The GABA shunt contributes to ROS homeostasis in guard cells of Arabidopsis.

γ-Aminobutyric acid (GABA) accumulates rapidly under stress via the GABA shunt pathway, which has been implicated in reducing the accumulation of stress-induced reactive oxygen species (ROS) in plants. γ-Aminobutyric acid has been demonstrated to act as a guard-cell signal in Arabidopsis thaliana, modulating stomatal opening. Knockout of the major GABA synthesis enzyme Glutamate Decarboxylase 2 (GAD2) increases the aperture of gad2 mutants, which results in greater stomatal conductance and reduces water-use efficiency compared with wild-type plants. Here, we found that the additional loss of GAD1, GAD4, and GAD5 in gad2 leaves increased GABA deficiency but abolished the more open stomatal pore phenotype of gad2, which we link to increased cytosolic calcium (Ca2+ ) and ROS accumulation in gad1/2/4/5 guard cells. Compared with wild-type and gad2 plants, glutamate was ineffective in closing gad1/2/4/5 stomatal pores, whereas lowering apoplastic calcium, applying ROS inhibitors or complementation with GAD2 reduced gad1/2/4/5 guard-cell ROS, restored the gad2-like greater stomatal apertures of gad1/2/4/5 beyond that of wild-type. We conclude that GADs are important contributors to ROS homeostasis in guard cells likely via a Ca2+ -mediated pathway. As such, this study reveals greater complexity in GABA's role as a guard-cell signal and the interactions it has with other established signals.

Lessons from relatives: C4 photosynthesis enhances CO2 assimilation during the low-light phase of fluctuations.

Despite the global importance of species with C4 photosynthesis, there is a lack of consensus regarding C4 performance under fluctuating light. Contrasting hypotheses and experimental evidence suggest that C4 photosynthesis is either less or more efficient in fixing carbon under fluctuating light than the ancestral C3 form. Two main issues have been identified that may underly the lack of consensus: neglect of evolutionary distance between selected C3 and C4 species and use of contrasting fluctuating light treatments. To circumvent these issues, we measured photosynthetic responses to fluctuating light across 3 independent phylogenetically controlled comparisons between C3 and C4 species from Alloteropsis, Flaveria, and Cleome genera under 21% and 2% O2. Leaves were subjected to repetitive stepwise changes in light intensity (800 and 100 µmol m-2 s-1 photon flux density) with 3 contrasting durations: 6, 30, and 300 s. These experiments reconciled the opposing results found across previous studies and showed that (i) stimulation of CO2 assimilation in C4 species during the low-light phase was both stronger and more sustained than in C3 species; (ii) CO2 assimilation patterns during the high-light phase could be attributable to species or C4 subtype differences rather than photosynthetic pathway; and (iii) the duration of each light step in the fluctuation regime can strongly influence experimental outcomes.

Compartmentation of photosynthesis gene expression in C4 maize depends on time of day.

Compared with the ancestral C3 state, C4 photosynthesis occurs at higher rates with improved water and nitrogen use efficiencies. In both C3 and C4 plants, rates of photosynthesis increase with light intensity and are maximal around midday. We determined that in the absence of light or temperature fluctuations, photosynthesis in maize (Zea mays) peaks in the middle of the subjective photoperiod. To investigate the molecular processes associated with these temporal changes, we performed RNA sequencing of maize mesophyll and bundle sheath strands over a 24-h time course. Preferential expression of C4 cycle genes in these cell types was strongest between 6 and 10 h after dawn when rates of photosynthesis were highest. For the bundle sheath, DNA motif enrichment and gene coexpression analyses suggested members of the DNA binding with one finger (DOF) and MADS (MINICHROMOSOME MAINTENANCE FACTOR 1/AGAMOUS/DEFICIENS/Serum Response Factor)-domain transcription factor families mediate diurnal fluctuations in C4 gene expression, while trans-activation assays in planta confirmed their ability to activate promoter fragments from bundle sheath expressed genes. The work thus identifies transcriptional regulators and peaks in cell-specific C4 gene expression coincident with maximum rates of photosynthesis in the maize leaf at midday.

