Leaf anatomy does not explain the large variability of mesophyll conductance across C3 crop species.

Increasing mesophyll conductance of CO2 (gm ) is a strategy to improve photosynthesis in C3 crops. However, the relative importance of different anatomical traits in determining gm 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 gm were evaluated using a one-dimensional anatomical CO2 diffusion model. The gm 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 gm across crops was not associated with any investigated leaf anatomical traits except chloroplast thickness. The gm 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 CO2 diffusion across the membrane and chloroplast stoma were taken into account, the model could output gm values very similar to those measured in vivo. These results indicate that gm variation across crops is probably also driven by the effective porosity of the cell wall and effects of facilitation of CO2 transport across the membrane and chloroplast stroma in addition to the thicknesses of the elements.

Elevated CO2 concentration increases maize growth under water deficit or soil salinity but with a higher risk of hydraulic failure.

Climate change presents a challenge for plants to acclimate their water relations under changing environmental conditions, and may increase the risks of hydraulic failure under stress. In this study, maize plants were acclimated to two different CO2 concentrations ([CO2]; 400 ppm and 700 ppm) while under either water stress (WS) or soil salinity (SS) treatments, and their growth and hydraulic traits were examined in detail. Both WS and SS inhibited growth and had significant impacts on hydraulic traits. In particular, the water potential at 50% loss of stem hydraulic conductance (P50) decreased by 1 MPa in both treatments at 400 ppm. When subjected to elevated [CO2], the plants under both WS and SS showed improved growth by 7-23%. Elevated [CO2] also significantly increased xylem vulnerability (measured as loss of conductivity with decreasing xylem pressure), resulting in smaller hydraulic safety margins. According to the plant desiccation model, the critical desiccation degree (time×vapor pressure deficit) that the plants could tolerate under drought was reduced by 43-64% under elevated [CO2]. In addition, sensitivity analysis showed that P50 was the most important trait in determining the critical desiccation degree. Thus, our results demonstrated that whilst elevated [CO2] benefited plant growth under WS or SS, it also interfered with hydraulic acclimation, thereby potentially placing the plants at a higher risk of hydraulic failure and increased mortality.

Influence of IAA and ABA on maize stem vessel diameter and stress resistance in variable environments.

The plasticity of the xylem and its associated hydraulic properties play crucial roles in plant acclimation to environmental changes, with vessel diameter (Dv) being the most functionally prominent trait. While the effects of external environmental factors on xylem formation and Dv are not fully understood, the endogenous hormones indole-3-acetic acid (IAA) and abscisic acid (ABA) are known to play significant signalling roles under stress conditions. This study investigates how these hormones impact Dv under various environmental changes. Experiments were conducted in maize plants subjected to drought, soil salinity, and high CO2 concentration treatments. We found that drought and soil salinity significantly reduced Dv at the same stem internode, while an elevated CO2 concentration can mitigate this decrease in Dv. Remarkably, significant negative correlations were observed between Dv and the contents of IAA and ABA when considering the different treatments. Moreover, appropriate foliar application of either IAA or ABA on well-watered and stressed plants led to a decrease in Dv, while the application of corresponding inhibitors resulted in an increase in Dv. This finding underscores the causal relationship between Dv and the levels of both IAA and ABA, offering a promising approach to manipulating xylem vessel size.

Photosynthesis of rice leaves with a parallel venation is highly tolerant to vein severing.

