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Quantifying the effect of factors controlling CO 2 assimilation is crucial for understanding plant functions and developing strategies to improve productivity. Methods exist in numerous variants and produce various indicators, such as limitations, contributions, and sensitivity, often causing confusion. Simplifications and common mistakes lead to overrating the importance of diffusion-whether across stomata or the mesophyll. This work develops a consistent set of definitions that integrates all previous methods, offering a generalised framework for quantifying restrictions. Ten worked examples are provided in a free downloadable spreadsheet, demonstrating the simplicity and applicability to a wide range of questions., Competing Interests: Declarations. Conflict of interest: The author declares no competing interests., (© 2025. The Author(s).)

Introduction: The identification of the physiological processes limiting carbon assimilation under water stress is crucial for improving model predictions and selecting drought-tolerant varieties. However, the influence of soil water availability on photosynthesis-limiting processes is still not fully understood. This study aimed to investigate the origins of photosynthesis limitations on potato ( Solanum tuberosum ) during a field drought experiment., Methods: Gas exchange and chlorophyll fluorescence measurements were performed at the leaf level to determine the response of photosynthesis-limiting factors to the decrease in the relative extractable water (REW) in the soil., Results: Drought induced a two-stage response with first a restriction of CO 2 diffusion to chloroplasts induced by stomatal closure and a decrease in mesophyll conductance, followed by a decrease in photosynthetic capacities under severe soil water restrictions. Limitation analysis equations were revisited and showed that mesophyll conductance was the most important constraint on carbon and water exchanges regardless of soil water conditions., Discussion: We provide a calibration of the response of stomatal and non-stomatal factors to REW to improve the representation of drought effects in models. These results emphasize the need to revisit the partitioning methods to unravel the physiological controls on photosynthesis and stomatal conductance under water stress., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Beauclaire, Vanden Brande and Longdoz.)

Plants have been affected by water stress ever since they settled on dry land. In severe and persisting drought, plant leaves are wilting. However, a documentation at the anatomical level of the minute changes that occur before wilting is challenging. On the other hand, understanding the anatomical alteration in plant leaves with respect to water stress provides a stronger basis to study molecular and submolecular processes through which plants enhance drought tolerance. In this work, we applied an affordable method to visualise mesophyll layers of Arabidopsis thaliana cell lines without preparation steps that would alter the volume of the cells. We rapidly plunge-froze the leaves in liquid nitrogen, cut them while in the N 2 bath, and immediately imaged the mesophyll cross sections in a scanning electron microscope. We applied a reduction of watering from 60 to 40 to 20 mL per day and investigated two time points, 7 and 12 days, respectively. Interestingly, the overall thickness of leaves increased in water stress conditions. Our results showed that the palisade and spongy layers behaved differently under varying watering regimes. Moreover, the results showed that this method can be used to image leaf sections after drought stress without the risk of artefacts or swelling caused by contact to liquids as during chemical fixation., (© 2024 The Author(s). Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society.)

Characterizing physiological and anatomical changes that underlie rapid evolution following climatic perturbation can broaden our understanding of how climate change is affecting biodiversity. It can also provide evidence of cryptic adaptation despite stasis at higher levels of biological organization. Here, we compared evolutionary changes in populations of Mimulus cardinalis from historically different climates in the north and south of the species' range following an exceptional drought. We grew seeds produced from predrought ancestral plants alongside peak-drought descendants in a common glasshouse and exposed them to wet and dry conditions. Before the drought, northern ancestral populations expressed traits contributing to drought escape, while southern ancestral populations expressed drought avoidance. Following the drought, both regions evolved to reduce water loss and maintain photosynthesis in dry treatments (drought avoidance), but via different anatomical alterations in stomata, trichomes, and palisade mesophyll. Additionally, southern populations lost the ability to take advantage of wet conditions. These results reveal rapid evolution towards drought avoidance at an anatomical level following an exceptional drought, but suggest that differences in the mechanisms between regions incur different trade-offs. This sheds light on the importance of characterizing underlying mechanisms for downstream life-history and macromorphological traits., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

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.)

Background: Leaf variegation is an intriguing phenomenon observed in many plant species. However, questions remain on its mechanisms causing patterns of different colours. In this study, we describe a tomato plant detected in an M 2 population of EMS mutagenised seeds, showing variegated leaves with sectors of dark green (DG), medium green (MG), light green (LG) hues, and white (WH). Cells and tissues of these classes, along with wild-type tomato plants, were studied by light, fluorescence, and transmission electron microscopy. We also measured chlorophyll a/b and carotene and quantified the variegation patterns with a machine-learning image analysis tool. We compared the genomes of pooled plants with wild-type-like and mutant phenotypes in a segregating F 2 population to reveal candidate genes responsible for the variegation., Results: A genetic test demonstrated a recessive nuclear mutation caused the variegated phenotype. Cross-sections displayed distinct anatomy of four-leaf phenotypes, suggesting a stepwise mesophyll degradation. DG sectors showed large spongy layers, MG presented intercellular spaces in palisade layers, and LG displayed deformed palisade cells. Electron photomicrographs of those mesophyll cells demonstrated a gradual breakdown of the chloroplasts. Chlorophyll a/b and carotene were proportionally reduced in the sectors with reduced green pigments, whereas white sectors have hardly any of these pigments. The colour segmentation system based on machine-learning image analysis was able to convert leaf variegation patterns into binary images for quantitative measurements. The bulk segregant analysis of pooled wild-type-like and variegated progeny enabled the identification of SNP and InDels via bioinformatic analysis. The mutation mapping bioinformatic pipeline revealed a region with three candidate genes in chromosome 4, of which the FtsH-like protein precursor (LOC100037730) carries an SNP that we consider the causal variegated phenotype mutation. Phylogenetic analysis shows the candidate is evolutionary closest to the Arabidopsis VAR1. The synonymous mutation created by the SNP generated a miRNA binding site, potentially disrupting the photoprotection mechanism and thylakoid development, resulting in leaf variegation., Conclusion: We described the histology, anatomy, physiology, and image analysis of four classes of cell layers and chloroplast degradation in a tomato plant with a variegated phenotype. The genomics and bioinformatics pipeline revealed a VAR1-related FtsH mutant, the first of its kind in tomato variegation phenotypes. The miRNA binding site of the mutated SNP opens the way to future studies on its epigenetic mechanism underlying the variegation., (© 2024. The Author(s).)