The long and tortuous path towards improving photosynthesis by engineering elevated mesophyll conductance.

The growing demand for global food production is likely to be a defining issue facing humanity over the next 50 years. To tackle this challenge, there is a desire to bioengineer crops with higher photosynthetic efficiencies, to increase yields. Recently, there has been a growing interest in engineering leaves with higher mesophyll conductance (gm), which would allow CO2 to move more efficiently from the substomatal cavities to the chloroplast stroma. However, if crop yield gains are to be realised through this approach, it is essential that the methodological limitations associated with estimating gm are fully appreciated. In this review, we summarise these limitations, and outline the uncertainties and assumptions that can affect the final estimation of gm. Furthermore, we critically assess the predicted quantitative effect that elevating gm will have on assimilation rates in crop species. We highlight the need for more theoretical modelling to determine whether altering gm is truly a viable route to improve crop performance. Finally, we offer suggestions to guide future research on gm, which will help mitigate the uncertainty inherently associated with estimating this parameter.

A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

Illuminating stomatal responses to red light: establishing the role of Ci-dependent vs -independent mechanisms in control of stomatal behaviour.

The stomatal response to red light appears to link stomatal conductance (gs) with photosynthetic rates. Initially, it was suggested that changes in intercellular CO2 (Ci) provide the main cue via a Ci-dependent response. However, evidence for Ci-independent mechanisms suggests an additional, more direct relationship with photosynthesis. While both Ci-dependent and -independent mechanisms clearly function in stomatal red-light responses, little is known regarding their relative contribution. The present study aimed to quantify the relative magnitude of Ci-dependent and -independent mechanisms on the stomatal red-light response, to characterise their interplay and to assess the putative link between plastoquinone (PQ) redox state and Ci-independent stomatal responses. Red light response curves measured at a range of Ci values for wild-type Arabidopsis thaliana (Col-0) and the CO2 hyposensitive mutant, ca1ca4, allowed deconvolution of Ci-dependent and -independent pathways. Surprisingly, we observed that both mechanisms contribute equally to stomatal red-light responses, but Ci-independent stomatal opening is suppressed at high Ci. The present data are also consistent with the involvement of PQ redox in coordinating the Ci-independent component. Overall, it seems that while Ci-independent mechanisms are distinct from responses to Ci, interplay between these two pathways is important to facilitate effective coordination between gs and photosynthesis.

GABA does not regulate stomatal CO2 signalling in Arabidopsis.

Optimal stomatal regulation is important for plant adaptation to changing environmental conditions and for maintaining crop yield. The guard-cell signal GABA is produced from glutamate by Glutamate Decarboxylase (GAD) during a reaction that generates carbon dioxide (CO2) as a by-product. Here, we investigated a putative connection between GABA signalling and the more clearly defined CO2 signalling pathway in guard cells. The GABA-deficient mutant lines gad2-1, gad2-2 and gad1/2/4/5 were examined for stomatal sensitivity to various CO2 concentrations. Our findings show a phenotypical discrepancy between the allelic mutant lines gad2-1 and gad2-2 - a weakened CO2 response in gad2-1 (GABI_474_E05) in contrast to a wild-type response in gad2-2 (SALK_028819) and gad1/2/4/5. Through transcriptomic and genomic investigation, we traced the response of gad2-1 to a deletion of full-length Mitogen-activated protein kinase 12 (MPK12) in the GABI-KAT line, thereafter as renamed gad2-1*. Guard cell-specific complementation of MPK12 restored the gad2-1* CO2 phenotype, which confirms the proposed importance of MPK12 to CO2 sensitivity. Additionally, we found that stomatal opening under low atmospheric CO2 occurs independently of the GABA-modulated opening-channel ALMT9. Our results confirm that GABA has a role in modulating the rate of stomatal opening and closing - but not in response to CO2  per se.

Photosynthesis-independent production of reactive oxygen species in the rice bundle sheath during high light is mediated by NADPH oxidase.

