Leaf diffusional capacity largely contributes to the reduced photosynthesis in rice plants under magnesium deficiency.

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 (gs and gm, 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 gs and gm. Our results showed that reduction of leaf photosynthesis under Mg deficiency was mainly caused by the decreased gm, followed by reduced leaf biochemical capacity and gs, and leaf outside-xylem hydraulic conductance (Kox) was the major factor restricting gs 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 Kox and cell wall composition play important roles in regulating gs and gm, respectively, in rice plants under Mg shortages.

The physiology and anatomy study in leaves of Saragolla and Svevo wheat cultivars under polyethylene glycol-simulated drought stress.

Drought stress is increasing in frequency and severity with the progression of global climate change, thereby becoming a major concern for the growth and yield of crop plants, including wheat. The current challenge is to explore different ways of developing wheat genotypes with increased tolerance to drought. Therefore, we renewed interest in 'ancient' varieties expected to be more tolerant to environmental stress than the few elite varieties nowadays cultivated. This study aimed to perform comparative analysis of the effect of drought-simulating polyethylene glycol (PEG-6000) treatment on morpho-anatomical and physiological foliar traits of two durum wheat seedlings cultivars, Saragolla and Svevo, as these can reflect the adaptability of the plant to the environment to a certain extent. Results demonstrated that drought-stressed Saragolla leaves exhibited a greater reduction of stomatal density, a minor reduction of stomatal pore width, a wider xylem vessel mean area, greater compactness of mesophyll cells, a minor loss of chlorophyll content, as well as better photosynthetic and growth performance compared to the other variety. From such behaviours, we consider the Saragolla cultivar more drought tolerant than Svevo and therefore probably very promising for cultivation in dry areas.

Predicting photosynthetic pathway from anatomy using machine learning.

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.

The multiple roles of trichomes in two Croton species.

Trichomes are common in plants from dry environments, and despite their recognized role in protection and defense, little is known about their role as absorptive structures and in other aspects of leaf ecophysiology. We combine anatomical and ecophysiological data to evaluate how trichomes affect leaf gas exchange and water balance during drought. We studied two congeneric species with pubescent leaves which co-occur in Brazilian Caatinga: Croton blanchetianus (dense trichomes) and Croton adenocalyx (sparse trichomes). We found a novel foliar water uptake (FWU) pathway in C. blanchetianus composed of stellate trichomes and underlying epidermal cells and sclereids that interconnect the trichomes from both leaf surfaces. The water absorbed by these trichomes is redistributed laterally by pectin protuberances on mesophyll cell walls. This mechanism enables C. blanchetianus leaves to absorb water more efficiently than C. adenocalyx. Consequently, the exposure of C. blanchetianus to dew during drought improved its leaf gas exchange and water status more than C. adenocalyx. C. blanchetianus trichomes also increase their leaf capacity to reflect light and maintain lower temperatures during drought. Our results emphasize the multiple roles that trichomes might have on plant functioning and the importance of FWU for the ecophysiology of Caatinga plants during drought.

GOLDEN2-like1 is sufficient but not necessary for chloroplast biogenesis in mesophyll cells of C4 grasses.

Chloroplasts are the site of photosynthesis. In land plants, chloroplast biogenesis is regulated by a family of transcription factors named GOLDEN2-like (GLK). In C4 grasses, it has been hypothesized that genome duplication events led to the sub-functionalization of GLK paralogs (GLK1 and GLK2) to control chloroplast biogenesis in two distinct cell types: mesophyll and bundle sheath cells. Although previous characterization of golden2 (g2) mutants in maize has demonstrated a role for GLK2 paralogs in regulating chloroplast biogenesis in bundle sheath cells, the function of GLK1 has remained elusive. Here we show that, contrary to expectations, GLK1 is not required for chloroplast biogenesis in mesophyll cells of maize. Comparisons between maize and Setaria viridis, which represent two independent C4 origins within the Poales, further show that the role of GLK paralogs in controlling chloroplast biogenesis in mesophyll and bundle sheath cells differs between species. Despite these differences, complementation analysis revealed that GLK1 and GLK2 genes from maize are both sufficient to restore functional chloroplast development in mesophyll and bundle sheath cells of S. viridis mutants. Collectively our results suggest an evolutionary trajectory in C4 grasses whereby both orthologs retained the ability to induce chloroplast biogenesis but GLK2 adopted a more prominent developmental role, particularly in relation to chloroplast activation in bundle sheath cells.

