Gas exchange measurements provide crucial insights into the complex mechanisms of photosynthesis. Responses of CO2 assimilation rate (A) to intercellular CO2 partial pressure (Ci) and irradiance (I) link gas exchange measurements to the underlying photosynthetic biochemistry of a leaf. The unique biochemistry and leaf anatomy which distinguish C4 photosynthesis make it necessary to apply models and fitting routines which appropriately parameterize and incorporate these characteristics. Here we provide updates to the C4 photosynthesis model by improving the parameterization of cyclic electron flow in C4 photosynthesis using experimentally derived values from Setaria viridis. We additionally describe two fitting routines for assessing C4 photosynthesis based on the updated model. Fitting of a CO2 response curve (A/Ci) provides estimates of maximum phosphoenolpyruvate carboxylase activity (Vpmax) and maximum Rubisco activity (Vcmax), and calculates the linear electron transport rate (J) needed to sustain the measured CO2 assimilation rate (A). Fitting of an irradiance response curve (A/I) provides estimates for the maximum linear electron transport rate (Jmax), day respiration rate (Rd), the quantum yield (ϕCO2), and light compensation point (Γlight). Values of the above output parameters are provided both at the measurement temperature and at 25 °C for ease of comparative reporting. The fitting tool has been designed in Microsoft Excel to minimize barriers to entry and enable simplicity of fitting while simultaneously catering to individuals with diverse expertise and experience in C4 gas exchange modelling.

This article is a Commentary on Sun et al . (2026), 249 : 24–38 .

Burgeoning global demand for crop products and the negative impact of climate change on crop production are driving the need to improve yield by developing new elite crop varieties without expanding planted area or increasing agronomic inputs. Improvement in photosynthesis is critical for enhancing crop productivity. Even though leaf photosynthesis is well-studied, the photosynthetic potential of non-foliar green tissues like pods in Brassicaceae and Fabaceae species remains underexplored. This review emphasizes pod photosynthesis in determining seed yield and quality in Brassicaceae and Fabaceae crops. At present, accurate and efficient phenotyping methods are unavailable, limiting understanding and genetic improvement of pod photosynthesis. Novel approaches like chlorophyll fluorescence and hyperspectral reflectance are promising for high-throughput phenotyping of pod photosynthetic traits. This review further discusses genetic targets and regulatory mechanisms for enhancing pod photosynthesis, including transcription factors like GOLDEN2-LIKE and GATA that may regulate photosynthetic capacity in pods, suggesting potential genetic manipulation strategies to boost crop productivity. In conclusion, unlocking the genetic and physiological bases of pod photosynthesis offers opportunities for advancing crop breeding to ensure sustainable food security amidst climate change and increasing global population pressures. Future research should focus on developing high-throughput phenotyping tools and elucidating genetic pathways to maximize pod photosynthesis in crops.

PROTON GRADIENT REGULATION 5 (PGR5) plays a critical role in generating proton motive force across thylakoid membranes and supporting photoprotection under fluctuating light in C3 plants. It is proposed that this function is achieved by regulating cyclic electron flow around Photosystem I. During the evolutionary transition from C3 to C4 photosynthesis, PGR5 abundance in leaves has increased, coinciding with an enhancement in cyclic electron flow rate. To investigate the role of PGR5 in C4 photosynthesis and photoprotection, we generated Setaria viridis (a model C4 monocot) lines with null pgr5 alleles. We demonstrate that loss of PGR5 severely impairs the establishment of proton motive force, photosynthetic control, and energy-dependent non-photochemical quenching at high irradiances, leading to a loss of Photosystem I activity under light stress. Furthermore, plants lacking PGR5 exhibit drastically reduced growth and photosynthesis when grown under fluctuating daylight; however, they are less severely affected than C3  pgr5 mutants. This relative tolerance arises from the ability of S. viridis lacking PGR5 to maintain significant levels of photosynthetic control, in contrast to C3 mutants. Additionally, in the absence of PGR5 and qE, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports survival under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation. Our findings highlight the essential role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by regulating photosynthetic control and energy-dependent non-photochemical quenching. This study also uncovers the interplay between multiple photoprotective mechanisms safeguarding C4 photosynthesis under light stress.

