°C, day/night), we observed co-linkages between quantitative trait loci intervals for photosynthetic light reactions and specific fatty acids, most strikingly, the thylakoid-specific fatty acid 16:1Δ3trans found exclusively in phosphatidylglycerol (PG 16:1t). By contrast, we did not observe co-associations with bulk polyunsaturated fatty acids or high-melting-point-PG (sum of PG 16:0, PG 18:0 and PG 16:1t) previously thought to be involved in chilling sensitivity. These results suggest that in cowpea, chilling sensitivity is modulated by specific lipid interactions rather than bulk properties. We were able to recapitulate the predicted impact of PG 16:1t levels on photosynthetic responses at low temperature using mutants and transgenic Arabidopsis lines. Because PG 16:1t synthesis requires the activity of peroxiredoxin-Q, which is activated by H2 O2 and known to be involved in redox signalling, we hypothesise that the accumulation of PG 16:1t occurs as a result of upstream effects on photosynthesis that alter redox status and production of reactive oxygen species.

Light potentials of photosynthetic energy storage in the field: what limits the ability to use or dissipate rapidly increased light energy?

The responses of plant photosynthesis to rapid fluctuations in environmental conditions are critical for efficient conversion of light energy. These responses are not well-seen laboratory conditions and are difficult to probe in field environments. We demonstrate an open science approach to this problem that combines multifaceted measurements of photosynthesis and environmental conditions, and an unsupervised statistical clustering approach. In a selected set of data on mint (Mentha sp.), we show that 'light potentials' for linear electron flow and non-photochemical quenching (NPQ) upon rapid light increases are strongly suppressed in leaves previously exposed to low ambient photosynthetically active radiation (PAR) or low leaf temperatures, factors that can act both independently and cooperatively. Further analyses allowed us to test specific mechanisms. With decreasing leaf temperature or PAR, limitations to photosynthesis during high light fluctuations shifted from rapidly induced NPQ to photosynthetic control of electron flow at the cytochrome b6f complex. At low temperatures, high light induced lumen acidification, but did not induce NPQ, leading to accumulation of reduced electron transfer intermediates, probably inducing photodamage, revealing a potential target for improving the efficiency and robustness of photosynthesis. We discuss the implications of the approach for open science efforts to understand and improve crop productivity.

The induction of pyrenoid synthesis by hyperoxia and its implications for the natural diversity of photosynthetic responses in Chlamydomonas.

In algae, it is well established that the pyrenoid, a component of the carbon-concentrating mechanism (CCM), is essential for efficient photosynthesis at low CO2. However, the signal that triggers the formation of the pyrenoid has remained elusive. Here, we show that, in Chlamydomonas reinhardtii, the pyrenoid is strongly induced by hyperoxia, even at high CO2 or bicarbonate levels. These results suggest that the pyrenoid can be induced by a common product of photosynthesis specific to low CO2 or hyperoxia. Consistent with this view, the photorespiratory by-product, H2O2, induced the pyrenoid, suggesting that it acts as a signal. Finally, we show evidence for linkages between genetic variations in hyperoxia tolerance, H2O2 signaling, and pyrenoid morphologies.

Inactivation of mitochondrial complex I stimulates chloroplast ATPase in Physcomitrium patens.

Light is the ultimate source of energy for photosynthetic organisms, but respiration is fundamental for supporting metabolism during the night or in heterotrophic tissues. In this work, we isolated Physcomitrella (Physcomitrium patens) plants with altered respiration by inactivating Complex I (CI) of the mitochondrial electron transport chain by independently targeting on two essential subunits. Inactivation of CI caused a strong growth impairment even in fully autotrophic conditions in tissues where all cells are photosynthetically active, demonstrating that respiration is essential for photosynthesis. CI mutants showed alterations in the stoichiometry of respiratory complexes while the composition of photosynthetic apparatus was substantially unaffected. CI mutants showed altered photosynthesis with high activity of both Photosystems I and II, likely the result of high chloroplast ATPase activity that led to smaller ΔpH formation across thylakoid membranes, decreasing photosynthetic control on cytochrome b6f in CI mutants. These results demonstrate that alteration of respiratory activity directly impacts photosynthesis in P. patens and that metabolic interaction between organelles is essential in their ability to use light energy for growth.

Improved photosynthetic capacity and photosystem I oxidation via heterologous metabolism engineering in cyanobacteria.

