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Sugars, produced through photosynthesis, are at the core of all organic compounds synthesized and used for plant growth and their response to environmental changes. Their production, transport, and utilization are highly regulated and integrated throughout the plant life cycle. The maintenance of sugar partitioning between the different subcellular compartments and between cells is important in adjusting the photosynthesis performance and response to abiotic constraints. We investigated the consequences of the disruption of four genes coding for SWEET sugar transporters in Arabidopsis (SWEET11, SWEET12, SWEET16, and SWEET17) on plant photosynthesis and the response to drought. Our results show that mutations in both SWEET11 and SWEET12 genes lead to an increase of cytosolic sugars in mesophyll cells and phloem parenchyma cells, which impacts several photosynthesis-related parameters. Further, our results suggest that in the swt11swt12 double mutant, the sucrose-induced feedback mechanism on stomatal closure is poorly efficient. On the other hand, changes in fructose partitioning in mesophyll and vascular cells, measured in the swt16swt17 double mutant, positively impact gas exchanges, probably through an increased starch synthesis together with higher vacuolar sugar storage. Finally, we propose that the impaired sugar partitioning, rather than the total amount of sugars observed in the quadruple mutant, is responsible for the enhanced sensitivity upon drought. This work highlights the importance of considering SWEET-mediated sugar partitioning rather than global sugar content in photosynthesis performance and plant response to drought. Such knowledge will pave the way to design new strategies to maintain plant productivity in a challenging environment., (© 2024 Scandinavian Plant Physiology Society.)

Nanomaterials as an emerging tool are being used to improve plant's net photosynthetic rate (A N ) when suffering salt stress, but the underlying mechanisms remain unclear. To clarify this, a hydroponic experiment was conducted to study the effects of polyacrylic acid coated nanoceria (PNC) on the A N of salt-stressed cotton and related intrinsic mechanisms. Results showed that the PNC-induced A N enhancement of salt-stressed leaves was strongly facilitated by the mesophyll conductance to CO 2 (g m ). Further analysis showed that the PNC-induced improvement of g m was related to the increased chloroplast surface area exposed to intercellular airspaces, which was attribute to the increased mesophyll surface area exposed to intercellular airspaces and chloroplast number due to the increased K + content and decreased reactive oxygen species level in salt-stressed leaves. Interestingly, our results also showed that PNC-induced variations in cell wall composition of salt-stressed cotton leaves strongly influenced g m , especially, hemicellulose and pectin. Moreover, the proportion of pectin in cell wall composition played a more important role in determining g m . Our study demonstrated for the first time that nanoceria, through alterations to anatomical traits and cell wall composition, drove g m enhancement, which ultimately increased A N of salt-stressed leaves., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Malate and fumarate constitute a significant fraction of the carbon fixed by photosynthesis, and they are at the crossroad of central metabolic pathways. In Arabidopsis thaliana, they are transiently stored in the vacuole to keep cytosolic homeostasis. The malate and fumarate transport systems of the vacuolar membrane are key players in the control of cell metabolism. Notably, the molecular identity of these transport systems remains mostly unresolved. We used a combination of imaging, electrophysiology and molecular physiology to identify an important molecular actor of dicarboxylic acid transport across the tonoplast. Here, we report the function of the A. thaliana Aluminium-Activated Malate Transporter 5 (AtALMT5). We characterised its ionic transport properties, expression pattern, localisation and function in vivo. We show that AtALMT5 is expressed in photosynthetically active tissues and localised in the tonoplast. Patch-clamp and in planta analyses demonstrated that AtALMT5 is an ion channel-mediating fumarate loading of the vacuole. We found in almt5 plants a reduced accumulation of fumarate in the leaves, in parallel with increased malate concentrations. These results identified AtALMT5 as an ion channel-mediating fumarate transport in the vacuoles of mesophyll cells and regulating the malate/fumarate balance in Arabidopsis., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Climate change not only leads to higher air temperatures but also increases the vapour pressure deficit (VPD) of the air. Understanding the direct effect of VPD on leaf gas exchange is crucial for precise modelling of stomatal functioning. We conducted combined leaf gas exchange and online isotope discrimination measurements on four common European tree species across a VPD range of 0.8-3.6 kPa, while maintaining constant temperatures without soil water limitation. In addition to applying the standard assumption of saturated vapour pressure inside leaves (e i ), we inferred e i from oxygen isotope discrimination of CO 2 and water vapour. e i desaturated progressively with increasing VPD, consistently across species, resulting in an intercellular relative humidity as low as 0.73 ± 0.11 at the highest tested VPD. Assuming saturation of e i overestimated the extent of reductions in stomatal conductance and CO 2 mole fraction inside leaves in response to increasing VPD compared with calculations that accounted for unsaturation. In addition, a significant decrease in mesophyll conductance with increasing VPD only occurred when the unsaturation of e i was considered. We suggest that the possibility of unsaturated e i should not be overlooked in measurements related to leaf gas exchange and in stomatal models, especially at high VPD., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

An Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinase (MAPK) cascade composed of YODA (YDA)-MKK4/MKK5-MPK3/MPK6 plays an essential role downstream of the ERECTA (ER)/ER-LIKE (ERL) receptor complex in regulating stomatal development in the leaf epidermis. STOMAGEN (STO), a peptide ligand produced in mesophyll cells, competes with EPIDERMAL PATTERNING FACTOR2 (EPF2) for binding ER/ERL receptors to promote stomatal formation. In this study, we found that activation of MPK3/MPK6 suppresses STO expression. Using MUTE and STO promoters that confer epidermis- and mesophyll-specific expression, respectively, we generated lines with cell-specific activation and suppression of MPK3/MPK6. The activation or suppression of MPK3/MPK6 in either epidermis or mesophyll cells is sufficient to alter stomatal differentiation. Epistatic analyses demonstrated that STO overexpression can rescue the suppression of stomatal formation conferred by the mesophyll-specific expression of the constitutively active MKK4DD or MKK5DD, but not by the epidermis-specific expression of these constitutively active MKKs. These data suggest that STO is downstream of MPK3/MPK6 in mesophyll cells, but upstream of MPK3/MPK6 in epidermal cells in stomatal development signaling. This function of the MPK3/MPK6 cascade allows it to coordinate plant epidermis development based on its activity in mesophyll cells during leaf development., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Terrestrial photosynthesis, or gross primary production (GPP), is the largest carbon flux in the biosphere, but its global magnitude and spatiotemporal dynamics remain uncertain 1 . The global annual mean GPP is historically thought to be around 120 PgC yr -1 (refs.  2-6 ), which is about 30-50 PgC yr -1 lower than GPP inferred from the oxygen-18 ( 18 O) isotope 7 and soil respiration 8 . This disparity is a source of uncertainty in predicting climate-carbon cycle feedbacks 9,10 . Here we infer GPP from carbonyl sulfide, an innovative tracer for CO 2 diffusion from ambient air to leaf chloroplasts through stomata and mesophyll layers. We demonstrate that explicitly representing mesophyll diffusion is important for accurately quantifying the spatiotemporal dynamics of carbonyl sulfide uptake by plants. From the estimate of carbonyl sulfide uptake by plants, we infer a global contemporary GPP of 157 (±8.5) PgC yr -1 , which is consistent with estimates from 18 O (150-175 PgC yr -1 ) and soil respiration ( 149 - 23 + 29  PgC yr -1 ), but with an improved confidence level. Our global GPP is higher than satellite optical observation-driven estimates (120-140 PgC yr -1 ) that are used for Earth system model benchmarking. This difference predominantly occurs in the pan-tropical rainforests and is corroborated by ground measurements 11 , suggesting a more productive tropics than satellite-based GPP products indicated. As GPP is a primary determinant of terrestrial carbon sinks and may shape climate trajectories 9,10 , our findings lay a physiological foundation on which the understanding and prediction of carbon-climate feedbacks can be advanced., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