When exposed to high light, plants produce reactive oxygen species (ROS). In Arabidopsis thaliana, local stress such as excess heat or light initiates a systemic ROS wave in phloem and xylem cells dependent on NADPH oxidase/respiratory burst oxidase homolog (RBOH) proteins. In the case of excess light, although the initial local accumulation of ROS preferentially takes place in bundle-sheath strands, little is known about how this response takes place. Using rice and the ROS probes diaminobenzidine and 2',7'-dichlorodihydrofluorescein diacetate, we found that, after exposure to high light, ROS were produced more rapidly in bundle-sheath strands than mesophyll cells. This response was not affected either by CO2 supply or photorespiration. Consistent with these findings, deep sequencing of messenger RNA (mRNA) isolated from mesophyll or bundle-sheath strands indicated balanced accumulation of transcripts encoding all major components of the photosynthetic apparatus. However, transcripts encoding several isoforms of the superoxide/H2O2-producing enzyme NADPH oxidase were more abundant in bundle-sheath strands than mesophyll cells. ROS production in bundle-sheath strands was decreased in mutant alleles of the bundle-sheath strand preferential isoform of OsRBOHA and increased when it was overexpressed. Despite the plethora of pathways able to generate ROS in response to excess light, NADPH oxidase-mediated accumulation of ROS in the rice bundle-sheath strand was detected in etiolated leaves lacking chlorophyll. We conclude that photosynthesis is not necessary for the local ROS response to high light but is in part mediated by NADPH oxidase activity.

Is chloroplast size optimal for photosynthetic efficiency?

Improving photosynthetic efficiency has recently emerged as a promising way to increase crop production in a sustainable manner. While chloroplast size may affect photosynthetic efficiency in several ways, we aimed to explore whether chloroplast size manipulation can be a viable approach to improving photosynthetic performance. Several tobacco (Nicotiana tabacum) lines with contrasting chloroplast sizes were generated via manipulation of chloroplast division genes to assess photosynthetic performance under steady-state and fluctuating light. A selection of lines was included in a field trial to explore productivity. Lines with enlarged chloroplasts underperformed in most of the measured traits. Lines with smaller and more numerous chloroplasts showed a similar efficiency compared with wild-type (WT) tobacco. Chloroplast size only weakly affected light absorptance and light profiles within the leaf. Increasing chloroplast size decreased mesophyll conductance (gm ) but decreased chloroplast size did not increase gm . Increasing chloroplast size reduced chloroplast movements and enhanced non-photochemical quenching. The chloroplast smaller than WT appeared to be no better than WT for photosynthetic efficiency and productivity under field conditions. The results indicate that chloroplast size manipulations are therefore unlikely to lead to higher photosynthetic efficiency or growth.

Excised leaves show limited and species-specific effects on photosynthetic parameters across crop functional types.

Photosynthesis is increasingly becoming a recognized target for crop improvement. Phenotyping photosynthesis-related traits on field-grown material is a key bottleneck to progress here due to logistical barriers and short measurement days. Many studies attempt to overcome these challenges by phenotyping excised leaf material in the laboratory. To date there are no demonstrated examples of the representative nature of photosynthesis measurements performed on excised leaves relative to attached leaves in crops. Here, we tested whether standardized leaf excision on the day prior to phenotyping affected a range of common photosynthesis-related traits across crop functional types using tomato (C3 dicot), barley (C3 monocot), and maize (C4 monocot). Potentially constraining aspects of leaf physiology that could be predicted to impair photosynthesis in excised leaves, namely leaf water potential and abscisic acid accumulation, were not different between attached and excised leaves. We also observed non-significant differences in spectral reflectance and chlorophyll fluorescence traits between the treatments across the three species. However, we did observe some significant differences between traits associated with gas exchange and photosynthetic capacity across all three species. This study represents a useful reference for those who perform measurements of this nature and the differences reported should be considered in associated experimental design and statistical analyses.

Can we improve the chilling tolerance of maize photosynthesis through breeding?