Stomatal closure under high VPD L (leaf to air vapour pressure deficit) is a primary means by which plants prevent large excursions in transpiration rate and leaf water potential (Ψ leaf ) that could lead to tissue damage. Yet, the drivers of this response remain controversial. Changes in Ψ leaf appear to drive stomatal VPD L response, but many argue that dynamic changes in soil-to-leaf hydraulic conductance (K s-l ) make an important contribution to this response pathway, even in well-hydrated soils. Here, we examined whether the regulation of whole plant stomatal conductance (g c ) in response to typical changes in daytime VPD L is influenced by dynamic changes in K s-l . We use well-watered plants of two species with contrasting ecological and physiological features: the herbaceous Arabidopsis thaliana (ecotype Columbia-0) and the dry forest conifer Callitris rhomboidea. The dynamics of K s-l and g c were continuously monitored by combining concurrent in situ measurements of Ψ leaf using an open optical dendrometer and whole plant transpiration using a balance. Large changes in VPD L were imposed to induce stomatal closure and observe the impact on K s-l . In both species, g c was observed to decline substantially as VPD L increased, while K s-l remained stable. Our finding suggests that stomatal regulation of transpiration is not contingent on a decrease in K s-l . Static K s-l provides a much simpler explanation for transpiration control in hydrated plants and enables simplified modelling and new methods for monitoring plant water use in the field., (© 2024 The Authors New Phytologist © 2024 New Phytologist Foundation.)

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.)

The mesophyll cells of grass leaves, such as rice, are traditionally viewed as displaying a relatively uniform pattern, in contrast to the clear distinctions of palisade and spongy layers in typical eudicot leaves. This quantitative analysis of mesophyll cell size and shape in rice leaves reveals that there is an inherent pattern in which cells in the middle layer of the mesophyll are larger and less circular and have a distinct orientation of their long axis compared to mesophyll cells in other layers. Moreover, this pattern was observed in a range of rice cultivars and species. The significance of this pattern with relation to potential photosynthetic function and the implication of the widespread use of middle layer mesophyll cells as typical of the rice leaf have been investigated and discussed., Competing Interests: No conflict of interest declared., (© 2023 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

The objective of this study was to evaluate the relationship between intercellular spaces and leaf gas exchange and the effect of total intercellular space on the growth of maize and sorghum under water restriction. The experiments were conducted in a greenhouse in a 2 × 3 factorial arrangement (two plant types and three water conditions: field capacity (FC = 100%), 75%FC, and 50%FC) with 10 replicates. The lack of water was a limiting factor for maize because it showed reductions in leaf area, leaf thickness, biomass, and gas exchange parameters, while sorghum remained unchanged, maintaining its water-use efficiency. This maintenance was correlated with the growth of intercellular spaces in sorghum leaves because the increased internal volume led to better CO 2 control and prevented excessive water loss under drought stress. In addition, sorghum had more stomata than maize. These characteristics contributed to the drought tolerance of sorghum, while maize could not make the same adjustments. Therefore, changes in intercellular spaces promoted adjustments to avoid water loss and may have improved CO 2 diffusion, characteristics that are important for drought-tolerant plants., (© 2023. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Little is known about long-distance mesophyll-driven signals that regulate stomatal conductance. Soluble and/or vapor-phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance in Arabidopsis thaliana by CO 2 /abscisic acid (ABA) was examined. We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll-dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene-signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO 2 ]-shifts. According to our research, higher [CO 2 ] causes Arabidopsis rosettes to produce more ethylene. An ACC-synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO 2 -induced stomatal movements. Ethylene-insensitive receptor (gain-of-function), etr1-1 and etr2-1, and signaling, ein2-5 and ein2-1, mutants showed intact stomatal responses to [CO 2 ]-shifts, whereas loss-of-function ethylene receptor mutants, including etr2-3;ein4-4;ers2-3, etr1-6;etr2-3 and etr1-6, showed markedly accelerated stomatal responses to [CO 2 ]-shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC-synthase octuple mutant and accelerated stomatal responses in the etr1-6;etr2-3, and etr1-6, but not in the etr2-3;ein4-4;ers2-3 mutants. These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO 2 and ABA., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

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.)

We aimed to efficiently enhance plant Hg(II) tolerance by the transgenic approach utilizing a bacterial mercury transporter MerC, an Arabidopsis mesophyll specific promoter pRBCS1A , and a vacuolar membrane targeting syntaxin AtVAM3/SYP22. We generated two independent homozygous Arabidopsis pRBCS1A-TCV lines expressing mT-Sapphire-MerC-AtVAM3 under the control of pRBCS1A . Quantitative RT-PCR showed that the transgene was expressed specifically in shoots of pRBCS1A-TCV lines. Confocal analyses further demonstrated the leaf mesophyll specific expression of mT-Sapphire-MerC-AtVAM3. Confocal observation of the protoplast derived from the F1 plants of the pRBCS1A-TCV line and the tonoplast marker line p35S-GFP-δTIP showed the tonoplast colocalization of mT-Sapphire-MerC-AtVAM3 and GFP-δTIP. These results clearly demonstrated that mT-Sapphire-MerC-AtVAM3 expression in Arabidopsis is spatially regulated as designed at the transcript and the membrane trafficking levels. We then examined the Hg(II) tolerance of the pRBCS1A-TCV lines as well as the p35S-driven MerC-AtVAM3 expressing line p35S-CV under the various Hg(II) stress conditions. Short-term (12 d) Hg(II) treatment indicated the enhanced Hg(II) tolerance of both pRBCS1A-TCV and p35S-CV lines. The longer (3 weeks) Hg(II) treatment highlighted the better shoot growth of the transgenic plants compared to the wild-type Col-0 and the pRBCS1A-TCV lines were more tolerant to Hg(II) stress than the p35S-CV line. These results suggest that mesophyll-specific expression of MerC-AtVAM3 is sufficient or even better to enhance the Arabidopsis Hg(II) tolerance. The Hg accumulation in roots and shoots did not differ between the wild-type Col-0 and the MerC-AtVAM3 expressing plants, suggesting that the boosted Hg(II) tolerance of the transgenic lines would be attributed to vacuolar Hg-sequestration by the tonoplast-localized MerC. Further perspectives of the MerC-based plant engineering are also discussed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Uraguchi, Ohshiro, Okuda, Kawakami, Yoneyama, Tsuchiya, Nakamura, Takanezawa and Kiyono.)