A fundamental assumption in plant science posits that leaf air spaces remain vapor saturated, leading to the predominant view that stomata alone control leaf water loss. This concept has been pivotal in photosynthesis and water-use efficiency research. However, recent evidence has refuted this longstanding assumption by providing evidence of unsaturation in the leaf air space of C 3 plants under relatively mild vapor pressure deficit (VPD) stress. This phenomenon represents a nonstomatal mechanism restricting water loss from the mesophyll. The potential ubiquity and physiological implications of this phenomenon, its driving mechanisms in different plant species and habitats, and its interaction with other ecological adaptations have. In this context, C 4 plants spark particular interest for their importance as crops, bundle sheath cells' unique anatomical characteristics and specialized functions, and notably higher water-use efficiency relative to C 3 plants. Here, we confirm reduced relative humidities in the substomatal cavity of the C 4 plants maize, sorghum, and proso millet down to 80% under mild VPD stress. We demonstrate the critical role of nonstomatal control in these plants, indicating that the role of the CO 2 concentration mechanism in CO 2 management at a high VPD may have been overestimated. Our findings offer a mechanistic reconciliation between discrepancies in CO 2 and VPD responses reported in C 4 species. They also reveal that nonstomatal control is integral to maintaining an advantageous microclimate of relatively higher CO 2 concentrations in the mesophyll air space of C 4 plants for carbon fixation, proving vital when these plants face VPD stress., Competing Interests: Competing interests statement:The authors declare no competing interest.

SO2/H2SO3 can damage plants. However, its toxic mechanism has still been controversial. Two models have been proposed, cytosolic acidification model and cellular oxidation model. Here, we assessed the toxic mechanism of H2SO3 in three cell types of Arabidopsis thaliana, mesophyll cells, guard cells (GCs), and petal cells. The sensitivity of GCs of Chloride channel a (CLCa)-knockout mutants to H2SO3 was significantly lower than those of wildtype plants. Expression of other CLC genes in mesophyll cells and petal cells were different from GCs. Treatment with antioxidant, disodium 4,5-dihydroxy-1,3-benzenedisulfonate (tiron), increased the median lethal concentration (LC50) of H2SO3 in GCs indicating the involvement of cellular oxidation, while the effect was negligible in mesophyll cells and petal cells. These results indicate that there are two toxic mechanisms of SO2 to Arabidopsis cells: cytosolic acidification and cellular oxidation, and the toxic mechanism may vary among cell types., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Salinity tolerance requires coordinated responses encompassing salt exclusion in roots and tissue/cellular compartmentation of salt in leaves. We investigated the possible control points for salt ions transport in roots and tissue tolerance to Na + and Cl - in leaves of two contrasting mungbean genotypes, salt-tolerant Jade AU and salt-sensitive BARI Mung-6, grown in nonsaline and saline (75 mM NaCl) soil. Cryo-SEM X-ray microanalysis was used to determine concentrations of Na, Cl, K, Ca, Mg, P, and S in various cell types in roots related to the development of apoplastic barriers, and in leaves related to photosynthetic performance. Jade AU exhibited superior salt exclusion by accumulating higher [Na] in the inner cortex, endodermis, and pericycle with reduced [Na] in xylem vessels and accumulating [Cl] in cortical cell vacuoles compared to BARI Mung-6. Jade AU maintained higher [K] in root cells than BARI Mung-6. In leaves, Jade AU maintained lower [Na] and [Cl] in chloroplasts and preferentially accumulated [K] in mesophyll cells than BARI Mung-6, resulting in higher photosynthetic efficiency. Salinity tolerance in Jade AU was associated with shoot Na and Cl exclusion, effective regulation of Na and Cl accumulation in chloroplasts, and maintenance of high K in root and leaf mesophyll cells., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The superior productivity of C 4 plants is achieved via a metabolic C 4 cycle which acts as a CO 2 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 C 4 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 CO 2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO 2 . 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 CO 2 fixation by the C 3 cycle in BS cells., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Understanding the short-term responses of mesophyll conductance (g m ) and stomatal conductance (g sc ) to environmental changes remains a challenging yet central aspect of plant physiology. This review synthesises our current knowledge of these short-term responses, which underpin CO 2 diffusion within leaves. Recent methodological advances in measuring g m using online isotopic discrimination and chlorophyll fluorescence have improved our confidence in detecting short-term g m responses, but results need to be carefully evaluated. Environmental factors like vapour pressure deficit and CO 2 concentration indirectly impact g m through g sc changes, highlighting some of the complex interactions between the two parameters. Evidence suggests that short-term responses of g m are not, or at least not fully, mechanistically linked to changes in g sc , cautioning against using g sc as a reliable proxy for g m . The overarching challenge lies in unravelling the mechanistic basis of short-term g m responses, which will contribute to the development of accurate models bridging laboratory insights with broader ecological implications. Addressing these gaps in understanding is crucial for refining predictions of g m behaviour under changing environmental conditions., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Mesophyll conductance (g m ) is a crucial plant trait that can significantly limit photosynthesis. Measurement of photosynthetic C 18 O 16 O discrimination (Δ 18 O) has proved to be the only viable means of resolving g m in both C 3 and C 4 plants. However, the currently available methods to exploit Δ 18 O for g m estimation are error prone due to their inadequacy in constraining the degree of oxygen isotope exchange (θ) during mesophyll CO 2 hydration. Here, we capitalized on experimental manipulation of leaf water isotopic dynamics to establish a novel, nonsteady state, regression-based approach for simultaneous determination of g m and θ from online Δ 18 O measurements. We demonstrated the methodological and theoretical robustness of this new Δ 18 O-g m estimation approach and showed through measurements on several C 3 and C 4 species that this approach can serve as a benchmark method against which to identify previously-unrecognized biases of the existing Δ 18 O-g m methods. Our results highlight the unique value of this nonsteady state-based approach for contributing to ongoing efforts toward quantitative understanding of mesophyll conductance for crop yield improvement and carbon cycle modeling., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

