Photosynthetic control at the cytochrome b6f complex.

Photosynthetic control (PCON) is a protective mechanism that prevents light-induced damage to photosystem I (PSI) by ensuring the rate of NADPH and ATP production via linear electron transfer (LET) is balanced by their consumption in the CO2 fixation reactions. Protection of PSI is a priority for plants since they lack a dedicated rapid-repair cycle for this complex, meaning that any damage leads to prolonged photoinhibition and decreased growth. The imbalance between LET and the CO2 fixation reactions is sensed at the level of the transthylakoid ΔpH, which increases when light is in excess. The canonical mechanism of PCON involves feedback control by ΔpH on the plastoquinol oxidation step of LET at cytochrome b6f. PCON thereby maintains the PSI special pair chlorophylls (P700) in an oxidized state, that allows excess electrons unused in the CO2 fixation reactions to be safely quenched via charge recombination. In this review we focus on angiosperms, considering how photo-oxidative damage to PSI comes about, explore the consequences of PSI photoinhibition on photosynthesis and growth, discuss recent progress in understanding PCON regulation, and finally consider the prospects for its future manipulation in crop plants to improve photosynthetic efficiency.

Proton Gradient Regulation 5 is required to avoid photosynthetic oscillations during light transitions.

The production of ATP and NADPH by the light reactions of photosynthesis and their consumption by the Calvin-Benson-Bassham (CBB) cycle and other downstream metabolic reactions requires careful regulation. Environmental shifts perturb this balance, leading to photo-oxidative stress and losses in CO2 assimilation. Imbalances in the production and consumption of ATP and NADPH manifest themselves as transient instability in the chlorophyll fluorescence, P700, electrochromic shift, and CO2 uptake signals recorded on leaves. These oscillations can be induced in wild-type plants by sudden shifts in CO2 concentration or light intensity; however, mutants exhibiting increased oscillatory behaviour have yet to be reported. This has precluded an understanding of the regulatory mechanisms employed by plants to suppress oscillations. Here we show that the Arabidopsis pgr5 mutant, which is deficient in Proton Gradient Regulation 5 (PGR5)-dependent cyclic electron transfer (CET), exhibits increased oscillatory behaviour. In contrast, mutants lacking the NADH-dehydrogenase-like-dependent CET are largely unaffected. The absence of oscillations in the hope2 mutant which, like pgr5, lacks photosynthetic control and exhibits high ATP synthase conductivity, ruled out loss of these photoprotective mechanisms as causes. Instead, we observed slower formation of the proton motive force and, by inference, ATP synthesis in pgr5 following environmental perturbation, leading to the transient reduction of the electron transfer chain and photosynthetic oscillations. PGR5-dependent CET therefore plays a major role in damping the effect of environmental perturbations on photosynthesis to avoid losses in CO2 fixation.

High cyclic electron transfer via the PGR5 pathway in the absence of photosynthetic control.

The light reactions of photosynthesis couple electron and proton transfers across the thylakoid membrane, generating NADPH, and proton motive force (pmf) that powers the endergonic synthesis of ATP by ATP synthase. ATP and NADPH are required for CO2 fixation into carbohydrates by the Calvin-Benson-Bassham cycle. The dominant ΔpH component of the pmf also plays a photoprotective role in regulating photosystem II light harvesting efficiency through nonphotochemical quenching (NPQ) and photosynthetic control via electron transfer from cytochrome b6f (cytb6f) to photosystem I. ΔpH can be adjusted by increasing the proton influx into the thylakoid lumen via upregulation of cyclic electron transfer (CET) or decreasing proton efflux via downregulation of ATP synthase conductivity (gH+). The interplay and relative contributions of these two elements of ΔpH control to photoprotection are not well understood. Here, we showed that an Arabidopsis (Arabidopsis thaliana) ATP synthase mutant hunger for oxygen in photosynthetic transfer reaction 2 (hope2) with 40% higher proton efflux has supercharged CET. Double crosses of hope2 with the CET-deficient proton gradient regulation 5 and ndh-like photosynthetic complex I lines revealed that PROTON GRADIENT REGULATION 5 (PGR5)-dependent CET is the major pathway contributing to higher proton influx. PGR5-dependent CET allowed hope2 to maintain wild-type levels of ΔpH, CO2 fixation and NPQ, however photosynthetic control remained absent and PSI was prone to photoinhibition. Therefore, high CET in the absence of ATP synthase regulation is insufficient for PSI photoprotection.

Inhibition of Arabidopsis stomatal development by plastoquinone oxidation.

Stomata are the pores in the epidermal surface of plant leaves that regulate the exchange of water and CO2 with the environment thus controlling leaf gas exchange.1 In the model dicot plant Arabidopsis thaliana, the transcription factors SPEECHLESS (SPCH) and MUTE sequentially control formative divisions in the stomatal lineage by forming heterodimers with ICE1.2 SPCH regulates entry into the stomatal lineage and its stability or activity is regulated by a mitogen-activated protein kinase (MAPK) signaling cascade, mediated by its interaction with ICE1.3-6 This MAPK pathway is regulated by extracellular epidermal patterning factor (EPFs) peptides, which bind a transmembrane receptor complex to inhibit (EPF1 and EPF2) or promote (STOMAGEN/EPFL9) stomatal development.7-9 MUTE controls the transition to guard mother cell identity and is regulated by the HD-ZIP transcription factor HDG2, which is expressed exclusively in stomatal lineage cells.10,11 Light signals acting through phytochrome and cryptochrome photoreceptors positively regulate stomatal development in response to increased irradiance.12,13 Here we report that stomatal development is also regulated by the redox state of the photosynthetic electron transport chain (PETC). Oxidation of the plastoquinone (PQ) pool inhibits stomatal development by negatively regulating SPCH and MUTE expression. This mechanism is dependent on MPK6 and forms part of the response to lowering irradiance, which is distinct to the photoreceptor dependent response to increasing irradiance. Our results show that environmental signals can act through the PETC, demonstrating that photosynthetic signals regulate the development of the pores through which CO2 enters the leaf.