Drought is a critical and increasingly common abiotic factor that has impacts on plant structures and functioning and is a challenge for the successful management of forest ecosystems. Here, we test the shifts in leaf morpho-anatomical or hydraulic traits and plant growth above ground caused by drought. A factorial experiment was conducted with two gymnosperms ( Larix gmelinii and Pinus koraiensis ) and two angiosperms ( Fraxinus mandshurica and Tilia amurensis ), tree species grown under three varying drought intensities in NE China. Considering all the species studied, the plant height (PH), root collar diameter (RCD), and plant biomass (PB) were significantly decreased by drought. The leaf thickness (LT) increased, while the leaf area (LA) decreased with drought intensity. In the gymnosperms, the mesophyll thickness (MT) increased, and the resin duct decreased, while in the angiosperms the palisade mesophyll thickness (PMT), the spongy mesophyll thickness (SMT), and the abaxial (ABE) and adaxial epidermis (ADE) thickness were increased by drought. The correlation analysis revealed that P. koraiensis and F. mandshurica had the higher RMF and total plant biomass, but the least LMF, suggesting drought tolerance. In contrast, the L. gmelinii had the least RMF and higher LMF, suggesting vulnerability to drought. Similarly, T. amurensis had the higher leaf size, which increased the evaporative demand and depleted the soil water quickly relative to the other species. The interrelation among the morpho-anatomical leaf traits was equally affected by drought across all the studied species, suggesting that there is no clear evidence to differentiate the taxa based on drought resistance vs. drought tolerance. Thus, we have identified some easily measurable traits (i.e., LMF, RMF, and PB) which evidenced the seedling's ability to cope with drought and which therefore could be used as proxies in the selection of drought tolerant species for reforestation in the temperate forest.

Competition assays are a simple phenotyping strategy that confront two bacterial strains to evaluate their relative fitness. Because they are more accurate than single-strain growth assays, competition assays can be used to highlight slight differences that would not otherwise be detectable. In the frame of host-pathogens interactions, they can be very useful to study the contribution of individual bacterial genes to bacterial fitness and lead to the identification of new adaptive traits. Here, we describe how to perform such competition assays by taking the example of the model phytopathogenic bacterium Xanthomonas campestris pv. campestris during infection of the mesophyll of its cauliflower host. This phenotypic assay is based on the use of a Competitive Index (CI) that compares the relative abundance of co-inoculated strains before and after inoculation. Since multiplication is a direct proxy for bacterial fitness, the evolution of the ratio between both strains in the mixed population is a direct way to assess differences in fitness in a given environment. In this protocol, we exploit the blue staining of GUS-expressing bacteria to count blue vs . white colonies on plates and estimate the competitiveness of the strains of interest in plant mesophyll., Competing Interests: Competing interestsThere are no competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

Many plant leaves have two layers of photosynthetic tissue: the palisade and spongy mesophyll. Whereas palisade mesophyll consists of tightly packed columnar cells, the structure of spongy mesophyll is not well characterized and often treated as a random assemblage of irregularly shaped cells. Using micro-computed tomography imaging, topological analysis, and a comparative physiological framework, we examined the structure of the spongy mesophyll in 40 species from 30 genera with laminar leaves and reticulate venation. A spectrum of spongy mesophyll diversity encompassed two dominant phenotypes: first, an ordered, honeycomblike tissue structure that emerged from the spatial coordination of multilobed cells, conforming to the physical principles of Euler's law; and second, a less-ordered, isotropic network of cells. Phenotypic variation was associated with transitions in cell size, cell packing density, mesophyll surface-area-to-volume ratio, vein density, and maximum photosynthetic rate. These results show that simple principles may govern the organization and scaling of the spongy mesophyll in many plants and demonstrate the presence of structural patterns associated with leaf function. This improved understanding of mesophyll anatomy provides new opportunities for spatially explicit analyses of leaf development, physiology, and biomechanics., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Iron (Fe) is an essential transition metal. Based on its redox-active nature under biological conditions, various Fe compounds serve as cofactors in redox enzymes. In plants, the photosynthetic machinery has the highest demand for Fe. In consequence, the delivery and incorporation of Fe into cofactors of the photosynthetic apparatus is the focus of Fe metabolism in leaves. Disturbance of foliar Fe homeostasis leads to impaired biosynthesis of chlorophylls and composition of the photosynthetic machinery. Nevertheless, mitochondrial function also has a significant demand for Fe. The proper incorporation of Fe into proteins and cofactors as well as a balanced intracellular Fe status in leaf cells require the ability to sense Fe, but may also rely on indirect signals that report on the physiological processes connected to Fe homeostasis. Although multiple pieces of information have been gained on Fe signalling in roots, the regulation of Fe status in leaves has not yet been clarified in detail. In this review, we give an overview on current knowledge of foliar Fe homeostasis, from the chemical forms to the allocation and sensing of Fe in leaves., (© 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.)

Background: Blue Native polyacrylamide gel electrophoresis (BN PAGE) followed by denaturing PAGE is a widely used, convenient and time efficient method to separate thylakoid complexes and study their composition, abundance, and interactions. Previous analyses unravelled multiple monomeric and dimeric/oligomeric thylakoid complexes but, in certain cases, the separation of complexes was not proper. Particularly, the resolution of super- and megacomplexes, which provides important information on functional interactions, still remained challenging., Results: Using a detergent mixture of 1% (w/V) n-dodecyl-β-D-maltoside plus 1% (w/V) digitonin for solubilisation and 4.3-8% gel gradients for separation as methodological improvements in BN PAGE, several large photosystem (PS) I containing bands were detected. According to BN(/BN)/SDS PAGE and mass spectrometry analyses, these PSI bands proved to be PSI-NADH dehydrogenase-like megacomplexes more discernible in maize bundle sheath thylakoids, and PSI complexes with different light-harvesting complex (LHC) complements (PSI-LHCII, PSI-LHCII*) more abundant in mesophyll thylakoids of lincomycin treated maize. For quantitative determination of the complexes and their comparison across taxa and physiological conditions, sample volumes applicable to the gel, correct baseline determination of the densitograms, evaluation methods to resolve complexes running together, calculation of their absolute/relative amounts and distribution among their different forms are proposed., Conclusions: Here we report our experience in Blue/Clear-Native polyacrylamide gel electrophoretic separation of thylakoid complexes, their identification, quantitative determination and comparison in different samples. The applied conditions represent a powerful methodology for the analysis of thylakoid mega- and supercomplexes., (© 2022. The Author(s).)