Chilling tolerance is necessary for crops to thrive in temperate regions where cold snaps and lower baseline temperatures place limits on life processes; this is particularly true for crops of tropical origin such as maize. Photosynthesis is often adversely affected by chilling stress, yet the maintenance of photosynthesis is essential for healthy growth and development, and most crucially for yield. In this review, we describe the physiological basis for enhancing chilling tolerance of photosynthesis in maize by examining nine key responses to chilling stress. We synthesize current knowledge of genetic variation for photosynthetic chilling tolerance in maize with respect to each of these traits and summarize the extent to which genetic mapping and candidate genes have been used to understand the genomic regions underpinning chilling tolerance. Finally, we provide perspectives on the future of breeding for photosynthetic chilling tolerance in maize. We advocate for holistic and high-throughput approaches to screen for chilling tolerance of photosynthesis in research and breeding programmes in order to develop resilient crops for the future.

Guard-cell-targeted overexpression of Arabidopsis Hexokinase 1 can improve water use efficiency in field-grown tobacco plants.

Water deficit currently acts as one of the largest limiting factors for agricultural productivity worldwide. Additionally, limitation by water scarcity is projected to continue in the future with the further onset of effects of global climate change. As a result, it is critical to develop or breed for crops that have increased water use efficiency and that are more capable of coping with water scarce conditions. However, increased intrinsic water use efficiency (iWUE) typically brings a trade-off with CO2 assimilation as all gas exchange is mediated by stomata, through which CO2 enters the leaf while water vapor exits. Previously, promising results were shown using guard-cell-targeted overexpression of hexokinase to increase iWUE without incurring a penalty in photosynthetic rates or biomass production. Here, two homozygous transgenic tobacco (Nicotiana tabacum) lines expressing Arabidopsis Hexokinase 1 (AtHXK1) constitutively (35SHXK2 and 35SHXK5) and a line that had guard-cell-targeted overexpression of AtHXK1 (GCHXK2) were evaluated relative to wild type for traits related to photosynthesis and yield. In this study, iWUE was significantly higher in GCHXK2 compared with wild type without negatively impacting CO2 assimilation, although results were dependent upon leaf age and proximity of precipitation event to gas exchange measurement.

Here comes the sun: How optimization of photosynthetic light reactions can boost crop yields.

Photosynthesis started to evolve some 3.5 billion years ago CO2 is the substrate for photosynthesis and in the past 200-250 years, atmospheric levels have approximately doubled due to human industrial activities. However, this time span is not sufficient for adaptation mechanisms of photosynthesis to be evolutionarily manifested. Steep increases in human population, shortage of arable land and food, and climate change call for actions, now. Thanks to substantial research efforts and advances in the last century, basic knowledge of photosynthetic and primary metabolic processes can now be translated into strategies to optimize photosynthesis to its full potential in order to improve crop yields and food supply for the future. Many different approaches have been proposed in recent years, some of which have already proven successful in different crop species. Here, we summarize recent advances on modifications of the complex network of photosynthetic light reactions. These are the starting point of all biomass production and supply the energy equivalents necessary for downstream processes as well as the oxygen we breathe.

Improving C4 photosynthesis to increase productivity under optimal and suboptimal conditions.

Although improving photosynthetic efficiency is widely recognized as an underutilized strategy to increase crop yields, research in this area is strongly biased towards species with C3 photosynthesis relative to C4 species. Here, we outline potential strategies for improving C4 photosynthesis to increase yields in crops by reviewing the major bottlenecks limiting the C4 NADP-malic enzyme pathway under optimal and suboptimal conditions. Recent experimental results demonstrate that steady-state C4 photosynthesis under non-stressed conditions can be enhanced by increasing Rubisco content or electron transport capacity, both of which may also stimulate CO2 assimilation at supraoptimal temperatures. Several additional putative bottlenecks for photosynthetic performance under drought, heat, or chilling stress or during photosynthetic induction await further experimental verification. Based on source-sink interactions in maize, sugarcane, and sorghum, alleviating these photosynthetic bottlenecks during establishment and growth of the harvestable parts are likely to improve yield. The expected benefits are also shown to be augmented by the increasing trend in planting density, which increases the impact of photosynthetic source limitation on crop yields.