An approach to improving radiation use efficiency (RUE) in wheat is to screen for variability in rates of leaf respiration in darkness (R-dark). We used a high-throughput system to quantify variation in R-dark among a diverse range of spring wheat genotypes (301 lines) grown in two countries (Mexico and Australia) and two seasons (2017 and 2018), and in doing so quantify the relative importance of genotype (G) and environment (E) in influencing variations in leaf R-dark. Through careful design, residual (unexplained) variation represented <10% of the total observed. Up to a third of the variation in R-dark (and related traits) was under genetic control. This suggests opportunities for breeders to use R-dark as a novel selection tool. In addition, E accounted for more than half of the total variation in area-based rates of R-dark. Here, the day of measurement was crucial, suggesting that day-to-day variations in the environment influence rates of R-dark measured at a common temperature. Overall, this study provides new insights into the role G and E play in determining variation in rates of leaf R-dark of one of the most important cereal crops, with implications for future improvements in carbon use efficiency and yield.

Our understanding of how photosynthetic capacity varies among C4 species and across growth and measurement conditions remains limited. We collated 1696 CO2 response curves of net CO2 assimilation rate (A/Ci curves) from C4 species grown and measured at various environmental conditions and used these data to estimate the apparent maximum carboxylation activity of phosphoenolpyruvate carboxylase (VpmaxA) and CO2-saturated net photosynthetic rate (Amax), two key parameters describing photosynthetic capacity. We examined how VpmaxA and Amax vary with species-specific traits, growth and measurement conditions. We found little systematic variation of VpmaxA and Amax across the classical C4 biochemical subtypes or growth forms, but showed that growth temperature and measurement conditions are major factors determining C4 photosynthetic capacity. We found no evidence that common C4 model species (e.g. maize, sorghum and Setaria viridis) differ in photosynthetic capacity from other C4 species when grown in controlled environments. However, C4 model species showed up to twice the photosynthetic capacity of other C4 species when grown in the field. Our multivariate model accounts for 47-51% of the variation reported in VpmaxA and Amax, and we argue that environmental conditions have a greater influence on C4 photosynthetic capacity than biochemical subtypes or growth forms.

Many C4 plants are used as food and fodder crops and often display improved resource use efficiency compared to C3 plants. However, the response of C4 plants to future extreme conditions such as heatwaves is less understood. Here, Setaria viridis, an emerging C4 model grass, was grown under long-term high-temperature stress for 2 wk (42 degrees C, compared to 28 degrees C). This resulted in stunted growth, but surprisingly had little impact on leaf thickness, leaf area-based photosynthetic rates, and bundle sheath leakiness. Dark respiration rates increased, and there were major alterations in carbon and nitrogen metabolism in the heat-stressed plants. Abscisic acid and indole-3-acetic acid-amino acid conjugates accumulated in the heat-stressed plants, consistent with transcriptional changes. Leaf transcriptomics, proteomics, and metabolomics analyses were carried out and mapped onto the metabolic pathways of photosynthesis, respiration, carbon/nitrogen metabolism, and phytohormone biosynthesis and signaling. An in-depth analysis of correlations between transcripts and their corresponding proteins revealed strong differences between groups in the strengths and signs of correlations. Overall, many stress signaling pathways were upregulated, consistent with multiple signals leading to reduced plant growth. A systems-based model of the plant response to long-term heat stress is presented based on the oxidative stress, phytohormone, and sugar signaling pathways. Long-term heat stress does not affect photosynthesis in the C4 grass Setaria viridis owing to an effective acclimation response of carbon assimilation, although it inhibits plant growth.

BACKGROUND:The need for rapid in-field measurement of key traits contributing to yield over many thousands of genotypes is a major roadblock in crop breeding. Recently, leaf hyperspectral reflectance data has been used to train machine learning models using partial least squares regression (PLSR) to rapidly predict genetic variation in photosynthetic and leaf traits across wheat populations, among other species. However, the application of published PLSR spectral models is limited by a fixed spectral wavelength range as input and the requirement of separate custom-built models for each trait and wavelength range. In addition, the use of reflectance spectra from the short-wave infrared region requires expensive multiple detector spectrometers. The ability to train a model that can accommodate input from different spectral ranges would potentially make such models extensible to more affordable sensors. Here we compare the accuracy of prediction of PLSR with various deep learning approaches and an ensemble model, each trained and tested using previously published data sets. RESULTS:We demonstrate that the accuracy of PLSR to predict photosynthetic and related leaf traits in wheat can be improved with deep learning-based and ensemble models without overfitting. Additionally, these models can be flexibly applied across spectral ranges without significantly compromising accuracy. CONCLUSION:The method reported provides an improved prediction of wheat leaf and photosynthetic traits from leaf hyperspectral reflectance and do not require a full range, high cost leaf spectrometer. We provide a web service for deploying these algorithms to predict physiological traits in wheat from a variety of spectral data sets, with important implications for wheat yield prediction and crop breeding.