Vegetative storage proteins (VSPs) are known to serve as nitrogen reserves in many dicot plants but remain undiscovered in grasses, most widely grown group of crops globally. We identified and characterized a VSP in maize and demonstrated that its overexpression improved drought tolerance. Nitrogen supplementation selectively induced a mesophyll lipoxygenase (ZmLOX6), which was targeted to chloroplasts by a novel N-terminal transit peptide of 62 amino acids. When ectopically expressed under the control of various tissue-specific promoters, it accumulated to a fivefold higher level upon expression in the mesophyll cells than the wild-type plants. Constitutive expression or targeted expression specifically to the bundle sheath cells increased its accumulation by less than twofold. The overexpressed ZmLOX6 was remobilized from the leaves like other major proteins during grain development. Evaluated in the field over locations and years, transgenic hybrids overexpressing ZmLOX6 in the mesophyll cells significantly outyielded nontransgenic sibs under managed drought stress imposed at flowering. Additional storage of nitrogen as a VSP in maize leaves ameliorated the effect of drought on grain yield., (© 2021 Pioneer Hi-Bred International, Inc (Corteva Agriscience). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

C 4 plants partition photosynthesis enzymes between the bundle sheath (BS) and the mesophyll (M) cells for the better delivery of CO 2 to RuBisCO and to reduce photorespiration. To better understand how C 4 photosynthesis is regulated at the transcriptional level, we performed RNA-seq, ATAC-seq, ChIP-seq and Bisulfite-seq (BS-seq) on BS and M cells isolated from maize leaves. By integrating differentially expressed genes with chromatin features, we found that chromatin accessibility coordinates with epigenetic features, especially H3K27me3 modification and CHH methylation, to regulate cell type-preferentially enriched gene expression. Not only the chromatin-accessible regions (ACRs) proximal to the genes (pACRs) but also the distal ACRs (dACRs) are determinants of cell type-preferentially enriched expression. We further identified cell type-preferentially enriched motifs, e.g. AAAG for BS cells and TGACC/T for M cells, and determined their corresponding transcription factors: DOFs and WRKYs. The complex interaction between cis and trans factors in the preferential expression of C 4 genes was also observed. Interestingly, cell type-preferentially enriched gene expression can be fine-tuned by the coordination of multiple chromatin features. Such coordination may be critical in ensuring the cell type-specific function of key C 4 genes. Based on the observed cell type-preferentially enriched expression pattern and coordinated chromatin features, we predicted a set of functionally unknown genes, e.g. Zm00001d042050 and Zm00001d040659, to be potential key C 4 genes. Our findings provide deep insight into the architectures associated with C 4 gene expression and could serve as a valuable resource to further identify the regulatory mechanisms present in C 4 species., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant organs are built of different cell types, characterized by specific transcription programs and metabolic profiles. The possibility of isolation of such cell types to perform differential transcriptomic, proteomic and metabolomic analyses is highly important to understand many aspects of plant physiology, namely, the structure and regulation of economically valuable specialized metabolic pathways. Here, we describe the isolation of idioblast leaf protoplasts of the medicinal plant Catharanthus roseus by fluorescence-activated cell sorting, taking advantage of the differential autofluorescence properties of those specialized cells., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Protoplast-based transient gene expression platforms can be used to study a range of questions concerning gene regulation. Crucial to the success of these studies is the isolation of large quantities of healthy protoplasts from the tissue of interest. Herein, we describe protocols for isolating and transfecting maize mesophyll protoplasts for gene expression studies. The isolation protocol yields approximately 1.8-1.9 × 10 7 protoplasts with 80-90% viability from 6 g of etiolated leaf tissue, and the polyethylene glycol-mediated transfection protocol results in 55-58% transfection efficiency. The transfection protocol describes the use of a dual-expression vector that carries the coding sequence for two fluorescent proteins (FPs), one driven by a constitutive promoter for normalization for transfection efficiency and the other driven by the construct of interest. The use of a dual-FP expression vector eliminates the need for co-transfection and separate steps for enzymatic/substrate processing as required for luciferase-based assays. These protocols have been tested on leaf tissue from the maize genotypes B73 and PHR03 and, as written, can be completed in 24 h., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O 3 triggered a drop in whole-plant CO 2 uptake. Within this early phase, O 3 pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O 3 induced cell death, systemic Ca 2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca 2+ ) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

The responses of stomatal aperture to light intensity and CO 2 concentration were studied in both Vicia faba (C 3 ) and Kalanchoë fedtschenkoi (Crassulacean acid metabolism; CAM), in material sampled from both light and dark periods. Direct comparison was made between intact leaf segments, epidermises grafted onto exposed mesophyll, and isolated epidermal peels, including transplantations between species and between diel periods. We reported the stomatal opening in response to darkness in isolated CAM peels from the light period, but not from the dark. Furthermore, we showed that C 3 mesophyll has stimulated CAM stomata in transplanted peels to behave as C 3 in response to light and CO 2 . By using peels and mesophyll from plants sampled in the dark and the light period, we provided clear evidence that CAM stomata behaved differently from C 3 . This might be linked to stored metabolites/ions and signalling pathway components within the guard cells, and/or a mesophyll-derived signal. Overall, our results provided evidence for both the involvement of guard cell metabolism and mesophyll signals in stomatal responses in both C 3 and CAM species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Santos, Davey, Hofmann, Borland, Hartwell and Lawson.)

Petals are typified by their conical epidermal cells that play a predominant role for the attraction and interaction with pollinators. However, cell identities in the petal can be very diverse, with different cell types in subdomains of the petal, in different cell layers, and depending on their adaxial-abaxial or proximo-distal position in the petal. In this mini-review, we give an overview of the main cell types that can be found in the petal and describe some of their functions. We review what is known about the genetic basis for the establishment of these cellular identities and their possible relation with petal identity and polarity specifiers expressed earlier during petal development, in an attempt to bridge the gap between organ identity and cell identity in the petal., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Cavallini-Speisser, Morel and Monniaux.)

Leaves have evolved to effectively harvest light, and, in parallel, to balance photosynthetic CO 2 assimilation with water losses. At times, leaves must operate under light limiting conditions while at other instances (temporally distant or even within seconds), the same leaves must modulate light capture to avoid photoinhibition and achieve a uniform internal light gradient. The light-harvesting capacity and the photosynthetic performance of a given leaf are both determined by the organization and the properties of its structural elements, with some of these having evolved as adaptations to stressful environments. In this respect, the present review focuses on the optical roles of particular leaf structural elements (the light capture module) while integrating their involvement in other important functional modules. Superficial leaf tissues (epidermis including cuticle) and structures (epidermal appendages such as trichomes) play a crucial role against light interception. The epidermis, together with the cuticle, behaves as a reflector, as a selective UV filter and, in some cases, each epidermal cell acts as a lens focusing light to the interior. Non glandular trichomes reflect a considerable part of the solar radiation and absorb mainly in the UV spectral band. Mesophyll photosynthetic tissues and biominerals are involved in the efficient propagation of light within the mesophyll. Bundle sheath extensions and sclereids transfer light to internal layers of the mesophyll, particularly important in thick and compact leaves or in leaves with a flutter habit. All of the aforementioned structural elements have been typically optimized during evolution for multiple functions, thus offering adaptive advantages in challenging environments. Hence, each particular leaf design incorporates suitable optical traits advantageously and cost-effectively with the other fundamental functions of the leaf.