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 (g m ), which would allow CO 2 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 g m are fully appreciated. In this review, we summarise these limitations, and outline the uncertainties and assumptions that can affect the final estimation of g m . Furthermore, we critically assess the predicted quantitative effect that elevating g m will have on assimilation rates in crop species. We highlight the need for more theoretical modelling to determine whether altering g m is truly a viable route to improve crop performance. Finally, we offer suggestions to guide future research on g m , which will help mitigate the uncertainty inherently associated with estimating this parameter., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Mesophyll conductance (g m ) describes the ease with which CO 2 passes from the sub-stomatal cavities of the leaf to the primary carboxylase of photosynthesis, Rubisco. Increasing g m is suggested as a means to engineer increases in photosynthesis by increasing [CO 2 ] at Rubisco, inhibiting oxygenation and accelerating carboxylation. Here, tobacco was transgenically up-regulated with Arabidopsis Cotton Golgi-related 3 (CGR3), a gene controlling methylesterification of pectin, as a strategy to increase CO 2 diffusion across the cell wall and thereby increase g m . Across three independent events in tobacco strongly expressing AtCGR3, mesophyll cell wall thickness was decreased by 7%-13%, wall porosity increased by 75% and g m measured by carbon isotope discrimination increased by 28%. Importantly, field-grown plants showed an average 8% increase in leaf photosynthetic CO 2 uptake. Up-regulating CGR3 provides a new strategy for increasing g m in dicotyledonous crops, leading to higher CO 2 assimilation and a potential means to sustainable crop yield improvement., (© 2024 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The response of mesophyll conductance (g m ) to CO 2 plays a key role in photosynthesis and ecosystem carbon cycles under climate change. Despite numerous studies, there is still debate about how g m responds to short-term CO 2 variations. Here we used multiple methods and looked at the relationship between stomatal conductance to CO 2 (g sc ) and g m to address this aspect. We measured chlorophyll fluorescence parameters and online carbon isotope discrimination (Δ) at different CO 2 mole fractions in sunflower (Helianthus annuus L.), cowpea (Vigna unguiculata L.), and wheat (Triticum aestivum L.) leaves. The variable J and Δ based methods showed that g m decreased with an increase in CO 2 mole fraction, and so did stomatal conductance. There were linear relationships between g m and g sc across CO 2 mole fractions. g m obtained from A-C i curve fitting method was higher than that from the variable J method and was not representative of g m under the growth CO 2 concentration. g m could be estimated by empirical models analogous to the Ball-Berry model and the USO model for stomatal conductance. Our results suggest that g m and g sc respond in a coordinated manner to short-term variations in CO 2 , providing new insight into the role of g m in photosynthesis modelling., (© 2024 John Wiley & Sons Ltd.)

In the face of anthropogenic warming, drought poses an escalating threat to food production. C 4 plants offer promise in addressing this threat. C 4 leaves operate a biochemical CO 2 concentrating mechanism that exchanges metabolites between two partially isolated compartments (mesophyll and bundle sheath), which confers high-productivity potential in hot climates boosting water use efficiency. However, when C 4 leaves experience dehydration, photosynthesis plummets. This paper explores the physiological mechanisms behind this decline. In a fast dehydration experiment, we measured the fluxes and isotopic composition of water and CO 2 in the gas exchanged by leaves, and we interpreted results using a novel biochemical model and analysis of elasticity. Our findings show that, while CO 2 supply to the mesophyll and to the bundle sheath persisted during dehydration, there was a decrease in CO 2 conductance at the bundle sheath-mesophyll interface. We interpret this as causing a slowdown of intercellular metabolite exchange - an essential feature of C 4 photosynthesis. This would impede the supply of reducing power to the bundle sheath, leading to phosphoglycerate accumulation and feedback inhibition of Rubisco carboxylation. The interplay between this rapid sensitivity and the effectiveness of coping strategies that C 4 plants deploy may be an overlooked driver of their competitive performance., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Certain species in the Brassicaceae family exhibit high photosynthesis rates, potentially providing a valuable route toward improving agricultural productivity. However, factors contributing to their high photosynthesis rates are still unknown. We compared Hirschfeldia incana, Brassica nigra, Brassica rapa and Arabidopsis thaliana, grown under two contrasting light intensities. Hirschfeldia incana matched B. nigra and B. rapa in achieving very high photosynthesis rates under high growth-light condition, outperforming A. thaliana. Photosynthesis was relatively more limited by maximum photosynthesis capacity in H. incana and B. rapa and by mesophyll conductance in A. thaliana and B. nigra. Leaf traits such as greater exposed mesophyll specific surface enabled by thicker leaf or high-density small palisade cells contributed to the variation in mesophyll conductance among the species. The species exhibited contrasting leaf construction strategies and acclimation responses to low light intensity. High-light plants distributed Chl deeper in leaf tissue, ensuring even distribution of photosynthesis capacity, unlike low-light plants. Leaf anatomy of H. incana, B. nigra and B. rapa facilitated effective CO 2 diffusion, efficient light use and provided ample volume for their high maximum photosynthetic capacity, indicating that a combination of adaptations is required to increase CO 2 -assimilation rates in plants., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Increases in shrub height, biomass and canopy cover are key whole-plant features of warming-induced vegetation change in tundra. We investigated leaf functional traits underlying photosynthetic capacity of Arctic shrub species, particularly its main limiting processes such as mesophyll conductance. In this nutrient-limited ecosystem, we expect leaf nitrogen concentration to be the main limiting factor for photosynthesis. We measured the net photosynthetic rate at saturated light (A sat ) in three Salix species throughout a glacial valley in High-Arctic tundra and used a causal approach to test relationships between leaf stomatal and mesophyll conductances (g sc , g m ), carboxylation capacity (Vc max ), nitrogen and phosphorus concentration (N area , P area ) and leaf mass ratio (LMA). Arctic Salix species showed no difference in A sat compared to a global data set, while being characterized by higher N area , P area and LMA. Vc max , g sc and g m independently increased A sat , with Vc max as its main limitation. We highlighted a nitrogen-influenced pathway for increasing photosynthesis in the two prostrate mesic habitat species. In contrast, the erect wetland habitat Salix richardsonii mainly increased A sat with increasing g sc . Overall, our study revealed high photosynthetic capacities of Arctic Salix species but contrasting regulatory pathways that may influence shrub ability to respond to environmental changes in High Arctic tundra., (© 2024 John Wiley & Sons Ltd.)