Plasmodesmal connectivity in C4 Gynandropsis gynandra is induced by light and dependent on photosynthesis.

In leaves of C4 plants, the reactions of photosynthesis become restricted between two compartments. Typically, this allows accumulation of C4 acids in mesophyll (M) cells and subsequent decarboxylation in the bundle sheath (BS). In C4 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 C4 dicotyledons also show this enhanced plasmodesmal connectivity and so whether this is a general requirement for C4 photosynthesis is not clear. How M and BS cells in C4 leaves become highly connected is also not known. We investigated these questions using 3D- and 2D-electron microscopy on the C4 dicotyledon Gynandropsis gynandra as well as phylogenetically close C3 relatives. The M-BS interface of C4 G. gynandra showed higher plasmodesmal frequency compared with closely related C3 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 C4 G. gynandra.

Cellular insights of beech leaf disease reveal abnormal ectopic cell division of symptomatic interveinal leaf areas.

The beech leaf disease nematode, Litylenchus crenatae subsp. mccannii, is recognized as a newly emergent nematode species that causes beech leaf disease (BLD) in beech trees (Fagus spp.) in North America. Changes of leaf morphology before emergence from the bud induced by BLD can provoke dramatic effects on the leaf architecture and consequently to tree performance and development. The initial symptoms of BLD appear as dark green, interveinal banding patterns of the leaf. Despite the fast progression of this disease, the cellular mechanisms leading to the formation of such aberrant leaf phenotype remains totally unknown. To understand the cellular basis of BLD, we employed several types of microscopy to provide an exhaustive characterization of nematode-infected buds and leaves. Histological sections revealed a dramatic cell change composition of these nematode-infected tissues. Diseased bud scale cells were typically hypertrophied and showed a high variability of size. Moreover, while altered cell division had no influence on leaf organogenesis, induction of cell proliferation on young leaf primordia led to a dramatic change in cell layer architecture. Hyperplasia and hypertrophy of the different leaf cell layers, coupled with an abnormal proliferation of chloroplasts especially in the mesophyll cell layers, resulted in the typical interveinal leaf banding. These discrepancies in leaf cell structure were depicted by an abnormal rate of cellular division of the leaf interveinal areas infected by the nematode, promoting significant increase of cell size and leaf thickness. The formation of symptomatic BLD leaves is therefore orchestrated by distinct cellular processes, to enhance the value of these feeding sites and to improve their nutrition status for the nematode. Our findings thus uncover relevant cellular events and provide a structural framework to understand this important disease.

Condensed tannin accretions specifically distributed in mesophyll cells of non-salt secretor mangroves help in salt tolerance.

MAIN CONCLUSION: Auto-fluorescent condensed tannins specifically accumulated in mesophyll cells of non-salt secretor mangroves are involved in the compartmentation of Na+ and osmotic regulation, contributing to their salt tolerance. Salinity is a major abiotic stress affecting the distribution and growth of mangrove plants. The salt exclusion mechanism from salt secretor mangrove leaves is quite known; however, salt management strategies in non-salt secretor leaves remain unclear. In this study, we reported the auto-fluorescent inclusions (AFIs) specifically accumulated in mesophyll cells (MCs) of four non-salt secretor mangroves but absent in three salt secretors. The AFIs increased with the leaf development under natural condition, and applied NaCl concentrations applied in the lab. The AFIs in MCs were isolated and identified as condensed tannin accretions (CTAs) using the dye dimethyl-amino-cinnamaldehyde (DMACA), specific for condensed tannin (CT), both in situ leaf cross sections and in the purified AFIs. Fluorescence microscopy and transmission electron microscope (TEM) analysis indicated that the CTAs originated from the inflated chloroplasts. The CTAs had an obvious membrane and could induce changes in shape and fluorescence intensity in hypotonic and hypertonic NaCl solutions, suggesting CTAs might have osmotic regulation ability and play an important role in the osmotic regulation in MCs. The purified CTAs were labeled by the fluorescent sodium-binding benzofuran isophthalate acetoxymethyl ester (SBFI-AM), confirming they were involved in the compartmentation of excess Na+ in MCs. This study provided a new view on the salt resistance-associated strategies in mangroves.