Understanding the mechanisms involved in host plant resistance opens the way for improved resistance breeding programs by using the traits involved as markers. Pest management is a major problem in cultivation of ornamentals. Gladiolus ( Gladiolus hybridus L.) is an economically important ornamental in the Netherlands. Gladiolus is especially sensitive to attack by western flower thrips ( Frankliniella occidentalis (Pergande) (Thysanoptera:Thripidae)). The objective of this study was, therefore, to investigate morphological and chemical markers for resistance breeding to western flower thrips in Gladiolus varieties. We measured thrips damage of 14 Gladiolus varieties in a whole-plant thrips bioassay and related this to morphological traits with a focus on papillae density. Moreover, we studied chemical host plant resistance to using an eco-metabolomic approach comparing the 1 H NMR profiles of thrips resistant and susceptible varieties representing a broad range of papillae densities. Thrips damage varied strongly among varieties: the most susceptible variety showed 130 times more damage than the most resistant one. Varieties with low thrips damage had shorter mesophylls and epidermal cells, as well as a higher density of epicuticular papillae. All three traits related to thrips damage were highly correlated with each other. We observed a number of metabolites related to resistance against thrips: two unidentified triterpenoid saponins and the amino acids alanine and threonine. All these compounds were highly correlated amongst each other as well as to the density of papillae. These correlations suggest that papillae are involved in resistance to thrips by producing and/or storing compounds causing thrips resistance. Although it is not possible to distinguish the individual effects of morphological and chemical traits statistically, our results show that papillae density is an easy marker in Gladiolus-breeding programs targeted at increased resistance to thrips.

Leaf function is influenced by leaf structure, which is itself related not only to the spatial arrangement of constituent mesophyll cells, but also their size and shape. In this study, we used confocal microscopy to image leaves of Triticum genotypes varying in ploidy level to extract 3D information on individual mesophyll cell size and geometry. Combined with X-ray Computed Tomography and gas exchange analysis, the effect of changes in wheat mesophyll cell geometry upon leaf structure and function were investigated. Mesophyll cell size and shape were found to have changed during the course of wheat evolution. An unexpected linear relationship between mesophyll cell surface area and volume was discovered, suggesting anisotropic scaling of mesophyll cell geometry with increasing ploidy. Altered mesophyll cell size and shape were demonstrated to be associated with changes in mesophyll tissue architecture. Under experimental growth conditions, CO 2 assimilation did not vary with ploidy, but stomatal conductance was lower in hexaploid plants, conferring a greater instantaneous water-use efficiency. We propose that as wheat mesophyll cells have become larger with increased ploidy, this has been accompanied by changes in cell geometry and packing which limit water loss while maintaining carbon assimilation., Competing Interests: The authors are not aware of any conflict of interest., (© 2021 The Authors. Plant Direct published by American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd.)

The partial or complete loss of chlorophylls, or albinism, is a rare phenomenon in plants. In the present study, we provide the first report of the occurrence in albino Delonix regia seedlings and describe the morpho-physiological changes associated with albinism. Wild-type (WT) and albino seedlings were characterized. Leaflets samples were processed following common procedures for analysis with light, scanning and transmission electron microscopy. The chlorophyll a fluorescence parameters and the carbohydrate, lipid and soluble protein content were also determined in leaf and cotyledon samples of both albino and WT seedlings. Albino seedlings showed reduced growth. They also had lower chlorophyll and protein content in foliar tissues than WT seedlings, in addition to lower concentrations of lipids and carbohydrates stored in cotyledons. The chloroplasts of albino seedlings were poorly developed, with an undefined internal membrane system and the presence of plastoglobules. Wild-type seedlings had a uniseriate and hypoestomatic epidermis. The mesophyll was dorsiventral, consisting of a layer of palisade parenchyma and two to four layers of spongy parenchyma. In albino seedlings, the spongy parenchyma was compact, with few intercellular spaces, and the thickness of the mesophyll was larger, resulting in increased thickness of the leaf blade. Albino seedlings had higher stomatal density and number of pavement cells, although the stomata had smaller dimensions. In addition to the partial loss of chlorophylls, albino D. regia showed changes at physiological and structural levels, demonstrating the crucial nature of photosynthetic pigments during the development and differentiation of plant leaf tissues/cells., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Plants transmit their experiences of environmental conditions to their progeny through epigenetic inheritance, improving their progeny's fitness under prevailing conditions. Though ABA is known to regulate epigenetic-modification genes, no strong phenotypic link between those genes and intergenerational "memory" has been shown. Previously, we demonstrated that mesophyll insensitivity to ABA (FBPase::abi1-1{fa} transgenic plants) results in a range of developmental phenotypes, including early growth vigor and early flowering (i.e., stress-escape behavior). Here, we show that null plants, used as controls (segregates of FBPase::abi1 that are homozygote descendants of a heterozygous transgenic plant, but do not contain the transformed abi1-1 gene) phenotypically resembled their FBPase::abi1-1 parents. However, in germination and early seedling development assays, null segregants resembled WT plants. These FBPase::abi1-1 null segregants mesophyll-related phenotypes were reproducible and stable for at least three generations. These results suggest that the heritability of stress response is linked to ABA's epigenetic regulatory effect through ABI1 and mesophyll-related traits. The discrepancy between the epigenetic heritability of seed and mesophyll-related traits is an example of the complexity of epigenetic regulation, which is both gene and process-specific, and may be attributed to the fine-tuning of tradeoffs between flowering time, growth rate and levels of risk that allow annual plants to optimize their fitness in uncertain environments., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background and Aims: It is commonly accepted that the leaf of a crassulacean acid metabolism (CAM) plant is thick, with large mesophyll cells and vacuoles that can accommodate the malic acid produced during the night. The link between mesophyll characteristics and CAM mode, whether obligate or C3/CAM, was evaluated., Methods: Published values of the carbon isotopic ratio (δ 13C) as an indicator of CAM, leaf thickness, leaf micrographs and other evidence of CAM operation were used to correlate cell density, cell area, the proportion of intercellular space in the mesophyll (IAS) and the length of cell wall facing the intercellular air spaces (Lmes/A) with CAM mode., Key Results: Based on 81 species and relatively unrelated families (15) belonging to nine orders, neither leaf thickness nor mesophyll traits helped explain the degree of CAM expression. A strong correlation was found between leaf thickness and δ 13C in some species of Crassulaceae and between leaf thickness and nocturnal acid accumulation in a few obligate CAM species of Bromeliaceae but, when all 81 species were pooled together, no significant changes with δ 13C were observed in cell density, cell area, IAS or Lmes/A., Conclusions: An influence of phylogeny on leaf anatomy was evidenced in a few cases but this precluded generalization for widely separate taxa containing CAM species. The possible relationships between leaf anatomy and CAM mode should be interpreted cautiously., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The hydraulic implications of stomatal positioning across leaf surfaces and the impact on internal water flow through amphistomatic leaves are not currently well understood. Amphistomaty potentially provides hydraulic efficiencies if the majority of hydraulic resistance in the leaf exists outside the xylem in the mesophyll. Such a scenario would mean that the same xylem network could equally supply a hypostomatic or amphistomatic leaf. Here we examine leaves of Helianthus annuus to determine whether amphistomaty in this species is associated with higher hydraulic efficiency compared with hypostomatic leaves. We identified asymmetry in the positioning of minor veins which were significantly closer to the abaxial than the adaxial leaf surface, combined with lower Kleaf when transpiration was driven through the adaxial rather than the abaxial surface. We also identified a degree of coordination in stomatal behaviour driven by leaf hydraulics, where the hydraulic conditions experienced by an individual leaf surface affected the stomatal behaviour on the opposite surface. We found no advantage to amphistomaty based on efficiencies in construction costs of the venous system, represented by vein density:stomatal density, only limited hydraulic independence between leaf surfaces. These results suggest that amphistomaty does not substantially increase whole-leaf hydraulic efficiency., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