Ozone (O 3 ) is one of the most harmful and widespread air pollutants, affecting crop yield and plant health worldwide. There is evidence that O 3 reduces the major limiting factor of photosynthesis, namely CO 2 mesophyll conductance (g m ), but there is little quantitative information of O 3 -caused changes in key leaf anatomical traits and their impact on g m . We exposed two O 3 -responsive clones of the economically important tree species Populus × canadensis Moench to 120 ppb O 3 for 21 days. An anatomical diffusion model within the leaf was used to analyse the entire CO 2 diffusion pathway from substomatal cavities to carboxylation sites and determine the importance of each structural and subcellular component as a limiting factor. g m decreased substantially under O 3 and was found to be the most important limitation of photosynthesis. This decrease was mostly driven by an increased cell wall thickness and length of subcellular diffusion pathway caused by altered interchloroplast spacing and chloroplast positioning. By contrast, the prominent leaf integrative trait leaf dry mass per area was neither affected nor related to g m under O 3 . The observed relationship between g m and anatomy, however, was clone-dependent, suggesting that mechanisms regulating g m may differ considerably between closely related plant lines. Our results confirm the need for further studies on factors constraining g m under stress conditions., (© 2024 John Wiley & Sons Ltd.)

Partial root-zone drying irrigation (PRD) can improve water-use efficiency (WUE) without reductions in photosynthesis; however, the mechanism by which this is attained is unclear. To amend that, PRD conditions were simulated by polyethylene glycol 6000 in a root-splitting system and the effects of PRD on cotton growth were studied. Results showed that PRD decreased stomatal conductance (g s ) but increased mesophyll conductance (g m ). Due to the contrasting effects on g s and g m , net photosynthetic rate (A N ) remained unaffected, while the enhanced g m /g s ratio facilitated a larger intrinsic WUE. Further analyses indicated that PRD-induced reduction of g s was related to decreased stomatal size and stomatal pore area in adaxial and abaxial surface which was ascribed to lower pore length and width. PRD-induced variation of g m was ascribed to the reduced liquid-phase resistance, due to increases in chloroplast area facing to intercellular airspaces and the ratio of chloroplast surface area to total mesophyll cell area exposed to intercellular airspaces, as well as to decreases in the distance between cell wall and chloroplast, and between adjacent chloroplasts. The above results demonstrate that PRD, through alterations to stomatal and mesophyll structures, decoupled g s and g m responses, which ultimately increased intrinsic WUE and maintained A N ., (© 2024 John Wiley & Sons Ltd.)

As an important warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) flourishes in warm areas around the world due to the existence of the C4 photosynthetic pathway. However, how C4 photosynthesis operates in bermudagrass leaves is still poorly understood. In this study, we performed single-cell RNA-sequencing on 5296 cells from bermudagrass leaf blades. Eight cell clusters corresponding to mesophyll, bundle sheath, epidermis and vascular bundle cells were successfully identified using known cell marker genes. Expression profiling indicated that genes encoding NADP-dependent malic enzymes (NADP-MEs) were highly expressed in bundle sheath cells, whereas NAD-ME genes were weakly expressed in all cell types, suggesting C4 photosynthesis of bermudagrass leaf blades might be NADP-ME type rather than NAD-ME type. The results also indicated that starch synthesis-related genes showed preferential expression in bundle sheath cells, whereas starch degradation-related genes were highly expressed in mesophyll cells, which agrees with the observed accumulation of starch-filled chloroplasts in bundle sheath cells. Gene co-expression analysis further revealed that different families of transcription factors were co-expressed with multiple C4 photosynthesis-related genes, suggesting a complex transcription regulatory network of C4 photosynthesis might exist in bermudagrass leaf blades. These findings collectively provided new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Vacuoles account for 90% of plant cell volume and play important roles in maintaining osmotic pressure, storing metabolites and lysosomes, compartmentalizing harmful ions, and storing and reusing minerals. These functions closely relay on the ion channels and transporters located on the tonoplast. The separation of intact vacuoles from plant cells is the key technology utilized in the study of tonoplast-located ion channels and transporters. However, the current vacuole separation methods are available for Arabidopsis and some other dicotyledons but are lacking for monocot crops. In this study, we established a new method for the vacuole separation from wheat mesophyll cells and investigated the transmembrane proton flux of tonoplasts with non-invasive micro-test technology (NMT). Moreover, our study provides a technology for the study of vacuole functions in monocot crops., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024. Published by Elsevier GmbH.)

Mesophyll resistance for CO 2 diffusion (r m ) is one of the main limitations for photosynthesis and plant growth. Breeding new varieties with lower r m requires knowledge of its distinct components. We tested new method for estimating the relative drawdowns of CO 2 concentration (c) across hypostomatous leaves of Fagus sylvatica. This technique yields values of the ratio of the internal CO 2 concentrations at the adaxial and abaxial leaf side, c d /c b , the drawdown in the intercellular air space (IAS), and intracellular drawdown between IAS and chloroplast stroma, c c /c bd . The method is based on carbon isotope composition of leaf dry matter and epicuticular wax isolated from upper and lower leaf sides. We investigated leaves from tree-canopy profile to analyse the effects of light and leaf anatomy on the drawdowns and partitioning of r m into its inter- (r IAS ) and intracellular (r liq ) components. Validity of the new method was tested by independent measurements of r m using conventional isotopic and gas exchange techniques. 73% of investigated leaves had adaxial epicuticular wax enriched in 13 C compared to abaxial wax (by 0.50‰ on average), yielding 0.98 and 0.70 for average of c d /c b and c c /c bd , respectively. The r IAS to r liq proportion were 5.5:94.5% in sun-exposed and 14.8:85.2% in shaded leaves. c c dropped to less than half of the atmospheric value in the sunlit and to about two-thirds of it in shaded leaves. This method shows that r IAS is minor but not negligible part of r m and reflects leaf anatomy traits, i.e. leaf mass per area and thickness., (© 2024 The Authors. Plant Biology published by John Wiley & Sons Ltd on behalf of German Society for Plant Sciences, Royal Botanical Society of the Netherlands.)