Vein severing in plants caused by leaf damage is common in fields where crops are cultivated. It is hypothesized that leaves with complex reticulate venation can withstand hydraulic disturbances caused by vein severing, thereby preserving leaf carbon assimilation. However, limited research focuses on vein damage of leaves with parallel venation. We studied how vein-severing affected the photosynthetic traits of rice (Oryza sativa) leaves in seconds, minutes and days, under varying water-demand conditions and differing extents of water supply disruption. Rice leaves completely lost their photosynthetic capacity within 2.5 minutes after excision. Severing the midrib resulted in reduced light-saturated photosynthetic rate (A), stomatal conductance (gsw ) and transpiration rate (E) by 2.6, 6.8 and 5.9%, respectively, already after thirty minutes. We further investigated the photosynthetic trait responses to various extents of leaf width severing, while keeping the midrib functional. Surprisingly, A, gsw and E in the downstream area of the severed leaves largely remained stable, showing minimal variation across different leaf width severing ratios. These traits declined only slightly even under increased ambient light intensity and leaf-to-air vapor pressure deficit. This sustained photosynthesis post-severing is attributed to the efficient lateral water transport. Long-term leaf damage slightly but not significantly, impacted the downstream photosynthetic traits within five days post-severing. However, a more pronounced reduction in gas exchange during leaf senescence was observed nine days after severing. These findings suggested that rice leaves can tolerate hydraulic disturbances from vein severing and maintain functionality under various conditions, which is crucial for crop yield stability. However, long-term consequences require further investigation.

How Important Are Functional and Developmental Constraints on Phenotypic Evolution? An Empirical Test with the Stomatal Anatomy of Flowering Plants.

AbstractQuantifying the relative contribution of functional and developmental constraints on phenotypic variation is a long-standing goal of macroevolution, but it is often difficult to distinguish different types of constraints. Alternatively, selection can limit phenotypic (co)variation if some trait combinations are generally maladaptive. The anatomy of leaves with stomata on both surfaces (amphistomatous) present a unique opportunity to test the importance of functional and developmental constraints on phenotypic evolution. The key insight is that stomata on each leaf surface encounter the same functional and developmental constraints but potentially different selective pressures because of leaf asymmetry in light capture, gas exchange, and other features. Independent evolution of stomatal traits on each surface imply that functional and developmental constraints alone likely do not explain trait covariance. Packing limits on how many stomata can fit into a finite epidermis and cell size-mediated developmental integration are hypothesized to constrain variation in stomatal anatomy. The simple geometry of the planar leaf surface and knowledge of stomatal development make it possible to derive equations for phenotypic (co)variance caused by these constraints and compare them with data. We analyzed evolutionary covariance between stomatal density and length in amphistomatous leaves from 236 phylogenetically independent contrasts using a robust Bayesian model. Stomatal anatomy on each surface diverges partially independently, meaning that packing limits and developmental integration are not sufficient to explain phenotypic (co)variation. Hence, (co)variation in ecologically important traits like stomata arises in part because there is a limited range of evolutionary optima. We show how it is possible to evaluate the contribution of different constraints by deriving expected patterns of (co)variance and testing them using similar but separate tissues, organs, or sexes.

Cell wall thickness has phylogenetically consistent effects on the photosynthetic nitrogen-use efficiency of terrestrial plants.

Leaf photosynthetic nitrogen-use efficiency (PNUE) diversified significantly among C3 species. To date, the morpho-physiological mechanisms and interrelationships shaping PNUE on an evolutionary time scale remain unclear. In this study, we assembled a comprehensive matrix of leaf morpho-anatomical and physiological traits for 679 C3 species, ranging from bryophytes to angiosperms, to comprehend the complexity of interrelationships underpinning PNUE variations. We discovered that leaf mass per area (LMA), mesophyll cell wall thickness (Tcwm ), Rubisco N allocation fraction (PR ), and mesophyll conductance (gm ) together explained 83% of PNUE variations, with PR and gm accounting for 65% of those variations. However, the PR effects were species-dependent on gm , meaning the contribution of PR on PNUE was substantially significant in high-gm species compared to low-gm species. Standard major axis (SMA) and path analyses revealed a weak correlation between PNUE and LMA (r2 = 0.1), while the SMA correlation for PNUE-Tcwm was robust (r2 = 0.61). PR was inversely related to Tcwm , paralleling the relationship between gm and Tcwm , resulting in the internal CO2 drawdown being only weakly proportional to Tcwm . The coordination of PR and gm in relation to Tcwm constrains PNUE during the course of evolution.

Leaf rolling precedes stomatal closure in rice (Oryza sativa) under drought conditions.