An indoor simulation experiment was conducted to explore the effects of cuticular wax content and specific leaf area (SLA) on accumulation and distribution of PAHs in different tissues of wheat leaf. Three levels (0, 1.25, 6.0 mg L -1 ) of mixed solution of five PAHs (Σ 5 PAHs) including phenanthrene (PHE), anthracene (ANT), pyrene (PYR), benz[a]anthracene (BaA), and benzo[a]pyrene (BaP) were sprayed on leaves of seven varieties of winter wheat for every other day during 20 consecutive days. Shoot and root biomass of wheat under 6.0 mg L -1 Σ 5 PAHs exposure were 5.87 and 0.33 g, which were significantly (p < 0.05) lower than those (7.14 and 0.65 g) without spraying Σ 5 PAHs solution, respectively. Elevated Σ 5 PAHs concentration in spraying solution significantly (p < 0.0001) decreased cuticular wax content (59.1 and 65.1 vs. 67.8 mg g -1 ) in leaves of wheat but exerted slight effects on SLA. Regardless of spraying Σ 5 PAHs or not, SLA in leaves of Jiaomai (269-276 cm 2  g -1 ) and Zhengmai (265-285 cm 2  g -1 ) and cuticular wax content (104-118 mg g -1 ) in leaves of Zhengmai were significantly higher than other varieties of wheat, respectively. Σ 5 PAHs concentration in cuticular waxes ranged from 24,616 to 106,353 μg kg -1 , which was 2~3 orders and 1~2 orders of magnitude higher than that in mesophylls (46.0-535 μg kg -1 ) and leaves (785-5366 μg kg -1 ). There was a significant (r = 0.46, p < 0.05, n = 28) positive correlation between SLA and Σ 5 PAHs concentration in wheat leaves when spraying 1.25 mg L -1 of Σ 5 PAHs. The present study indicated that cuticular wax content was significantly (p < 0.01) positive correlated with Σ 5 PAHs concentration in the leaves and the translocation factor (TF w-m ) of PHE, ANT, PYR, and Σ 5 PAHs from cuticular wax to mesophyll. Based on principal component analysis (PCA), cuticular wax content was the main limiting factor for folia uptake of PAHs in winter wheat. The present study suggested that cuticular wax could play significant roles in foliar uptake of PAHs of wheat via affecting their accumulation in cuticular wax and translocation to mesophyll.

The control of gaseous exchange between the leaf and external atmosphere is governed by stomatal conductance ( g s ); therefore, stomata play a critical role in photosynthesis and transpiration and overall plant productivity. Stomatal conductance is determined by both anatomical features and behavioral characteristics. Here we review some of the osmoregulatory pathways in guard cell metabolism, genes and signals that determine stomatal function and patterning, and the recent work that explores coordination between g s and carbon assimilation ( A ) and the influence of spatial distribution of functional stomata on underlying mesophyll anatomy. We also evaluate the current literature on mesophyll-driven signals that may coordinate stomatal behavior with mesophyll carbon assimilation and explore stomatal kinetics as a possible target to improve A and water use efficiency. By understanding these processes, we can start to provide insight into manipulation of these regulatory pathways to improve stomatal behavior and identify novel unexploited targets for altering stomatal behavior and improving crop plant productivity.