Measurements of respiratory properties have often been made at a single time point either during daytime using dark-adapted leaves or during nighttime. The influence of the day-night cycle on respiratory metabolism has received less attention but is crucial to understand photosynthesis and photorespiration. Here, we examined how CO2- and O-2-based rates of leaf dark respiration (R-dark) differed between midday (after 30-min dark adaptation) and midnight in 8 C-3 and C-4 grasses. We used these data to calculate the respiratory quotient (RQ; ratio of CO2 release to O-2 uptake), and assessed relationships between R-dark and leaf metabolome. R-dark was higher at midday than midnight, especially in C-4 species. The day-night difference in R-dark was more evident when expressed on a CO2 than O-2 basis, with the RQ being higher at midday than midnight in all species, except in rice (Oryza sativa). Metabolomic analyses showed little correlation of R-dark or RQ with leaf carbohydrates (sucrose, glucose, fructose, or starch) but strong multivariate relationships with other metabolites. The results suggest that rates of R-dark and differences in RQ were determined by several concurrent CO2-producing and O-2-consuming metabolic pathways, not only the tricarboxylic acid cycle (organic acids utilization) but also the pentose phosphate pathway, galactose metabolism, and secondary metabolism. As such, R-dark was time-, type- (C-3/C-4) and species-dependent, due to the use of different substrates.

Abstract The PGR5-PGRL1 pathway protects plants from photodamage by regulating electron flow to maintain Photosystem I in an oxidised state. Grasses possess two PGRL1 paralogs, but their functional roles have remained unknown. Here, we show that the ancestral PGRL1 paralog, PGRL1α, which is conserved across algae and land plants, is enriched in the mesophyll cells of grasses that perform the NADP-ME subtype of C 4 photosynthesis. In contrast, the grass-specific paralog PGRL1β is enriched in bundle sheath cells. To investigate the functional significance of this cell-specific expression, we generated gene-edited lines of the NADP-ME C 4 grass Setaria viridis lacking either PGRL1 paralog. We found that PGRL1β in bundle sheath cells was required for rapid photoprotection, enabling Photosystem I oxidation five seconds faster during transitions from darkness to high light. In contrast, PGRL1α in mesophyll cells was essential for maintaining Photosystem I oxidation under steady-state high-light conditions. We propose that these complementary functions arise from structural differences within the lumen-facing regions of the two paralogs, providing a mechanistic basis for their distinct roles in regulating photoprotection. The conservation of this dual PGRL1 system across grasses suggests that functional specialisation of the paralogs expands the dynamic range of protective responses, enhancing photosynthetic performance under fluctuating and high-light stress conditions.

To ensure global food security, crop breeders conduct extensive trials across various locations to discover new crop varieties that grow more robustly, have higher yields, and are resilient to local stress factors. These trials consist of thousands of plots, each containing a unique crop variety monitored at intervals during the growing season, requiring considerable manual effort. In this study, we combined satellite imagery and deep learning techniques to automatically collect plot-level phenotypes from plant breeding trials in South Australia and Sonora, Mexico. We implemented two novel methods, utilising state-of-the-art computer vision architectures, to predict plot-level phenotypes: flowering, canopy cover, greenness, height, biomass, and normalised difference vegetation index (NDVI). The first approach uses a classification model to predict for just the centred plot. The second approach predicts per-pixel and then aggregates predictions to determine a value per-plot. Using a modified ResNet18 model to predict the centred plot was found to be the most effective method. These results highlight the exciting potential for improving crop trials with remote sensing and machine learning.

The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase-like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C4 photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell-level electron fluxes remained unknown. We have created gene-edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells. We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO2. Our results indicate that NDH-mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO2 fixation by the C3 cycle in BS cells.