Key Message: Immunofluorescence staining with frozen sections of plant tissues and a nest tube is convenient and effective, and broadens the applicability of immunofluorescence staining. Immunofluorescence staining is an indispensable and extensively employed technique for determining the subcellular localization of chloroplast division proteins. At present, it is difficult to effectively observe the localization of target proteins in leaves that are hard, or very thin, or have epidermal hair or glands with the current immunofluorescence staining methods. Moreover, signals of target proteins were predominantly detected in mesophyll cells, not the cells of other types. Thus, the method of immunofluorescence staining was further explored for improvement in this study. The plant tissue was embedded with 50% PEG4000 at -60℃, which was then cut into sections by a cryomacrotome. The sections were immediately immersed in fixation solution. Then, the sample was transferred into a special nested plastic tube, which facilitated the fixation and immunofluorescence staining procedures. The use of frozen sections in this method enabled a short processing time and reduced material requirements. By optimizing the thickness of the sections, a large proportion of the cells could be well stained. With this method, we observed the localization of a chloroplast division protein FtsZ1 in the wild-type Arabidopsis and various chloroplast division mutants. Meanwhile, the localization of FtsZ1 was also observed not only in mesophyll cells, but also in guard cells and epidermal cells in a lot of other plant species, including many species with hard leaf tissues. This method is not only easy to use, but also expands the scope of applicability for immunofluorescence staining., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

As the main location of photosynthesis, leaf mesophyll cells are one of the most abundant and essential cell types on earth. Forming the bulk of the internal tissues of the leaf, their size, shape, and patterns of interconnectivity define the internal structure and surface area of the leaf, which in turn determines the efficiency of light capture and carbon fixation. Understanding how these cellular traits are controlled and translated into tissue- and organ-scale traits, and how they influence photosynthetic performance will be key to our ability to improve crop plants in the face of a changing climate. In contrast to the extensive literature on the anatomical and physiological aspects of mesophyll function, our understanding of the cell-level morphogenetic processes underpinning mesophyll cell growth and differentiation is scant. In this review, we focus on how cell division, expansion, and separation are coordinated to create the intricate architecture of the spongy mesophyll., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K + /H + antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K + /H + antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Plants with Crassulacean acid metabolism (CAM) have long been associated with a specialized anatomy, including succulence and thick photosynthetic tissues. Firm, quantitative boundaries between non-CAM and CAM plants have yet to be established - if they indeed exist. Using novel computer vision software to measure anatomy, we combined new measurements with published data across flowering plants. We then used machine learning and phylogenetic comparative methods to investigate relationships between CAM and anatomy. We found significant differences in photosynthetic tissue anatomy between plants with differing CAM phenotypes. Machine learning-based classification was over 95% accurate in differentiating CAM from non-CAM anatomy, and had over 70% recall of distinct CAM phenotypes. Phylogenetic least squares regression and threshold analyses revealed that CAM evolution was significantly correlated with increased mesophyll cell size, thicker leaves, and decreased intercellular airspace. Our findings suggest that machine learning may be used to aid the discovery of new CAM species and that the evolutionary trajectory from non-CAM to strong, obligate CAM requires continual anatomical specialization., (© 2024 The Authors. New Phytologist © 2024 New Phytologist Foundation.)

Numerous studies have clarified the impacts of magnesium (Mg) on leaf photosynthesis from the perspectives of protein synthesis, enzymes activation and carbohydrate partitioning. However, it still remains largely unknown how stomatal and mesophyll conductances (g s and g m , respectively) are regulated by Mg. In the present study, leaf gas exchanges, leaf hydraulic parameters, leaf structural traits and cell wall composition were examined in rice plants grown under high and low Mg treatments to elucidate the impacts of Mg on g s and g m . Our results showed that reduction of leaf photosynthesis under Mg deficiency was mainly caused by the decreased g m , followed by reduced leaf biochemical capacity and g s , and leaf outside-xylem hydraulic conductance (K ox ) was the major factor restricting g s under Mg deficiency. Moreover, increased leaf hemicellulose, lignin and pectin contents and decreased cell wall effective porosity were observed in low Mg plants relative to high Mg plants. These results suggest that K ox and cell wall composition play important roles in regulating g s and g m , respectively, in rice plants under Mg shortages., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