Variation in photosynthetic induction between rice accessions and its potential for improving productivity.

Photosynthetic induction describes the transient increase in leaf CO2 uptake with an increase in light. During induction, efficiency is lower than at steady state. Under field conditions of fluctuating light, this lower efficiency during induction may cost > 20% of potential crop assimilation. Accelerating induction would boost photosynthetic and resource-use efficiencies. Variation between rice accessions and potential for accelerating induction was analysed by gas exchange. Induction during shade to sun transitions of 14 accessions representing five subpopulations from the 3000 Rice Genome Project Panel (3K RGP) was analysed. Differences of 109% occurred in the CO2 fixed during the first 300 s of induction, 117% in the half-time to completion of induction, and 65% in intrinsic water-use efficiency during induction, between the highest and lowest performing accessions. Induction in three accessions with contrasting responses (AUS 278, NCS 771 A and IR64-21) was compared for a range of [CO2 ] to analyse limitations. This showed in vivo capacity for carboxylation at Rubisco (Vc,max ), and not stomata, as the primary limitation to induction, with significant differences between accessions. Variation in nonsteady-state efficiency greatly exceeded that at steady state, suggesting a new and more promising opportunity for selection of greater crop photosynthetic efficiency in this key food crop.

Photosynthetic efficiency and mesophyll conductance are unaffected in Arabidopsis thaliana aquaporin knock-out lines.

Improving photosynthetic efficiency is widely regarded as a major route to achieving much-needed yield gains in crop plants. In plants with C3 photosynthesis, increasing the diffusion conductance for CO2 transfer from substomatal cavity to chloroplast stroma (gm) could help to improve the efficiencies of CO2 assimilation and photosynthetic water use in parallel. The diffusion pathway from substomatal cavity to chloroplast traverses cell wall, plasma membrane, cytosol, chloroplast envelope membranes, and chloroplast stroma. Specific membrane intrinsic proteins of the aquaporin family can facilitate CO2 diffusion across membranes. Some of these aquaporins, such as PIP1;2 in Arabidopsis thaliana, have been suggested to exert control over gm and the magnitude of the CO2 assimilation flux, but the evidence for a direct physiological role of aquaporins in determining gm is limited. Here, we estimated gm with four different methods under a range of light intensities and CO2 concentrations in two previously characterized pip1;2 knock-out lines as well as pip1;3 and pip2;6 knock-out lines, which have not been previously evaluated for a role in gm. This study presents the most in-depth analysis of gm in Arabidopsis aquaporin knock-out mutants to date. Surprisingly, all methods failed to show any significant differences between the pip1;2, pip1;3, or pip2;6 mutants and the Col-0 control.

Predicting light-induced stomatal movements based on the redox state of plastoquinone: theory and validation.

Prediction of stomatal conductance is a key element to relate and scale up leaf-level gas exchange processes to canopy, ecosystem and land surface models. The empirical models that are typically employed for this purpose are simple and elegant formulations which relate stomatal conductance on a leaf area basis to the net rate of CO2 assimilation, humidity and CO2 concentration. Although light intensity is not directly modelled as a stomatal opening cue, it is well-known that stomata respond strongly to light. One response mode depends specifically on the blue-light part of the light spectrum, whereas the quantitative or 'red' light response is less spectrally defined and relies more on the quantity of incident light. Here, we present a modification of an empirical stomatal conductance model which explicitly accounts for the stomatal red-light response, based on a mesophyll-derived signal putatively initiated by the chloroplastic plastoquinone redox state. The modified model showed similar prediction accuracy compared to models using a relationship between stomatal conductance and net assimilation rate. However, fitted parameter values with the modified model varied much less across different measurement conditions, lessening the need for frequent re-parameterization to different conditions required of the current model. We also present a simple and easy to parameterize extension to the widely used Farquhar-Von Caemmerer-Berry photosynthesis model to facilitate coupling with the modified stomatal conductance model, which should enable use of the new stomatal conductance model to simulate ecosystem water vapour exchange in terrestrial biosphere models.