PROTON GRADIENT REGULATION 5 (PGR5) is essential for generating proton motive force across thylakoid membranes in C3 plants and supporting photoprotection under fluctuating light conditions. It is proposed that this function is achieved by regulating cyclic electron flow around Photosystem I. During the evolutionary transition from C3 to C4 photosynthesis, the leaf abundance of PGR5 has increased, coinciding with a rise in the cyclic electron flow rate. To investigate the contribution of PGR5 to photoprotection in C4 photosynthesis, we generated model C4 monocot Setaria viridis with null pgr5 alleles. We show that plants lacking PGR5 struggle to establish proton motive force and energy-dependent non-photochemical quenching (qE) at higher irradiances during instantaneous measurements. This leads to a progressive decline in maximum Photosystem I activity when leaves are exposed to repeated cycles of high irradiance. Additionally, plants without PGR5 exhibit severely reduced growth and photosynthesis compared to wild type plants when grown under fluctuating daylight but not under constant daylight. In the absence of PGR5, a slower-relaxing, zeaxanthin-dependent form of non-photochemical quenching supports growth under fluctuating light, albeit at the cost of reduced photochemical efficiency and assimilation rate. Our findings underscore the role of PGR5 in enabling efficient C4 photosynthesis under fluctuating light by establishing proton motive force for the rapid upregulation of qE and preventing photodamage to the electron transport machinery. This study highlights the importance of various non-photochemical quenching mechanisms for C4 photosynthesis and emphasises the role of PGR5 in the evolution of C4 plants. ### Competing Interest Statement The authors have declared no competing interest.

Plasmodesmata (PD) are nanochannels that facilitate cell-to-cell transport in plants. More productive and photosynthetically efficient C4 plants form more PD at the mesophyll (M)-bundle sheath (BS) interface in their leaves than their less efficient C3 relatives. In C4 leaves, PD play an essential role in facilitating the rapid metabolite exchange between the M and BS cells to operate a biochemical CO2 concentrating mechanism, which increases the CO2 partial pressure at the site of Rubisco in the BS cells and hence photosynthetic efficiency. The genetic mechanism controlling PD formation in C3 and C4 leaves is largely unknown, especially in monocot crops, due to the technical challenge of quantifying these nanostructures with electron microscopy. To address this issue, we have generated stably transformed lines of Oryza sativa (rice, C3) and Setaria viridis (setaria, C4) with fluorescent protein-tagged PD to build the first spatiotemporal atlas of leaf pit field (cluster of PD) density in monocots without the need for electron microscopy. Across leaf development, setaria had consistently more PD connections at the M-BS wall interface than rice while the difference in M-M pit field density varied. While light was a critical trigger of PD formation, cell type and function determined leaf pit field density. Complementary temporal mRNA sequencing and gene co-expression network analysis revealed that the pattern of pit field density correlated with differentially expressed PD-associated genes and photosynthesis-related genes. PD-associated genes identified from our co-expression network analysis are related to cell wall expansion, translation and chloroplast signalling.

One of the major events in plant history is the emergence of grasses (Poaceae), which colonised previously uninhabitable open land areas. Some grasses later evolved C4 photosynthesis - operating between mesophyll and bundle sheath cells - becoming the most productive and resilient plants on Earth. We investigated whether a key regulator of photoprotection PGR5-LIKE PHOTOSYNTHETIC PHENOTYPE 1 (PGRL1), essential for survival of plants under fluctuating light, facilitated the adaptation of grasses to the open environments. We found that, in addition to PGRL1α present in all photosynthetic organisms, grasses evolved a new paralog, PGRL1β . AlphaFold3 modelling of predicted PGRL1 dimers suggests that the lumenal regions of PGRL1α monomers are linked by specific hydrogen bonds whereas lumenal regions of PGRL1β show primarily hydrophobic interactions. These differences likely result in distinct modes of regulation of photoprotection offered by the two paralogs which is supported by predominant expression of PGRL1α in mesophyll cells and PGRL1β in bundle sheath cells of NADP-ME C4 grasses maize, sorghum, and Setaria viridis . We propose that the mixed PGRL1α/β system enabled a better balancing between Photosystem I protection and Photosystem II activity depending on environmental conditions. Therefore, the emergence of PGRL1β contributed to the ecological dominance of grasses and the superior efficiency of NADP-ME C4 photosynthesis. ### Competing Interest Statement The authors have declared no competing interest.

Plant growth depends on sugar production and export by photosynthesizing source leaves and sugar allocation and import by sink tissues (grains, roots, stems, and young leaves). Photosynthesis and sink demand are tightly coordinated through metabolic (substrate, allosteric) feedback and signalling (sugar, hormones) mechanisms. Sugar signalling integrates sugar production with plant development and environmental cues. In C3 plants (e.g. wheat and rice), it is well documented that sugar accumulation in source leaves, due to source-sink imbalance, negatively feeds back on photosynthesis and plant productivity. However, we have a limited understanding about the molecular mechanisms underlying those feedback regulations, especially in C4 plants (e.g. maize, sorghum, and sugarcane). Recent work with the C4 model plant Setaria viridis suggested that C4 leaves have different sugar sensing thresholds and behaviours relative to C3 counterparts. Addressing this research priority is critical because improving crop yield requires a better understanding of how plants coordinate source activity with sink demand. Here we review the literature, present a model of action for sugar sensing in C4 source leaves, and suggest ways forward.