Cyanobacteria must prevent imbalances between absorbed light energy (source) and the metabolic capacity (sink) to utilize it to protect their photosynthetic apparatus against damage. A number of photoprotective mechanisms assist in dissipating excess absorbed energy, including respiratory terminal oxidases and flavodiiron proteins, but inherently reduce photosynthetic efficiency. Recently, it has been hypothesized that some engineered metabolic pathways may improve photosynthetic performance by correcting source/sink imbalances. In the context of this subject, we explored the interconnectivity between endogenous electron valves, and the activation of one or more heterologous metabolic sinks. We coexpressed two heterologous metabolic pathways that have been previously shown to positively impact photosynthetic activity in cyanobacteria, a sucrose production pathway (consuming ATP and reductant) and a reductant-only consuming cytochrome P450. Sucrose export was associated with improved quantum yield of phtotosystem II (PSII) and enhanced electron transport chain flux, especially at lower illumination levels, while cytochrome P450 activity led to photosynthetic enhancements primarily observed under high light. Moreover, coexpression of these two heterologous sinks showed additive impacts on photosynthesis, indicating that neither sink alone was capable of utilizing the full "overcapacity" of the electron transport chain. We find that heterologous sinks may partially compensate for the loss of photosystem I (PSI) oxidizing mechanisms even under rapid illumination changes, although this compensation is incomplete. Our results provide support for the theory that heterologous metabolism can act as a photosynthetic sink and exhibit some overlapping functionality with photoprotective mechanisms, while potentially conserving energy within useful metabolic products that might otherwise be "lost."

The roles of photorespiration and alternative electron acceptors in the responses of photosynthesis to elevated temperatures in cowpea.

We explored the effects, on photosynthesis in cowpea (Vigna unguiculata) seedlings, of high temperature and light-environmental stresses that often co-occur under field conditions and can have greater impact on photosynthesis than either by itself. We observed contrasting responses in the light and carbon assimilatory reactions, whereby in high temperature, the light reactions were stimulated while CO2 assimilation was substantially reduced. There were two striking observations. Firstly, the primary quinone acceptor (QA ), a measure of the regulatory balance of the light reactions, became more oxidized with increasing temperature, suggesting increased electron sink capacity, despite the reduced CO2 fixation. Secondly, a strong, O2 -dependent inactivation of assimilation capacity, consistent with down-regulation of rubisco under these conditions. The dependence of these effects on CO2 , O2 and light led us to conclude that both photorespiration and an alternative electron acceptor supported increased electron flow, and thus provided photoprotection under these conditions. Further experiments showed that the increased electron flow was maintained by rapid rates of PSII repair, particularly at combined high light and temperature. Overall, the results suggest that photodamage to the light reactions can be avoided under high light and temperatures by increasing electron sink strength, even when assimilation is strongly suppressed.

Mesoscopic to Macroscopic Electron Transfer by Hopping in a Crystal Network of Cytochromes.

Rapid and directed electron transfer (ET) is essential for biological processes. While the rates of ET over 1-2 nm in proteins can largely be described by simplified nonadiabatic theory, it is not known how these processes scale to microscopic distances. We generated crystalline lattices of Small Tetraheme Cytochromes (STC) forming well-defined, three-dimensional networks of closely spaced redox centers that appear to be nearly ideal for multistep ET. Electrons were injected into specific locations in the STC crystals by direct photoreduction, and their redistribution was monitored by imaging. The results demonstrate ET over mesoscopic to microscopic (∼100 µm) distances through sequential hopping in a biologically based heme network. We estimate that a hypothetical "nanowire" composed of crystalline STC with a cross-section of about 100 cytochromes could support the anaerobic respiration of a Shewanella cell. The crystalline lattice insulates mobile electrons from oxidation by O2, as compared to those in cytochromes in solution, potentially allowing for efficient delivery of current without production of reactive oxygen species. The platform allows direct tests of whether the assumptions based on short-range ET hold for sequential ET over mesoscopic distances. We estimate that the interprotein ET across 6 Å between hemes in adjacent proteins was about 105 s-1, about 100-fold slower than expectations based on simplified theory. More detailed analyses implied that additional factors, possibly contributed by the crystal lattice, may strongly impact mesoscale ET mainly by increasing the reorganizational energy of interprotein ET, which suggests design strategies for engineering improved nanowires suitable for future bioelectronic materials.