Leaf rolling is a physiological response to drought that may help to reduce water loss, but its significance as a contribution to drought tolerance is uncertain. We scored the leaf rolling of four rice genotypes along an experimental drought gradient using an improved cryo-microscopy method. Leaf water potential (Ψleaf), gas exchange, chlorophyll fluorescence, leaf hydraulic conductance, rehydration capacity, and the bulk turgor loss point were also analysed. During the drought process, stomatal conductance declined sharply to reduce water loss, and leaves rolled up before the stomata completely closed. The leaf water loss rate of rolled leaves was significantly reduced compared with artificially flattened leaves. The Ψleaf threshold of initial leaf rolling ranged from -1.95 to -1.04 MPa across genotypes. When a leaf rolled so that the leaf edges were touching, photosynthetic rate and stomatal conductance declined more than 80%. Across genotypes, leaf hydraulic conductance declined first, followed by gas exchange and chlorophyll fluorescence parameters. However, the Ψleaf threshold for a given functional trait decline differed significantly among genotypes, with the exception of leaf hydraulic conductance. Our results suggested that leaf rolling was mechanistically linked to drought avoidance and tolerance traits and might serve as a useful phenotypic trait for rice breeding in future drought scenarios.

Mesophyll conductance variability of rice aquaporin knockout lines at different growth stages and growing environments.

The plasma membrane subfamily of aquaporins [plasma membrane intrinsic proteins (PIPs)], which facilitates the CO2 diffusion across membranes, is proposed to play an important role in mesophyll conductance to CO2 (gm ), a major limiting factor of photosynthesis. However, recent studies implied no causal relationship between gm and PIPs because they failed to repeat the previous observed differences in gm between PIP knockout lines and their wild-type. The contrasting results on the role of PIPs in gm were interpreted as the different growth conditions among studies, which has never been tested. Here, we assessed whether the differences in gm between wild-type and PIP knockout lines, Ospip1;1, Ospip1;2 and Ospip2;1, varied with growth condition (field versus pot condition) and growth stages in rice. Under field conditions, no differences were observed in plant performance, photosynthetic rate (A) and gm between PIP knockout lines and the wild-type. However, in agreement with previous studies, two out of three knockout lines showed significant declines in tiller number, plant height, A and gm under pot conditions. Moreover, we found that the differences in A and gm between PIP knockout lines and the wild-type varied with the growth stage of the plants. Our results showed that the differences in gm between PIP knockout lines and wild-type were depending on the growth environments and stage of the plants, and further efforts are required to reveal the underlying mechanisms.

Effects of contrasting N supplies on leaf photosynthetic induction under fluctuating light in rice (Oryza sativa L.).

Nitrogen (N) is one of the most important nutrients for crop growth and yield formation, as it is an important constituent in a large amount of proteins, cell walls, and membranes related to photosynthesis. Recently, increasing studies have suggested the important roles of photosynthetic induction and stomatal movement under fluctuating light in regulating plant carbon assimilation and water use efficiency. How leaf N content affects photosynthetic induction remains uncertain. Here, we observed a significantly faster photosynthetic induction with the increasing supply of N under fluctuating light conditions. Photosynthetic induction was mainly limited by biochemical processes but not stomatal opening after a stepwise increase in irradiance across different N supplies. Higher N supply enhanced photosynthetic efficiency under constant and fluctuating light conditions but reduced leaf intrinsic water use efficiency (WUEi ). This study is mainly focused on clarifying the crucial limitation of photosynthetic induction under different N treatments, which may facilitate the improvement of photosynthetic efficiency under complicated environments in the future.

Safety-efficiency tradeoffs? Correlations of photosynthesis, leaf hydraulics, and dehydration tolerance across species.