Plasticity of mesophyll cell density and cell wall thickness and composition play a pivotal role in regulating plant growth and photosynthesis under shading in rapeseed.

BACKGROUND AND AIMS: Plasticity of leaf growth and photosynthesis is an important strategy of plants to adapt to shading stress; however, their strategy of leaf development to achieve a simultaneous increase in leaf area and photosynthesis under shading remains unknown. METHODS: In the present study, a pot experiment was conducted using three rapeseed genotypes of Huayouza 50 (HYZ50), Zhongshuang 11 (ZS11) and Huayouza 62 (HYZ62), and the responses of plant growth, leaf morphoanatomical traits, cell wall composition and photosynthesis to shading were investigated. KEY RESULTS: Shading significantly increased leaf area per plant (LAplant) in all genotypes, but the increase in HYZ62 was greater than that in HYZ50 and ZS11. The greater increment of LAplant in HYZ62 was related to the larger decrease in leaf mass per area (LMA) and leaf density (LD), which were in turn related to less densely packed mesophyll cells and thinner cell walls (Tcw). Moreover, shading significantly increased photosynthesis in HYZ62 but significantly decreased it in HYZ50. The enhanced photosynthesis in HYZ62 was related to increased mesophyll conductance (gm) due primarily to thinner cell walls. CONCLUSIONS: The data presented indicate that the different plasticity of mesophyll cell density, cell wall thickness and cell wall composition in response to shading can dramatically affect leaf growth and photosynthesis.

Metabolic modelling revealed source-sink interactions between four segments of Setaria viridis leaves.

As in most plants, during their growth from immature to mature stages, the leaves of Setaria viridis, a model C4 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 C4 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.

Altered cell wall hydroxycinnamate composition impacts leaf- and canopy-level CO2 uptake and water use in rice.

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.

Scalable Transfection of Maize Mesophyll Protoplasts.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here, we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

Unveiling the dark side of guard cell metabolism.

Evidence suggests that guard cells have higher rate of phosphoenolpyruvate carboxylase (PEPc)-mediated dark CO2 assimilation than mesophyll cells. However, it is unknown which metabolic pathways are activated following dark CO2 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 a13C-HCO3 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 CO2 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 13C-enrichment in sugars and metabolites associated to the TCA cycle. Sucrose was labelled in the dark, but light exposure increased the 13C-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 13C-enrichment in pyruvate, succinate and glutamate. Only one 13C was incorporated into malate and citrate in either dark or light conditions. Our results indicate that several metabolic pathways are redirected following PEPc-mediated CO2 assimilation in the dark, including gluconeogenesis and the TCA cycle. We further showed that the PEPc-mediated CO2 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.

Leaf hydraulic maze: Abscisic acid effects on bundle sheath, palisade, and spongy mesophyll conductance.

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.

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.

Unpredicted, rapid and unintended structural and functional changes occurred during early domestication of Silphium integrifolium, a perennial oilseed.

MAIN CONCLUSION: Selection for increased yield changed structure, physiology and overall resource-use strategy from conservative towards acquisitive leaves. Alternative criteria can be considered, to increase yield with less potentially negative traits. We compared the morphology, anatomy and physiology of wild and semi-domesticated (SD) accessions of Silphium integrifolium (Asteraceae), in multi-year experiments. We hypothesized that several cycles of selection for seed-yield would result in acquisitive leaves, including changes predicted by the leaf economic spectrum. Early-selection indirectly resulted in leaf structural and functional changes. Leaf anatomy changed, increasing mesophyll conductance and the size of xylem vessels and mesophyll cells increased. Leaves of SD plants were larger, heavier, with lower stomatal conductance, lower internal CO2 concentration, and lower resin concentration than those of wild types. Despite increased water use efficiency, SD plants transpired 25% more because their increase in leaf area. Unintended and undesired changes in functional plant traits could quickly become fixed during domestication, shortening the lifespan and increasing resource consumption of the crop as well as having consequences in the provision and regulation of ecosystem services.