In leaves of C 4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C 4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C 4 grasses, proliferation of plasmodesmata between these cell types is thought to increase cell-to-cell connectivity to allow efficient metabolite movement. However, it is not known whether C 4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C 4 photosynthesis is not clear. How M and BS cells in C 4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C 4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C 3 relatives. The M-BS interface of C 4 G. gynandra showed higher plasmodesmal frequency compared with closely related C 3 species. Formation of these plasmodesmata was induced by light. Pharmacological agents that perturbed photosynthesis reduced the number of plasmodesmata, but this inhibitory effect could be reversed by the provision of exogenous sucrose. We conclude that enhanced formation of plasmodesmata between M and BS cells is wired to the induction of photosynthesis in C 4 G. gynandra., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a "BAHD" CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (-14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance., Competing Interests: Conflict of interest statement. The authors declare no conflict of interests., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Leaf hydraulic conductance (Kleaf) facilitates the supply of water, enabling continual CO2 uptake while maintaining plant water status. We hypothesized that bundle sheath and mesophyll cells play key roles in regulating the radial flow of water out of the xylem by responding to abscisic acid (ABA). Thus, we generated transgenic Arabidopsis thaliana plants that are insensitive to ABA in their bundle sheath (BSabi) and mesophyll (MCabi) cells. We also introduced tissue-specific fluorescent markers to distinguish between cells of the palisade mesophyll, spongy mesophyll, and bundle sheath. Both BSabi and MCabi plants showed greater Kleaf and transpiration under optimal conditions. MCabi plants had larger stomatal apertures, higher stomatal index, and greater vascular diameter and biomass relative to the wild-type (WT) and BSabi plants. In response to xylem-fed ABA, both transgenic and WT plants reduced their Kleaf and transpiration. The membrane osmotic water permeability (Pf) of the WT's spongy mesophyll was higher than that of the WT's palisade mesophyll. While the palisade mesophyll maintained a low Pf in response to high ABA, the spongy mesophyll Pf was reduced. Compared to the WT, BSabi bundle sheath cells had a higher Pf, but MCabi spongy mesophyll had an unexpected lower Pf. These results suggest that tissue-specific regulation of Pf by ABA may be confounded by whole-leaf hydraulics and transpiration. ABA increased the symplastic permeability, but its contribution to Kleaf was negligible. We suggest that the bundle sheath spongy mesophyll pathway dynamically responds to the fluctuations in water availability, while the palisade mesophyll serves as a hydraulic buffer., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO 2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO 2 assimilation in guard cells. Furthermore, it remains unclear how the metabolic fluxes throughout the tricarboxylic acid (TCA) cycle and associated pathways are regulated in illuminated guard cells. Here we carried out a 13 C-HCO 3 labelling experiment in tobacco guard cells harvested under continuous dark or during the dark-to-light transition to elucidate principles of metabolic dynamics downstream of CO 2 assimilation. Most metabolic changes were similar between dark-exposed and illuminated guard cells. However, illumination altered the metabolic network structure of guard cells and increased the 13 C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13 C-labelling and leads to more drastic reductions in the content of this metabolite. Fumarate was strongly labelled under both dark and light conditions, while illumination increased the 13 C-enrichment in pyruvate, succinate and glutamate. Only one 13 C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO 2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO 2 assimilation provides carbons for gluconeogenesis, the TCA cycle and glutamate synthesis and that previously stored malate and citrate are used to underpin the specific metabolic requirements of illuminated guard cells., Competing Interests: Declaration of competing interest The authors declare no potential conflict of interest., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C 4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C 4 photosynthesis., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Increasing mesophyll conductance of CO 2 (g m ) is a strategy to improve photosynthesis in C 3 crops. However, the relative importance of different anatomical traits in determining g m in crops is unclear. Mesophyll conductance measurements were performed on 10 crops using the online carbon isotope discrimination method and the 'variable J' method in parallel. The influences of crucial leaf anatomical traits on g m were evaluated using a one-dimensional anatomical CO 2 diffusion model. The g m values measured using two independent methods were compatible, although significant differences were observed in their absolute values. Quantitative analysis showed that cell wall thickness and chloroplast stroma thickness are the most important elements along the diffusion pathway. Unexpectedly, the large variability of g m across crops was not associated with any investigated leaf anatomical traits except chloroplast thickness. The g m values estimated using the anatomical model differed remarkably from the values measured in vivo in most species. However, when the species-specific effective porosity of the cell wall and the species-specific facilitation effect of CO 2 diffusion across the membrane and chloroplast stoma were taken into account, the model could output g m values very similar to those measured in vivo. These results indicate that g m variation across crops is probably also driven by the effective porosity of the cell wall and effects of facilitation of CO 2 transport across the membrane and chloroplast stroma in addition to the thicknesses of the elements., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Photosynthesis is the most temperature-sensitive process in the plant kingdom, but how the photosynthetic pathway responds during low-temperature exposure remains unclear. Herein, cold stress (4°C) induced widespread damage in the form DNA double-stranded breaks (DSBs) in the mesophyll cells of rice (Oryza sativa), subsequently causing a global inhibition of photosynthetic carbon metabolism (PCM) gene expression. Topoisomerase genes TOP6A3 and TOP6B were induced at 4°C and their encoded proteins formed a complex in the nucleus. TOP6A3 directly interacted with KU70 to inhibit its binding to cold-induced DSBs, which was facilitated by TOP6B, finally blocking the loading of LIG4, a component of the classic non-homologous end joining (c-NHEJ) pathway. The repression of c-NHEJ repair imposed by cold extended DSB damage signaling, thus prolonging the inhibition of photosynthesis in leaves. Furthermore, the TOP6 complex negatively regulated 13 crucial PCM genes by directly binding to their proximal promoter regions. Phenotypically, TOP6A3 overexpression exacerbated the γ-irradiation-triggered suppression of PCM genes and led to the hypersensitivity of photosynthesis parameters to cold stress, dependent on the DSB signal transducer ATM. Globally, the TOP6 complex acts as a signal integrator to control PCM gene expression and synchronize cold-induced photosynthesis inhibition, which modulates carbon assimilation rates immediately in response to changes in ambient temperature., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Leaf phosphorus (P) comprises four major fractions: inorganic phosphate (P i ), nucleic acids, phospholipids, P-containing metabolites and a residual fraction. In this review paper, we investigated whether allocation of P fractions varies among groups of terrestrial vascular plants, and is indicative of a species' strategy to use P efficiently. We found that as leaf total P concentration increases, the P i fraction increases the most, without a plateau, while other fractions plateau. Variability of the concentrations of leaf P fractions is greatest among families > species(family) > regions > plant life forms. The percentage of total P allocated to nucleic acid-P (20-35%) and lipid-P (14-34%) varies less among families/species. High photosynthetic P-use efficiency is associated with low concentrations of all P fractions, and preferential allocation of P to metabolite-P and mesophyll cells. Sequential resorption of P from senescing leaves starts with P i , followed by metabolite-P, and then other organic P fractions. Allocation of P to leaf P fractions varies with season. Leaf phytate concentrations vary considerably among species, associated with variation in photosynthesis and defence. Plasticity of P allocation to its fractions is important for acclimation to low soil P availability, and species-specific P allocation is needed for co-occurrence with other species., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

As in most plants, during their growth from immature to mature stages, the leaves of Setaria viridis , a model C 4 bioenergy plant, have differential growth rates from the base (immature or growing) to the tip (most mature). In this study, we constructed a multi-segment C 4 leaf metabolic model of S. viridis with two cell types (bundle sheath and mesophyll cells) across four leaf segments (base to tip). We incorporated differential growth rates for each leaf segment as constraints and integrated transcriptomic data as the objective function for our model simulation using flux balance analysis. The model was able to predict the exchanges of metabolites between immature and mature segments of the leaf and the distribution of the activities of biomass synthesis across those segments. Our model demonstrated the use of a modelling approach in studying the source-sink relationship within an organ and provided insights into the metabolic interactions across different parts of a leaf.

Leaf structure plays an important role in photosynthesis. However, the causal relationship and the quantitative importance of any single structural parameter to the overall photosynthetic performance of a leaf remains open to debate. In this paper, we report on a mechanistic model, eLeaf, which successfully captures rice leaf photosynthetic performance under varying environmental conditions of light and CO 2 . We developed a 3D reaction-diffusion model for leaf photosynthesis parameterised using a range of imaging data and biochemical measurements from plants grown under ambient and elevated CO 2 and then interrogated the model to quantify the importance of these elements. The model successfully captured leaf-level photosynthetic performance in rice. Photosynthetic metabolism underpinned the majority of the increased carbon assimilation rate observed under elevated CO 2 levels, with a range of structural elements making positive and negative contributions. Mesophyll porosity could be varied without any major outcome on photosynthetic performance, providing a theoretical underpinning for experimental data. eLeaf allows quantitative analysis of the influence of morphological and biochemical properties on leaf photosynthesis. The analysis highlights a degree of leaf structural plasticity with respect to photosynthesis of significance in the context of attempts to improve crop photosynthesis., (© 2022 The Authors New Phytologist © 2022 New Phytologist Foundation.)