To study the effects of slightly elevated temperature and ozone (O3) on leaf structural characteristics of silver birch (Betula pendula Roth), saplings of four clonal genotypes of this species were exposed to elevated temperature (ambient air temperature +0.8-1.0 °C) and elevated O3 (1.3-1.4× ambient O3), alone and in combination, in an open-air exposure field over two growing seasons (2007 and 2008). So far, the impacts of moderate elevation of temperature or the combination of elevated temperature and O3 on leaf structure of silver birch have not been intensively studied, thus showing the urgent need for this type of studies. Elevated temperature significantly increased leaf size, reduced non-glandular trichome density, decreased epidermis thickness and increased plastoglobuli size in birch leaves during one or both growing seasons. During the second growing season, O3 elevation reduced leaf size, increased palisade layer thickness and decreased the number of plastoglobuli in spongy cells. Certain leaf structural changes observed under a single treatment of elevated temperature or O3, such as increase in the amount of chloroplasts or vacuole, were no longer detected at the combined treatment. Leaf structural responses to O3 and rising temperature may also depend on timing of the exposure during the plant and leaf development as indicated by the distinct changes in leaf structure along the experiment. Genotype-dependent cellular responses to the treatments were detected particularly in the palisade cells. Overall, this study showed that even a slight but realistic elevation in ambient temperature can notably modify leaf structure of silver birch saplings. Leaf structure, in turn, influences leaf function, thus potentially affecting acclimation capacity under changing climate., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Plants experience changes in light intensity and quality due to variations in solar angle and shading from clouds and overlapping leaves. Stomatal opening to increasing irradiance is often an order of magnitude slower than photosynthetic responses, which can result in CO2 diffusional limitations on leaf photosynthesis, as well as unnecessary water loss when stomata continue to open after photosynthesis has reached saturation. Stomatal opening to light is driven by two distinct pathways; the 'red' or photosynthetic response that occurs at high fluence rates and saturates with photosynthesis, and is thought to be the main mechanism that coordinates stomatal behaviour with photosynthesis; and the guard cell-specific 'blue' light response that saturates at low fluence rates, and is often considered independent of photosynthesis, and important for early morning stomatal opening. Here we review the literature on these complicated signal transduction pathways and osmoregulatory processes in guard cells that are influenced by the light environment. We discuss the possibility of tuning the sensitivity and magnitude of stomatal response to blue light which potentially represents a novel target to develop ideotypes with the 'ideal' balance between carbon gain, evaporative cooling, and maintenance of hydraulic status that is crucial for maximizing crop performance and productivity., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Leaf function is intimately related to the size, shape, abundance and position of cells and chloroplasts. Anatomy has long been assessed and quantified in two dimensions with 3D structure inferred from 2D micrographs. Serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct 95 cells and 1173 chloroplasts from three wheat and nine chickpea leaves (three samples each from three chickpea genotypes). Wheat chloroplast volume was underestimated by 61% in mesophyll cells and 45% in bundle sheath cells from 2D micrographs, whereas chickpea mesophyll chloroplast volume was underestimated by 60% using simple geometrical models. Models of chickpea spongy and palisade cells both under- and overestimated surface area and volume by varying degrees. These models did not adequately capture irregular shapes such as flattening of chloroplasts or lobed spongy mesophyll cells. It is concluded that simple geometrical models to estimate chloroplast and cell 3D volume and surface area from 2D micrographs are inadequate, and that SBF-SEM has strong potential to contribute to improved understanding of leaf form and function., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

The quantitative and spatial coordination of stomatal pores in the epidermis and airspaces in the underlying mesophyll tissue is vital for efficient gas exchange in the leaf. The mechanisms that determine the distribution of stomata in the epidermis have been studied extensively, but how this relates to the regulation of mesophyll airspace configuration is poorly understood. Recent studies have investigated how development is coordinated between these tissue layers. The evidence suggests that multiple mechanisms are likely to work concurrently to coordinate stomatal and mesophyll development for optimal leaf gas exchange, and that both genetic and physiological factors contribute to this regulation. Such advances in our understanding of leaf development have important implications for potential improvement of crop water use efficiency., (© 2019 The Authors New Phytologist © 2019 New Phytologist Trust.)

Laser Capture Microdissection is a powerful tool that allows thin slices of specific cell types to be separated from one another. However, the most commonly used protocol, which involves embedding tissue in paraffin wax, results in severely degraded RNA. Yields from low abundance cell types of leaves are particularly compromised. We reasoned that the relatively high temperature used for sample embedding, and aqueous conditions associated with sample preparation prior to microdissection contribute to RNA degradation. Here, we describe an optimized procedure to limit RNA degradation that is based on the use of low-melting-point wax as well as modifications to sample preparation prior to dissection, and isolation of paradermal, rather than transverse sections. Using this approach, high-quality RNA suitable for down-stream applications such as quantitative reverse transcriptase-polymerase chain reactions or RNA-sequencing is recovered from microdissected bundle sheath strands and mesophyll cells of leaf tissue., Competing Interests: The authors declare no conflict of interest associated with the work described in this manuscript.

The highly efficient C 4 photosynthetic pathway is facilitated by 'Kranz' leaf anatomy. In Kranz leaves, closely spaced veins are encircled by concentric layers of photosynthetic bundle sheath (inner) and mesophyll (outer) cells. Here, we demonstrate that, in the C 4 monocot maize, Kranz patterning is regulated by redundant function of SCARECROW 1 (ZmSCR1) and a previously uncharacterized homeologue: ZmSCR1h. ZmSCR1 and ZmSCR1h transcripts accumulate in ground meristem cells of developing leaf primordia and in Zmscr1;Zmscr1h mutant leaves, most veins are separated by one rather than two mesophyll cells; many veins have sclerenchyma above and/or below instead of mesophyll cells; and supernumerary bundle sheath cells develop. The mutant defects are unified by compromised mesophyll cell development. In addition to Kranz defects, Zmscr1;Zmscr1h mutants fail to form an organized endodermal layer in the root. Collectively, these data indicate that ZmSCR1 and ZmSCR1h redundantly regulate cell-type patterning in both the leaves and roots of maize. Leaf and root pathways are distinguished, however, by the cell layer in which they operate - mesophyll at a two-cell distance from leaf veins versus endodermis immediately adjacent to root vasculature., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Compartmentation of photosynthetic reactions between mesophyll and bundle sheath cells is a key feature of C 4 photosynthesis and depends on the cell-specific accumulation of major C 4 enzymes, such as phosphoenolpyruvate carboxylase 1. The ZmPEPC1 upstream region, which drives light-inducible and mesophyll-specific gene expression in maize, has been shown to keep the same properties when introduced into rice (C 3 plant), indicating that rice has the transcription factors (TFs) needed to confer C 4 -like gene expression. Using a yeast one-hybrid approach, we identified OsbHLH112, a rice basic Helix-Loop-Helix (bHLH) TF that interacts with the maize ZmPEPC1 upstream region. Moreover, we found that maize OsbHLH112 homologues, ZmbHLH80, and ZmbHLH90, also interact with the ZmPEPC1 upstream region, suggesting that these C 4 regulators were co-opted from C 3 plants. A transactivation assay in maize mesophyll protoplasts revealed that ZmbHLH80 represses, whereas ZmbHLH90 activates, ZmPEPC1 expression. In addition, ZmbHLH80 was shown to impair the ZmPEPC1 promoter activation caused by ZmbHLH90. We showed that ZmbHLH80 and ZmbHLH90 bind to the same cis-element within the ZmPEPC1 upstream region either as homodimers or heterodimers. The formation of homo- and heterodimers with higher oligomeric forms promoted by ZmbHLH80 may explain its negative effect on gene transcription. Gene expression analysis revealed that ZmbHLH80 is preferentially expressed in bundle sheath cells, whereas ZmbHLH90 does not show a clear cell-specific expression pattern. Altogether, our results led us to propose a model in which ZmbHLH80 contributes to mesophyll-specific ZmPEPC1 gene expression by impairing ZmbHLH90-mediated ZmPEPC1 activation in the bundle sheath cells., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

The role of the mesophyll in stomatal functioning in thin amphistomatous leaves was investigated by altering gas exchange for one surface and observing the effects on stomatal conductance for the other surface. Three methods of perturbing gas exchange on the adaxial surface were used. First, gas exchange for the adaxial surface was completely blocked with plastic wrap or vacuum grease. Second, leaves were inverted to induce closure of the adaxial stomata. And third, ambient humidity for the adaxial surface was reduced to induce stomatal closure on that surface. Experiments were performed at low light intensity and three different CO 2 concentrations to test the idea that stomatal responses in thin amphistomatous leaves are partially controlled by a signal from the mesophyll that varies with light and CO 2 . In general, stomata on the abaxial surface opened when gas exchange on the adaxial surface was reduced, with the largest increases in conductance occurring at high CO 2 concentration. The data are discussed with respect to role of a purported signal from the mesophyll and the partitioning of that signal between the two surfaces of the leaf., (© 2018 John Wiley & Sons Ltd.)