CO2 mesophyll conductance regulated by light: a review.

MAIN CONCLUSION: Light quality and intensity regulate plant mesophyll conductance, which has played an essential role in photosynthesis by controlling leaf structural and biochemical properties. Mesophyll conductance (gm), a crucial physiological factor influencing the photosynthetic rate of leaves, is used to describe the resistance of CO2 from the sub-stomatal cavity into the chloroplast up to the carboxylation site. Leaf structural and biochemical components, as well as external environmental factors such as light, temperature, and water, all impact gm. As an essential factor of plant photosynthesis, light affects plant growth and development and plays a vital role in regulating gm as well as determining photosynthesis and yield. This review aimed to summarize the mechanisms of gm response to light. Both structural and biochemical perspectives were combined to reveal the effects of light quality and intensity on the gm, providing a guide for selecting the optimal conditions for intensifying photosynthesis in plants.

Physiological responses of plants to in vivo X-ray damage from X-ray fluorescence measurements: insights from anatomical, elemental, histochemical, and ultrastructural analyses.

X-ray fluorescence spectroscopy (XRF) is a powerful technique for the in vivo assessment of plant tissues. However, the potential X-ray exposure damages might affect the structure and elemental composition of living plant tissues, leading to artefacts in the recorded data. Herein, we exposed in vivo soybean (Glycine max (L.) Merrill) leaves to several X-ray doses through a polychromatic benchtop microprobe X-ray fluorescence spectrometer, modulating the photon flux density by adjusting either the beam size, current, or exposure time. Changes in the irradiated plant tissues' structure, ultrastructure, and physiology were investigated through light and transmission electron microscopy (TEM). Depending on X-ray exposure dose, decreased K and X-ray scattering intensities and increased Ca, P, and Mn signals on soybean leaves were recorded. Anatomical analysis indicated the necrosis of epidermal and mesophyll cells on the irradiated spots, where TEM images revealed the collapse of cytoplasm and cell wall breaking. Furthermore, the histochemical analysis detected the production of reactive oxygen species and the inhibition of chlorophyll autofluorescence in these areas. Under certain X-ray exposure conditions, e.g. high photon flux density and long exposure time, XRF measurements may affect the soybean leaves structures, elemental composition, and cellular ultrastructure, inducing programmed cell death. Our characterization shed light on the plant's responses to the X-ray-induced radiation damage and might help to establish proper X-ray radiation limits and novel strategies for in vivo benchtop-XRF analysis of vegetal materials.

Stomatal, mesophyll and biochemical limitations to photosynthesis and their relationship with leaf structure over an elevation gradient in two conifers.

Photosynthetic responses across complex elevational gradients provides insight into fundamental processes driving responses of plant growth and net primary production to environmental change. Gas exchange of needles and twig water potential were measured in two widespread coniferous tree species, Pinus contorta and Picea engelmannii, over an 800-m elevation gradient in southeastern Wyoming, USA. We hypothesized that limitations to photosynthesis imposed by mesophyll conductance (gm) would be greatest at the highest elevation sites due to higher leaf mass per area (LMA) and that estimations of maximum rate of carboxylation (Vcmax) without including gm would obscure elevational patterns of photosynthetic capacity. We found that gm decreased with elevation for P. contorta and remained constant for P. engelmannii, but in general, limitation to photosynthesis by gm was small. Indeed, estimations of Vcmax when including gm were equivalent to those estimated without including gm and no correlation was found between gm and LMA nor between gm and leaf N. Stomatal conductance (gs) and biochemical demand for CO2 were by far the most limiting processes to photosynthesis at all sites along the elevation gradient. Photosynthetic capacity (A) and gs were influenced strongly by differences in soil water availability across the elevation transect, while gm was less responsive to water availability. Based on our analysis, variation in gm plays only a minor role in driving patterns of photosynthesis in P. contorta and P. engelmannii across complex elevational gradients in dry, continental environments of the Rocky Mountains and accurate modeling of photosynthesis, growth and net primary production in these forests may not require detailed estimation of this trait value.