In the developing seeds of all higher plants, filial cells are symplastically isolated from the maternal tissue supplying photosynthate to the reproductive structure. Photoassimilates must be transported apoplastically, crossing several membrane barriers, a process facilitated by sugar transporters. Sugars Will Eventually be Exported Transporters (SWEETs) have been proposed to play a crucial role in apoplastic sugar transport during phloem unloading and the post-phloem pathway in sink tissues. Evidence for this is presented here for developing seeds of the C4 model grass Setaria viridis. Using immunolocalization, SvSWEET4 was detected in various maternal and filial tissues within the seed along the sugar transport pathway, in the vascular parenchyma of the pedicel, and in the xylem parenchyma of the stem. Expression of SvSWEET4a in Xenopus laevis oocytes indicated that it functions as a high-capacity glucose and sucrose transporter. Carbohydrate and transcriptional profiling of Setaria seed heads showed that there were some developmental shifts in hexose and sucrose content and consistent expression of SvSWEET4 homologues. Collectively, these results provide evidence for the involvement of SWEETs in the apoplastic transport pathway of sink tissues and allow a pathway for post-phloem sugar transport into the seed to be proposed.

BACKGROUND AND AIMS:The mechanisms of sugar sensing in grasses remain elusive, especially those using C4 photosynthesis even though a large proportion of the world's agricultural crops utilize this pathway. We addressed this gap by comparing the expression of genes encoding components of sugar sensors in C3 and C4 grasses, with a focus on source tissues of C4 grasses. Given C4 plants evolved into a two-cell carbon fixation system, it was hypothesized this may have also changed how sugars were sensed.METHODS:For six C3 and eight C4 grasses, putative sugar sensor genes were identified for target of rapamycin (TOR), SNF1-related kinase 1 (SnRK1), hexokinase (HXK) and those involved in the metabolism of the sugar sensing metabolite trehalose-6-phosphate (T6P) using publicly available RNA deep sequencing data. For several of these grasses, expression was compared in three ways: source (leaf) versus sink (seed), along the gradient of the leaf, and bundle sheath versus mesophyll cells.KEY RESULTS:No positive selection of codons associated with the evolution of C4 photosynthesis was identified in sugar sensor proteins here. Expressions of genes encoding sugar sensors were relatively ubiquitous between source and sink tissues as well as along the leaf gradient of both C4 and C3 grasses. Across C4 grasses, SnRK1β1 and TPS1 were preferentially expressed in the mesophyll and bundle sheath cells, respectively. Species-specific differences of gene expression between the two cell types were also apparent.CONCLUSIONS:This comprehensive transcriptomic study provides an initial foundation for elucidating sugar-sensing genes within major C4 and C3 crops. This study provides some evidence that C4 and C3 grasses do not differ in how sugars are sensed. While sugar sensor gene expression has a degree of stability along the leaf, there are some contrasts between the mesophyll and bundle sheath cells.

Summary Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C 4 pathway of photosynthesis in which a biochemical carbon‐concentrating mechanism results in high CO 2 assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b 6 f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO 2 assimilation in the model C 4 plant Setaria viridis . To test whether productivity of C 4 crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady‐state rates of electron transport and CO 2 assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b 6 f is not the only factor limiting electron transport in sorghum at high light and high CO 2 . However, faster responses of non‐photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO 2 assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C 4 crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.

Hexokinases (HXK) were the first sugar signalling proteins identified in plants and are well known for their feedback regulation of photosynthetic gene expression. In some C3 plants, HXKs have been found to regulate stomatal function. However, the role of HXK in C4 photosynthesis, which is inherently more water use efficient than C3 metabolism, remains poorly understood. Here, we report on the first tissue-specific modification of HXK in a C4 plant. SvHXK6 was expressed in the model C4 grass Setaria viridis under the control of the ZmPEPC promoter ( ZmPEPC pro ), which directs expression in the leaf mesophyll tissue. Three S. viridis transgenic lines with increased abundance of SvHXK6 transcripts in the leaf tissue showed significant reduction in stomatal conductance with minimal effects on leaf CO2 assimilation rate. Consequently, the transgenic lines had higher leaf-level water use efficiency relative to the control (wild-type and null) plants. Overexpression of SvHXK6 had no effect on shoot biomass or seed yield of the S. viridis plants. Our study shows conserved function of HXK in regulating stomatal conductance in a C4 grass, demonstrating possible widespread utility in improving water use efficiency in C4 as well as C3 species.