Chloroplast envelope K+/H+ antiporters are involved in cytosol pH regulation.

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

Single-cell RNA-sequencing provides new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass leaf blades.

As an important warm-season turfgrass species, bermudagrass (Cynodon dactylon L.) flourishes in warm areas around the world due to the existence of the C4 photosynthetic pathway. However, how C4 photosynthesis operates in bermudagrass leaves is still poorly understood. In this study, we performed single-cell RNA-sequencing on 5296 cells from bermudagrass leaf blades. Eight cell clusters corresponding to mesophyll, bundle sheath, epidermis and vascular bundle cells were successfully identified using known cell marker genes. Expression profiling indicated that genes encoding NADP-dependent malic enzymes (NADP-MEs) were highly expressed in bundle sheath cells, whereas NAD-ME genes were weakly expressed in all cell types, suggesting C4 photosynthesis of bermudagrass leaf blades might be NADP-ME type rather than NAD-ME type. The results also indicated that starch synthesis-related genes showed preferential expression in bundle sheath cells, whereas starch degradation-related genes were highly expressed in mesophyll cells, which agrees with the observed accumulation of starch-filled chloroplasts in bundle sheath cells. Gene co-expression analysis further revealed that different families of transcription factors were co-expressed with multiple C4 photosynthesis-related genes, suggesting a complex transcription regulatory network of C4 photosynthesis might exist in bermudagrass leaf blades. These findings collectively provided new insights into the cell-specific expression patterns and transcriptional regulation of photosynthetic genes in bermudagrass.

Immunofluorescence staining of chloroplast proteins with frozen sections of plant tissues.

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

Partial root-zone drying irrigation improves intrinsic water-use efficiency and maintains high photosynthesis by uncoupling stomatal and mesophyll conductance in cotton leaves.

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

A method for separating tonoplast from wheat.

Vacuoles account for 90% of plant cell volume and play important roles in maintaining osmotic pressure, storing metabolites and lysosomes, compartmentalizing harmful ions, and storing and reusing minerals. These functions closely relay on the ion channels and transporters located on the tonoplast. The separation of intact vacuoles from plant cells is the key technology utilized in the study of tonoplast-located ion channels and transporters. However, the current vacuole separation methods are available for Arabidopsis and some other dicotyledons but are lacking for monocot crops. In this study, we established a new method for the vacuole separation from wheat mesophyll cells and investigated the transmembrane proton flux of tonoplasts with non-invasive micro-test technology (NMT). Moreover, our study provides a technology for the study of vacuole functions in monocot crops.

Effect of light-induced changes in leaf anatomy on intercellular and cellular components of mesophyll resistance for CO2 in Fagus sylvatica.

Mesophyll resistance for CO2 diffusion (rm) is one of the main limitations for photosynthesis and plant growth. Breeding new varieties with lower rm requires knowledge of its distinct components. We tested new method for estimating the relative drawdowns of CO2 concentration (c) across hypostomatous leaves of Fagus sylvatica. This technique yields values of the ratio of the internal CO2 concentrations at the adaxial and abaxial leaf side, cd/cb, the drawdown in the intercellular air space (IAS), and intracellular drawdown between IAS and chloroplast stroma, cc/cbd. The method is based on carbon isotope composition of leaf dry matter and epicuticular wax isolated from upper and lower leaf sides. We investigated leaves from tree-canopy profile to analyse the effects of light and leaf anatomy on the drawdowns and partitioning of rm into its inter- (rIAS) and intracellular (rliq) components. Validity of the new method was tested by independent measurements of rm using conventional isotopic and gas exchange techniques. 73% of investigated leaves had adaxial epicuticular wax enriched in 13C compared to abaxial wax (by 0.50‰ on average), yielding 0.98 and 0.70 for average of cd/cb and cc/cbd, respectively. The rIAS to rliq proportion were 5.5:94.5% in sun-exposed and 14.8:85.2% in shaded leaves. cc dropped to less than half of the atmospheric value in the sunlit and to about two-thirds of it in shaded leaves. This method shows that rIAS is minor but not negligible part of rm and reflects leaf anatomy traits, i.e. leaf mass per area and thickness.

Beauty is more than epidermis deep: How cell division and expansion sculpt the leaf spongy mesophyll.

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.

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.

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.

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.

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.