The tradeoffs between carbon assimilation and hydraulic efficiencies and drought-tolerance traits on different scales are considered a central tenet in plant ecophysiology; however, no clear tradeoff between these traits has emerged in previous studies using woody angiosperms or grasses by investigating several hydraulic tolerance and gas exchange efficiency and/or water transport efficiency traits. In this study, we measured numerous efficiency, resistance, and leaf anatomical traits, including light-saturated gas exchange, leaf hydraulic vulnerability curves, pressure-volume curves, and leaf anatomical traits, in seven species with diverse drought tolerance. A substantial variation in photosynthetic rate, stomatal conductance, mesophyll conductance, maximum leaf hydraulic conductance (Kmax), mesophyll anatomical traits, and leaf vein density across species was observed. Both mesophyll conductance and Kmax were related to leaf anatomical traits, but other gas exchange traits were decoupled from Kmax. Although the efficiency and tolerance traits varied widely across estimated species, no clear trade-off between safety traits and efficiency traits was observed. These findings suggested that postulated leaf-level drought tolerance-carbon assimilation and hydraulic efficiency tradeoff does not exist among distant species and that the fact that different leaf anatomical traits determine efficiency and tolerance capacity might contribute to the lack of such tradeoffs.

Linking water relations and hydraulics with photosynthesis.

For land plants, water is the principal governor of growth. Photosynthetic performance is highly dependent on the stable and suitable water status of leaves, which is balanced by the water transport capacity, the water loss rate as well as the water capacitance of the plant. This review discusses the links between leaf water status and photosynthesis, specifically focussing on the coordination of CO2 and water transport within leaves, and the potential role of leaf capacitance and elasticity on CO2 and water transport.

Leaf anatomical characteristics are less important than leaf biochemical properties in determining photosynthesis responses to nitrogen top-dressing.

The photosynthetic capacity of leaves is dramatically influenced by nitrogen (N) availability in the soil, as CO2 concentration in chloroplasts and photosynthetic biochemical capacity are related to leaf N content. The relationship between mesophyll conductance (gm) and leaf N content was expected to be shaped by leaf anatomical traits. However, the increased gm in mature leaves achieved by N top-dressing is unlikely to be caused by changes in leaf anatomy. Here, we assessed the impacts of N supply on leaf anatomical, biochemical, and photosynthetic features, specifically, the dynamic responses of leaf anatomy, biochemistry, and photosynthesis to N top-dressing in tobacco. Plant performance was substantially affected by soil N status. In comparison with the leaves of plants subjected to low N treatment, leaves of plants with high N treatment photosynthesized significantly more, due to higher CO2 diffusion conductance and photosynthetic biochemical capacity. The high gm in high N-treated leaves apparently related to modifications in the leaf anatomy; however, the rapid response of gm to N top-dressing cannot be fully explained by leaf anatomical modifications.

Cell wall thickness and composition are involved in photosynthetic limitation.

The key role of cell walls in setting mesophyll conductance to CO2 (gm) and, consequently, photosynthesis is reviewed. First, the theoretical properties of cell walls that can affect gm are presented. Then, we focus on cell wall thickness (Tcw) reviewing empirical evidence showing that Tcw varies strongly among species and phylogenetic groups in a way that correlates with gm and photosynthesis; that is, the thicker the mesophyll cell walls, the lower the gm and photosynthesis. Potential interplays of gm, Tcw, dehydration tolerance, and hydraulic properties of leaves are also discussed. Dynamic variations of Tcw in response to the environment and their implications in the regulation of photosynthesis are discussed, and recent evidence suggesting an influence of cell wall composition on gm is presented. We then propose a hypothetical mechanism for the influence of cell walls on photosynthesis, combining the effects of thickness and composition, particularly pectins. Finally, we discuss the prospects for using biotechnology for enhancing photosynthesis by altering cell wall-related genes.

Leaf photosynthetic plasticity does not predict biomass responses to growth irradiance in rice.