Water shortage strongly affects plants' physiological performance. Since tomato (Solanum lycopersicum) non-long shelf-life (nLSL) and long shelf-life (LSL) genotypes differently face water deprivation, we subjected a nLSL and a LSL genotype to four treatments: control (well watering), short-term water deficit stress at 40% field capacity (FC) (ST 40% FC), short-term water deficit stress at 30% FC (ST 30% FC), and short-term water deficit stress at 30% FC followed by recovery (ST 30% FC-Rec). Treatments promoted genotype-dependent elastic adjustments accompanied by distinct photosynthetic responses. While the nLSL genotype largely modified mesophyll conductance (g m ) across treatments, it was kept within a narrow range in the LSL genotype. However, similar g m values were achieved under ST 30% FC conditions. Particularly, modifications in the relative abundance of cell wall components and in sub-cellular anatomic parameters such as the chloroplast surface area exposed to intercellular air space per leaf area (S c /S) and the cell wall thickness (T cw ) regulated g m in the LSL genotype. Instead, only changes in foliar structure at the supra-cellular level influenced g m in the nLSL genotype. Even though further experiments testing a larger range of genotypes and treatments would be valuable to support our conclusions, we show that even genotypes of the same species can present different elastic, anatomical, and cell wall composition-mediated mechanisms to regulate g m when subjected to distinct water regimes., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Mesophyll CO 2 conductance (g m ) in C 3 species responds to short-term (minutes) changes in environment potentially due to changes in leaf anatomical and biochemical properties and measurement artefacts. Compared with C 3 species, there is less information on g m responses to short-term changes in environmental conditions such as partial pressure of CO 2 (pCO 2 ) across diverse C 4 species and the potential determinants of these responses. Using 16 C 4 grasses we investigated the response of g m to short-term changes in pCO 2 and its relationship with leaf anatomy and biochemistry. In general, g m increased as pCO 2 decreased (statistically significant increase in 12 species), with percentage increases in g m ranging from +13% to +250%. Greater increase in g m at low pCO 2 was observed in species exhibiting relatively thinner mesophyll cell walls along with greater mesophyll surface area exposed to intercellular air spaces, leaf N, photosynthetic capacity and activities of phosphoenolpyruvate carboxylase and Rubisco. Species with greater CO 2 responses of g m were also able to maintain their leaf water-use efficiencies (TE i ) under low CO 2 . Our study advances understanding of CO 2 response of g m in diverse C 4 species, identifies the key leaf traits related to this response and has implications for improving C 4 photosynthetic models and TE i through modification of g m ., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Mesophyll conductance (g m ) limits photosynthesis by restricting CO 2 diffusion between the substomatal cavities and chloroplasts. Although it is known that g m is determined by both leaf anatomical and biochemical traits, their relative contribution across plant functional types (PFTs) is still unclear. We compiled a dataset of g m measurements and concomitant leaf traits in unstressed plants comprising 563 studies and 617 species from all major PFTs. We investigated to what extent g m limits photosynthesis across PFTs, how g m relates to structural, anatomical, biochemical, and physiological leaf properties, and whether these relationships differ among PFTs. We found that g m imposes a significant limitation to photosynthesis in all C 3 PFTs, ranging from 10-30% in most herbaceous annuals to 25-50% in woody evergreens. Anatomical leaf traits explained a significant proportion of the variation in g m (R 2  > 0.3) in all PFTs except annual herbs, in which g m is more strongly related to biochemical factors associated with leaf nitrogen and potassium content. Our results underline the need to elucidate mechanisms underlying the global variability of g m . We emphasise the underestimated potential of g m for improving photosynthesis in crops and identify modifications in leaf biochemistry as the most promising pathway for increasing g m in these species., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Leaf growth relies on photosynthesis and hydraulics to provide carbohydrates and expansion power; in turn, leaves intercept light and construct organism systems for functioning. Under potassium (K) deficiency stress, leaf area, photosynthesis and hydraulics are all affected by alterations in leaf structure. However, the connection between changes in leaf growth and function caused by the structure under K regulation is unclear. Consequently, the leaf hydraulic conductance (K leaf ) and photosynthetic rate (A) combined with leaf anatomical characteristics of Brassica napus were continuously observed during leaf growth under different K supply levels. The results showed that K leaf and A decreased simultaneously after leaf area with the increasing K deficiency stress. K deficiency significantly increased longitudinal mesophyll cell investment, leading to a reduced volume fraction of intercellular air-space (f ias ) and decreased leaf expansion rate. Furthermore, reduced f ias decreased mesophyll and chloroplast surfaces exposed to intercellular airspace and gas phase H 2 O transport, which induced coordinated changes in CO 2 mesophyll conductance and hydraulic conductance in extra-xylem pathways. Adequate K supply facilitated higher f ias through smaller palisade tissue cell density (loose mesophyll cell arrangement) and smaller spongy tissue cell size, which coordinated CO 2 and H 2 O conductance and promoted leaf area expansion., (© 2022 John Wiley & Sons Ltd.)

Studies investigating the mechanisms underlying the variation of photosynthesis along plant phylogeny and especially during domestication are of great importance, and may provide new insights to further improve crop photosynthesis. In the present study, we compiled a database including 542 sets of data of leaf gas exchange parameters and leaf structural and chemical traits in ferns and fern allies, gymnosperms, non-crop angiosperms, and crops. We found that photosynthesis was dramatically improved from ferns and fern allies to non-crop angiosperms, and further increased in crops. The improvement of photosynthesis during phylogeny and domestication was related to increases in carbon dioxide diffusional capacities and, to a lesser extent, biochemical capacity. Cell wall thickness rather than chloroplast surface area facing intercellular airspaces drives the variation of mesophyll conductance. The variation of the maximum carboxylation rate was not related to leaf nitrogen content. The slope of the relationship between mass-based photosynthesis and nitrogen was lower in crops than in non-crop angiosperms. These findings suggest that the manipulation of cell wall thickness is the most promising approach to further improve crop photosynthesis, and that an increase of leaf nitrogen will be less efficient in improving photosynthesis in crops than in non-crop angiosperms., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Bryophytes are the group of land plants with the lowest photosynthetic rates, which was considered to be a consequence of their higher anatomical CO2 diffusional limitation compared with tracheophytes. However, the most recent studies assessing limitations due to biochemistry and mesophyll conductance in bryophytes reveal discrepancies based on the methodology used. In this study, we compared data calculated from two different methodologies for estimating mesophyll conductance: variable J and the curve-fitting method. Although correlated, mesophyll conductance estimated by the curve-fitting method was on average 4-fold higher than the conductance obtained by the variable J method; a large enough difference to account for the scale of differences previously shown between the biochemical and diffusional limitations to photosynthesis. Biochemical limitations were predominant when the curve-fitting method was used. We also demonstrated that variations in bryophyte relative water content during measurements can also introduce errors in the estimation of mesophyll conductance, especially for samples which are overly desiccated. Furthermore, total chlorophyll concentration and soluble proteins were significantly lower in bryophytes than in tracheophytes, and the percentage of proteins quantified as Rubisco was also significantly lower in bryophytes (<6.3% in all studied species) than in angiosperms (>16% in all non-stressed cases). Photosynthetic rates normalized by Rubisco were not significantly different between bryophytes and angiosperms. Our data suggest that the biochemical limitation to photosynthesis in bryophytes is more relevant than so far assumed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Main Conclusion: This study suggests that stomatal and leaf structures are highly correlated, and mesophyll cell size is an important anatomical trait determining the coordination between stomatal size and mesophyll porosity. A comprehensive study of the correlations between the structural traits and on their relationships with gas exchange parameters may provide some useful information into leaf development and improvement in efficiencies of photosynthetic CO 2 fixation and transpirational water loss. In the present study, nine plant materials from eight crop species were pot grown in a growth chamber. Leaf structural traits, gas exchange, and leaf nitrogen content were measured. We found that stomatal size, mesophyll cell size (MCS), and mesophyll porosity were positively correlated and that the surface areas of mesophyll cells and chloroplasts facing intercellular air spaces were positively correlated with both stomatal density and stomatal area per leaf area (SA). These results suggested that the developments of stomata and mesophyll cells are highly correlated among different crop species. Additionally, MCS was positively correlated with leaf thickness and negatively correlated with leaf density and leaf mass per area, which indicated that MCS might play an important role in leaf structural investments and physiological functions among species. In summary, this study illustrates the correlations between stomatal and mesophyll structures, and it highlights the importance of considering the covariations among leaf traits with the intent of improving photosynthesis and iWUE., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