Efforts to breed salt tolerant crops could benefit from investigating previously unexplored traits. One of them is a tissue succulency. In this work, we have undertaken an electrophysiological and biochemical comparison of properties of mesophyll and storage parenchyma leaf tissues of a succulent halophyte species Carpobrotus rosii ("pigface"). We show that storage parenchyma cells of C. rossii act as Na + sink and possessed both higher Na + sequestration (298 vs. 215 mM NaCl in mesophyll) and better K + retention ability. The latter traits was determined by the higher rate of H + -ATPase operation and higher nonenzymatic antioxidant activity in this tissue. Na + uptake in both tissues was insensitive to either Gd 3+ or elevated Ca 2+ ruling out involvement of nonselective cation channels as a major path for Na + entry. Patch-clamp experiments have revealed that Caprobrotus plants were capable to downregulate activity of fast vacuolar channels when exposed to saline environment; this ability was higher in the storage parenchyma cells compared with mesophyll. Also, storage parenchyma cells have constitutively lower number of open slow vacuolar channels, whereas in mesophyll, this suppression was inducible by salt. Taken together, these results provide a mechanistic basis for efficient Na + sequestration in the succulent leaf tissues., (© 2018 John Wiley & Sons Ltd.)

Cell differentiation is driven by changes in the activity of transcription factors (TFs) and subsequent alterations in transcription. To study this process, differences in TF binding between cell types can be deduced by probing chromatin accessibility. We used cell type-specific nuclear purification followed by the assay for transposase-accessible chromatin (ATAC-seq) to delineate differences in chromatin accessibility and TF regulatory networks between stem cells of the shoot apical meristem (SAM) and differentiated leaf mesophyll cells in Arabidopsis thaliana. Chromatin accessibility profiles of SAM stem cells and leaf mesophyll cells were very similar at a qualitative level, yet thousands of regions having quantitatively different chromatin accessibility were also identified. Analysis of the genomic regions preferentially accessible in each cell type identified hundreds of overrepresented TF-binding motifs, highlighting sets of TFs that are probably important for each cell type. Within these sets, we found evidence for extensive co-regulation of target genes by multiple TFs that are preferentially expressed in each cell type. Interestingly, the TFs within each of these cell type-enriched sets also showed evidence of extensively co-regulating each other. We further found that preferentially accessible chromatin regions in mesophyll cells tended to also be substantially accessible in the stem cells, whereas the converse was not true. This observation suggests that the generally higher accessibility of regulatory elements in stem cells might contribute to their developmental plasticity. This work demonstrates the utility of cell type-specific chromatin accessibility profiling for the rapid development of testable models of regulatory control differences between cell types., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Cell-type-specific transcription factors are key to deducing the distinct functions of specialized cells from gene expression profiles. Mesophyll is a major tissue for photosynthesis, and contributes about 80% of total RNA from leaves. Palisade and spongy mesophyll cells are sub-tissues that have different morphologies and physiologies. Thus, determining the palisade and spongy mesophyll-specific transcription factors from the respective sub-tissue-specific transcriptomes is vital to understanding or verifying functions of major plant tissues. One way in which gene expression profiles can be addressed is through direct isolation. Here, we present rapid and simple methods to isolate palisade and spongy mesophyll cells mechanically and enzymatically. This method provides a good yield of each isolated cell type, and the isolated cells can be used for various downstream applications.

The mesophyll surface area exposed to intercellular air space per leaf area (S m ) is closely associated with CO 2 diffusion and photosynthetic rates. S m is typically estimated from two-dimensional (2D) leaf sections and corrected for the three-dimensional (3D) geometry of mesophyll cells, leading to potential differences between the estimated and actual cell surface area. Here, we examined how 2D methods used for estimating S m compare with 3D values obtained from high-resolution X-ray microcomputed tomography (microCT) for 23 plant species, with broad phylogenetic and anatomical coverage. Relative to 3D, uncorrected 2D S m estimates were, on average, 15-30% lower. Two of the four 2D S m methods typically fell within 10% of 3D values. For most species, only a few 2D slices were needed to accurately estimate S m within 10% of the whole leaf sample median. However, leaves with reticulate vein networks required more sections because of a more heterogeneous vein coverage across slices. These results provide the first comparison of the accuracy of 2D methods in estimating the complex 3D geometry of internal leaf surfaces. Because microCT is not readily available, we provide guidance for using standard light microscopy techniques, as well as recommending standardization of reporting S m values., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Arabidopsis thaliana cation exchangers (CAX1 and CAX3) are closely related tonoplast-localized calcium/proton (Ca2+/H+) antiporters that contribute to cellular Ca2+ homeostasis. CAX1 and CAX3 were previously shown to interact in yeast; however, the function of this complex in plants has remained elusive. Here, we demonstrate that expression of CAX1 and CAX3 occurs in guard cells. Additionally, CAX1 and CAX3 are co-expressed in mesophyll tissue in response to wounding or flg22 treatment, due to the induction of CAX3 expression. Having shown that the transporters can be co-expressed in the same cells, we demonstrate that CAX1 and CAX3 can form homomeric and heteromeric complexes in plants. Consistent with the formation of a functional CAX1-CAX3 complex, CAX1 and CAX3 integrated into the yeast genome suppressed a Ca2+-hypersensitive phenotype of mutants defective in vacuolar Ca2+ transport, and demonstrated enzyme kinetics different from those of either CAX protein expressed by itself. We demonstrate that the interactions between CAX proteins contribute to the functioning of stomata, because stomata were more closed in cax1-1, cax3-1, and cax1-1/cax3-1 loss-of-function mutants due to an inability to buffer Ca2+ effectively. We hypothesize that the formation of CAX1-CAX3 complexes may occur in the mesophyll to affect intracellular Ca2+ signaling during defense responses., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)