Photosynthetic induction and its diffusional, carboxylation and electron transport processes as affected by CO2 partial pressure, temperature, air humidity and blue irradiance.

BACKGROUND AND AIMS: Plants depend on photosynthesis for growth. In nature, factors such as temperature, humidity, CO2 partial pressure, and spectrum and intensity of irradiance often fluctuate. Whereas irradiance intensity is most influential and has been studied in detail, understanding of interactions with other factors is lacking. METHODS: We tested how photosynthetic induction after dark-light transitions was affected by CO2 partial pressure (20, 40, 80 Pa), leaf temperatures (15·5, 22·8, 30·5 °C), leaf-to-air vapour pressure deficits (VPDleaf-air; 0·5, 0·8, 1·6, 2·3 kPa) and blue irradiance (0-20 %) in tomato leaves (Solanum lycopersicum). KEY RESULTS: Rates of photosynthetic induction strongly increased with CO2 partial pressure, due to increased apparent Rubisco activation rates and reduced diffusional limitations. High leaf temperature produced slightly higher induction rates, and increased intrinsic water use efficiency and diffusional limitation. High VPDleaf-air slowed down induction rates and apparent Rubisco activation and (at 2·3 kPa) induced damped stomatal oscillations. Blue irradiance had no effect. Slower apparent Rubisco activation in elevated VPDleaf-air may be explained by low leaf internal CO2 partial pressure at the beginning of induction. CONCLUSIONS: The environmental factors CO2 partial pressure, temperature and VPDleaf-air had significant impacts on rates of photosynthetic induction, as well as on underlying diffusional, carboxylation and electron transport processes. Furthermore, maximizing Rubisco activation rates would increase photosynthesis by at most 6-8 % in ambient CO2 partial pressure (across temperatures and humidities), while maximizing rates of stomatal opening would increase photosynthesis by at most 1-3 %.

One crop breeding cycle from starvation? How engineering crop photosynthesis for rising CO2 and temperature could be one important route to alleviation.

Global climate change is likely to severely impact human food production. This comes at a time when predicted demand for primary foodstuffs by a growing human population and changing global diets is already outpacing a stagnating annual rate of increase in crop productivity. Additionally, the time required by crop breeding and bioengineering to release improved varieties to farmers is substantial, meaning that any crop improvements needed to mitigate food shortages in the 2040s would need to start now. In this perspective, the rationale for improvements in photosynthetic efficiency as a breeding objective for higher yields is outlined. Subsequently, using simple simulation models it is shown how predicted changes in temperature and atmospheric [CO2] affect leaf photosynthetic rates. The chloroplast accounts for the majority of leaf nitrogen in crops. Within the chloroplast about 25% of nitrogen is invested in the carboxylase, Rubisco, which catalyses the first step of CO2 assimilation. Most of the remaining nitrogen is invested in the apparatus to drive carbohydrate synthesis and regenerate ribulose-1:5-bisphosphate (RuBP), the CO2-acceptor molecule at Rubisco. At preindustrial [CO2], investment in these two aspects may have been balanced resulting in co-limitation. At today's [CO2], there appears to be over-investment in Rubisco, and despite the counter-active effects of rising temperature and [CO2], this imbalance is predicted to worsen with global climate change. By breeding or engineering restored optimality under future conditions increased productivity could be achieved in both tropical and temperate environments without additional nitrogen fertilizer. Given the magnitude of the potential shortfall, better storage conditions, improved crop management and better crop varieties will all be needed. With the short time-scale at which food demand is expected to outpace supplies, all available technologies to improve crop varieties, from classical crop breeding to crop genetic engineering should be employed. This will require vastly increased public and private investment to support translation of first discovery in laboratories to replicated field trials, and an urgent re-evaluation of regulation of crop genetic engineering.