Phenotypic plasticity, the capacity of an organism to generate alternative phenotypes in response to different environments, is a particularly important characteristic to enable sessile plants to adapt to rapid changes in their surroundings. Leaf anatomical and physiological traits exhibit plasticity in response to growth irradiances, but it is relatively unclear if the plasticity varies among genotypes for a species. Equally importantly, empirical results on how leaf-level plasticity influences whole-plant growth are largely absent. We conducted an integrated investigation into the light-introduced plasticity by measuring 48 traits involving plant growth, leaf anatomy, leaf biochemistry, and leaf physiology of five rice genotypes grown under two irradiances. More than half of the estimated traits were significantly affected by growth light intensities, and the sizes of the cumulative effect of growth light ranged from -25.04% (stomatal conductance at high measurement light) to 135.2% (tiller number). Growth irradiance levels dramatically shifted the relationship between photosynthetic rate and stomatal conductance. However, the relationship between photosynthetic rate and mesophyll conductance was rarely influenced by growth light levels. Importantly, the present study highlights the significant variation in trait plasticity across rice genotypes and that the light-introduced biomass changes were rarely predicted by leaf photosynthetic plasticity. Our findings imply that the genotypes with high productivity at the low growth light conditions do not necessarily have high productivity under high light conditions.

From one side to two sides: the effects of stomatal distribution on photosynthesis.

The functions of stomata have been studied for a long time; however, a clear understanding of the influences of stomatal distribution on photosynthesis, especially the CO2 diffusion, is still unclear. Here, we investigated the stomatal morphology, distribution on leaf surfaces, vein traits and gas exchange parameters of 61 species, of which 29 were amphistomatous, spanning 32 families. Photosynthesis (A) was tightly coupled with operational stomatal conductance (gs ) and mesophyll conductance (gm ) regardless of whether phylogenetic relationships were accounted for. Although the enhancement of gs from ferns and gymnosperms to angiosperms could largely be explained by the increase in leaf vein density (VLA) and stomatal density (SD), the gs was decoupled from VLA and SD across angiosperm species. Instead, A in angiosperms was further influenced by the allocation of stomatal pores on leaf surfaces, which dramatically increased gs and gm . Moreover, the ratio of gs to anatomically based maximum gs was, on average, 0.12 across species. Our results show that the shift of stomatal pores from one leaf side to both sides played an important role in regulating CO2 diffusion via both stomata and mesophyll tissues. Modifications of stomata distribution have potential as a functional trait for photosynthesis improvement.

Differential coordination of stomatal conductance, mesophyll conductance, and leaf hydraulic conductance in response to changing light across species.

Stomatal conductance (gs ) and mesophyll conductance (gm ) represent major constraints to photosynthetic rate (A), and these traits are expected to coordinate with leaf hydraulic conductance (Kleaf ) across species, under both steady-state and dynamic conditions. However, empirical information about their coordination is scarce. In this study, Kleaf , gas exchange, stomatal kinetics, and leaf anatomy in 10 species including ferns, gymnosperms, and angiosperms were investigated to elucidate the correlation of H2 O and CO2 diffusion inside leaves under varying light conditions. Gas exchange, Kleaf , and anatomical traits varied widely across species. Under light-saturated conditions, the A, gs , gm , and Kleaf were strongly correlated across species. However, the response patterns of A, gs , gm , and Kleaf to varying light intensities were highly species dependent. Moreover, stomatal opening upon light exposure of dark-adapted leaves in the studied ferns and gymnosperms was generally faster than in the angiosperms; however, stomatal closing in light-adapted leaves after darkening was faster in angiosperms. The present results show that there is a large variability in the coordination of leaf hydraulic and gas exchange parameters across terrestrial plant species, as well as in their responses to changing light.

Leaf economics spectrum in rice: leaf anatomical, biochemical, and physiological trait trade-offs.