Heat-induced changes in the abundance of wheat Rubisco activase isoforms.

The Triticum aestivum (wheat) genome encodes three isoforms of Rubisco activase (Rca) differing in thermostability, which could be exploited to improve the resilience of this crop to global warming. We hypothesized that elevated temperatures would cause an increase in the relative abundance of heat-stable Rca1ß. Wheat plants were grown at 25° C : 18°C (day : night) and exposed to heat stress (38° C : 22°C) for up to 5 d at pre-anthesis. Carbon (C) assimilation, Rubisco activity, CA1Pase activity, transcripts of Rca1ß, Rca2ß, and Rca2α, and the quantities of the corresponding protein products were measured during and after heat stress. The transcript of Rca1ß increased 40-fold in 4 h at elevated temperatures and returned to the original level after 4 h upon return of plants to control temperatures. Rca1ß comprised up to 2% of the total Rca protein in unstressed leaves but increased three-fold in leaves exposed to elevated temperatures for 5 d and remained high at 4 h after heat stress. These results show that elevated temperatures cause rapid changes in Rca gene expression and adaptive changes in Rca isoform abundance. The improved understanding of the regulation of C assimilation under heat stress will inform efforts to improve wheat productivity and climate resilience.

An isoleucine residue acts as a thermal and regulatory switch in wheat Rubisco activase.

The regulation of Rubisco, the gatekeeper of carbon fixation into the biosphere, by its molecular chaperone Rubisco activase (Rca) is essential for photosynthesis and plant growth. Using energy from ATP hydrolysis, Rca promotes the release of inhibitors and restores catalytic competence to Rubisco-active sites. Rca is sensitive to moderate heat stress, however, and becomes progressively inhibited as the temperature increases above the optimum for photosynthesis. Here, we identify a single amino acid substitution (M159I) that fundamentally alters the thermal and regulatory properties of Rca in bread wheat (Triticum aestivum L.). Using site-directed mutagenesis, we demonstrate that the M159I substitution extends the temperature optimum of the most abundant Rca isoform by 5°C in vitro, while maintaining the efficiency of Rubisco activation by Rca. The results suggest that this single amino acid substitution acts as a thermal and regulatory switch in wheat Rca that can be exploited to improve the climate resilience and efficiency of carbon assimilation of this cereal crop as temperatures become warmer and more volatile.

Rubisco activation by wheat Rubisco activase isoform 2ß is insensitive to inhibition by ADP.

Rubisco activase (Rca) is a catalytic chaperone that remodels the active site, promotes the release of inhibitors and restores catalytic competence to Rubisco. Rca activity and its consequent effect on Rubisco activation and photosynthesis are modulated by changes to the chloroplast environment induced by fluctuations in light levels that reach the leaf, including redox status and adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio. The Triticum aestivum (wheat) genome encodes for three Rca protein isoforms: 1ß (42.7 kDa), 2ß (42.2 kDa) and 2α (46.0 kDa). The regulatory properties of these isoforms were characterised by measuring rates of Rubisco activation and ATP hydrolysis by purified recombinant Rca proteins in the presence of physiological ADP/ATP ratios. ATP hydrolysis by all three isoforms was sensitive to inhibition by increasing amounts of ADP in the assay. In contrast, Rubisco activation activity of Rca 2ß was insensitive to ADP inhibition, while Rca 1ß and 2α were inhibited. Two double and one quadruple site-directed mutants were designed to elucidate if differences in the amino acid sequences between Rca 1ß and 2ß could explain the differences in ADP sensitivity. Changing two amino acids in Rca 2ß to the corresponding residues in 1ß (T358K & Q362E) resulted in significant inhibition of Rubisco activation in presence of ADP. The results show that the wheat Rca isoforms differ in their regulatory properties and that amino acid changes in the C domain influence ADP sensitivity. Advances in the understanding of Rubisco regulation will aid efforts to improve the efficiency of photosynthetic CO2 assimilation.

Spectrophotometric Determination of RuBisCO Activity and Activation State in Leaf Extracts.

RuBisCO plays a central role in photosynthesis and, due to its catalytic inefficiencies, frequently limits CO2 assimilation in fully illuminated leaves at the top of unstressed crop canopies. The CO2-fixing enzyme is heavily regulated and not all the enzyme present in the leaf is active at any given moment. In this chapter, a spectrophotometric assay is described for measuring RuBisCO activity and activation state in leaf extracts. Most of the assay components are available commercially and others can be produced by established protocols, making adoption of the assay achievable by most plant biochemistry laboratories. Its relative high-throughput capacity enables large-scale experiments aimed at screening germplasm for improved RuBisCO function.

Increasing metabolic potential: C-fixation.

Due to the growing world population, crop yields must increase to meet the rising demand. Crop plants also require adaptation to optimize performance in the changing environments caused by climate change. Improving photosynthetic carbon fixation is a promising, albeit technically challenging, strategy whose potential has only just begun to be considered in breeding programmes. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a fundamental enzyme of carbon fixation, is extremely inefficient and many strategies to improve photosynthesis focus on overcoming the limitations of this enzyme, either by improving Rubisco activity and regulation or by improving the supply of substrates. Although progress is being made, the need to tailor solutions for each crop and their respective environments has been highlighted. Even so, continuing research will be required to achieve these objectives and to grow crops more sustainably in the future.