In plants with C3 photosynthesis, increasing the diffusion conductance for CO2 from the substomatal cavity to chloroplast stroma (mesophyll conductance) can improve the efficiencies of both CO2 assimilation and photosynthetic water use. In the diffusion pathway from substomatal cavity to chloroplast stroma, the plasmalemma and chloroplast envelope membranes impose a considerable barrier to CO2 diffusion, limiting photosynthetic efficiency. In an attempt to improve membrane permeability to CO2, and increase photosynthesis in tobacco, we generated transgenic lines in Nicotiana tabacum L. cv Petite Havana carrying either the Arabidopsis PIP1;2 (AtPIP1;2) or PIP1;4 (AtPIP1;4) gene driven by the constitutive dual 2x35S CMV promoter. From a collection of independent T0 transgenics, two T2 lines from each gene were characterized, with western blots confirming increased total aquaporin protein abundance in the AtPIP1;2 tobacco lines. Transient expression of AtPIP1;2-mGFP6 and AtPIP1;4-mGFP6 fusions in Nicotiana benthamiana identified that both AtPIP1;2 and AtPIP1;4 localize to the plasmalemma. Despite achieving ectopic production and correct localization, gas exchange measurements combined with carbon isotope discrimination measurements detected no increase in mesophyll conductance or CO2 assimilation rate in the tobacco lines expressing AtPIP. We discuss the complexities associated with trying to enhance gm through modified aquaporin activity., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Starting as small, densely packed boxes, leaf mesophyll cells expand to form an intricate mesh of interconnected cells and air spaces, the organization of which dictates the internal surface area of the leaf for light capture and gas exchange during photosynthesis. Despite their importance, little is known about the basic patterns of mesophyll cell division, and how they contribute to cell and intercellular space organization. To address this, we tracked divisions within individual cell lineages in three dimensions over time in Arabidopsis spongy mesophyll. We found that early on, successive cell division planes switch their orientation such that each new cell wall intersects the previous at a right angle, creating a new multi-cell junction (the intersection of three or more cells). These junctions then open to create intercellular spaces. During subsequent enlargement of the spaces, the division planes of the surrounding cells show an increasing tendency to tilt in the direction of their adjacent intercellular spaces. This disrupts the alternating pattern, and by extension, halts the initiation of new multi-cell junctions and intercellular spaces, but allows the expansion of existing spaces. Both division patterns are specified before mitosis by the orientation of interphase cortical microtubules, which gradually narrow to form a preprophase band in the same orientation to establish the future plane of cell division. In the absence of the microtubule-associated protein CLASP, the early alternating division plane and microtubule patterns are compromised, whereas space-oriented divisions are exacerbated. This results in large distortions of the topological relations between cells and intercellular spaces, as well as changes in their relative abundance. Our data reveal the existence of two competing cell division mechanisms that are balanced by CLASP to specify the distribution of cells and intercellular spaces in spongy mesophyll tissue., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Leaf imaging via microscopy has provided critical insights into research on photosynthesis at multiple junctures, from the early understanding of the role of stomata, through elucidating C4 photosynthesis via Kranz anatomy and chloroplast arrangement in single cells, to detailed explorations of diffusion pathways and light utilization gradients within leaves. In recent decades, the original two-dimensional (2D) explorations have begun to be visualized in three-dimensional (3D) space, revising our understanding of structure-function relationships between internal leaf anatomy and photosynthesis. In particular, advancing new technologies and analyses are providing fresh insight into the relationship between leaf cellular components and improving the ability to model net carbon fixation, water use efficiency, and metabolite turnover rate in leaves. While ground-breaking developments in imaging tools and techniques have expanded our knowledge of leaf 3D structure via high-resolution 3D and time-series images, there is a growing need for more in vivo imaging as well as metabolite imaging. However, these advances necessitate further improvement in microscopy sciences to overcome the unique challenges a green leaf poses. In this review, we discuss the available tools, techniques, challenges, and gaps for efficient in vivo leaf 3D imaging, as well as innovations to overcome these difficulties., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Leaf photosynthesis is highly correlated with CO 2 -diffusion capacities, which are determined by both leaf anatomical traits and environmental stimuli. In the present study, leaf photosynthetic rate ( A ), stomatal conductance ( g s ), mesophyll conductance ( g m ) and the related leaf anatomical traits were studied on rice plants at two growth stages and with two different N supplies, and the response of photosynthesis to temperature ( T ) was also studied. We found that g m was significantly higher at mid-tillering stage and at high N treatment. The larger g m was related to a larger chloroplast surface area facing intercellular air spaces and a thinner cell wall in comparison with booting stage and zero N treatment. At mid-tillering stage and at high N treatment, g m showed a stronger temperature response. The modelling of the g m - T relationships suggested that, in comparison with booting stage and zero N treatment, the stronger temperature response of g m was related to the higher activation energy of the membrane at mid-tillering stage and at high N treatment. The findings in the present study can enhance our knowledge on the physiological and environmental determinants of photosynthesis.