The leaf economics spectrum (LES) is an ecophysiological concept describing the trade-offs of leaf structural and physiological traits, and has been widely investigated on multiple scales. However, the effects of the breeding process on the LES in crops, as well as the mechanisms of the trait trade-offs underlying the LES, have not been thoroughly elucidated to date. In this study, a dataset that included leaf anatomical, biochemical, and functional traits was constructed to evaluate the trait covariations and trade-offs in domesticated species, namely rice (Oryza species). The slopes and intercepts of the major bivariate correlations of the leaf traits in rice were significantly different from the global LES dataset (Glopnet), which is based on multiple non-crop species in natural ecosystems, although the general patterns were similar. The photosynthetic traits responded differently to leaf structural and biochemical changes, and mesophyll conductance was the most sensitive to leaf nitrogen (N) status. A further analysis revealed that the relative limitation of mesophyll conductance declined with leaf N content; however, the limitation of the biochemistry increased relative to leaf N content. These findings indicate that breeding selection and high-resource agricultural environments lead crops to deviate from the leaf trait covariation in wild species, and future breeding to increase the photosynthesis of rice should primarily focus on improvement of the efficiency of photosynthetic enzymes.

Leaf hydraulic vulnerability triggers the decline in stomatal and mesophyll conductance during drought in rice.

Understanding the physiological responses of crops to drought is important for ensuring sustained crop productivity under climate change, which is expected to exacerbate the frequency and intensity of periods of drought. Drought responses involve multiple traits, and the correlations between these traits are poorly understood. Using a variety of techniques, we estimated the changes in gas exchange, leaf hydraulic conductance, and leaf turgor in rice (Oryza sativa) in response to both short- and long-term soil drought. We performed a photosynthetic limitation analysis to quantify the contributions of each limiting factor to the resultant overall decrease in photosynthesis during drought. Biomass, leaf area, and leaf width significantly decreased during the 2-week drought treatment, but leaf mass per area and leaf vein density increased. Light-saturated photosynthetic rate declined dramatically during soil drought, mainly due to the decrease in stomatal conductance (gs) and mesophyll conductance (gm). Stomatal modeling suggested that the decline in leaf hydraulic conductance explained most of the decrease in stomatal closure during the drought treatment, and may also trigger the drought-related decrease of stomatal conductance and mesophyll conductance. The results of this study provide insight into the regulation of carbon assimilation under drought conditions.

Diffusional conductance to CO2 is the key limitation to photosynthesis in salt-stressed leaves of rice (Oryza sativa).

Salinity significantly limits leaf photosynthesis but the factors causing the limitation in salt-stressed leaves remain unclear. In the present work, photosynthetic and biochemical traits were investigated in four rice genotypes under two NaCl concentration (0 and 150 mM) to assess the stomatal, mesophyll and biochemical contributions to reduced photosynthetic rate (A) in salt-stressed leaves. Our results indicated that salinity led to a decrease in A, leaf osmotic potential, electron transport rate and CO2 concentrations in the chloroplasts (Cc ) of rice leaves. Decreased A in salt-stressed leaves was mainly attributable to low Cc , which was determined by stomatal and mesophyll conductance. The increased stomatal limitation was mainly related to the low leaf osmotic potential caused by soil salinity. However, the increased mesophyll limitation in salt-stressed leaves was related to both osmotic stress and ion stress. These findings highlight the importance of considering mesophyll conductance when developing salinity-tolerant rice cultivars.

Leaf anatomy mediates coordination of leaf hydraulic conductance and mesophyll conductance to CO2 in Oryza.

Leaf hydraulic conductance (Kleaf ) and mesophyll conductance (gm ) both represent major constraints to photosynthetic rate (A), and previous studies have suggested that Kleaf and gm is correlated in leaves. However, there is scarce empirical information about their correlation. In this study, Kleaf , leaf hydraulic conductance inside xylem (Kx ), leaf hydraulic conductance outside xylem (Kox ), A, stomatal conductance (gs ), gm , and anatomical and structural leaf traits in 11 Oryza genotypes were investigated to elucidate the correlation of H2 O and CO2 diffusion inside leaves. All of the leaf functional and anatomical traits varied significantly among genotypes. Kleaf was not correlated with the maximum theoretical stomatal conductance calculated from stomatal dimensions (gsmax ), and neither gs nor gsmax were correlated with Kx . Moreover, Kox was linearly correlated with gm and both were closely related to mesophyll structural traits. These results suggest that Kleaf and gm are related to leaf anatomical and structural features, which may explain the mechanism for correlation between gm and Kleaf .