Intercellular Air Spaces Research Articles

Nanoceria-induced variations in leaf anatomy and cell wall composition drive the increase in mesophyll conductance of salt-stressed cotton leaves

Nanomaterials as an emerging tool are being used to improve plant′s net photosynthetic rate (AN) when suffering salt stress, but the underlying mechanisms remain unclear. To clarify this, a hydroponic experiment was conducted to study the effects of polyacrylic acid coated nanoceria (PNC) on the AN of salt-stressed cotton and related intrinsic mechanisms. Results showed that the PNC-induced AN enhancement of salt-stressed leaves was strongly facilitated by the mesophyll conductance to CO2 (gm). Further analysis showed that the PNC-induced improvement of gm was related to the increased chloroplast surface area exposed to intercellular airspaces, which was attribute to the increased mesophyll surface area exposed to intercellular airspaces and chloroplast number due to the increased K+ content and decreased reactive oxygen species level in salt-stressed leaves. Interestingly, our results also showed that PNC-induced variations in cell wall composition of salt-stressed cotton leaves strongly influenced gm, especially, hemicellulose and pectin. Moreover, the proportion of pectin in cell wall composition played a more important role in determining gm. Our study demonstrated for the first time that nanoceria, through alterations to anatomical traits and cell wall composition, drove gm enhancement, which ultimately increased AN of salt-stressed leaves.

Into the spongy-verse: structural differences between leaf and flower mesophyll.

As the site of almost all terrestrial carbon fixation, the mesophyll tissue is critical to leaf function. However, mesophyll tissue is not restricted only to leaves but also occurs in the laminar, heterotrophic organs of the floral perianth, providing a powerful test of how metabolic differences are linked to differences in tissue structure. Here, we compared mesophyll tissues of leaves and flower perianths of six species using high-resolution X-ray computed microtomography (microCT) imaging. Consistent with previous studies, stomata were nearly absent from flowers, and flowers had a significantly lower vein density compared to leaves. However, mesophyll porosity was significantly higher in flowers than in leaves, and higher mesophyll porosity was associated with more aspherical mesophyll cells. Despite these differences in cell and tissue structure between leaf and flower mesophyll, modeled intercellular airspace conductance did not differ significantly between organs, regardless of differences in stomatal density between organs. These results suggest that in addition to differences between leaves and flowers in vein and stomatal densities, the mesophyll cells and tissues inside these organs also exhibit marked differences that may allow for flowers to be relatively cheaper in terms of biomass investment per unit of flower surface area.

Growing at the arid edge: Anatomical variations in leaves are more extensive than in stems of five Mediterranean species across contrasting moisture regimes.

Increasing aridity in the Mediterranean region affects ecosystems and plant life. Various anatomical changes in plants help them cope with dry conditions. This study focused on anatomical differences in leaves and xylem of five co-occurring Mediterranean plant species namely Quercus calliprinos, Pistacia palaestina, Pistacia lentiscus, Rhamnus lycioides, and Phillyrea latifolia in wet and dry sites. Stomatal density, stomatal length, leaf mass area, lamina composition, percentage of intercellular air spaces, and mesophyll cell area in leaves of plants in wet and dry sites were analyzed. Xylem anatomy was assessed through vessel length and area in branches. In the dry site, three species had increased stomatal density and decreased stomatal length. Four species had increased palisade mesophyll and reduced air space volume. In contrast, phenotypic changes in the xylem were less pronounced; vessel length was unaffected by site conditions, but vessel diameter decreased in two species. Intercellular air spaces proved to be the most dynamic anatomical feature. Quercus calliprinos had the most extensive anatomical changes; Rhamnus lycioides had only minor changes. All these changes were observed in comparison to the species in the wet site. This study elucidated variations in anatomical responses in leaves among co-occurring Mediterranean plant species and identified the most dynamic traits. Understanding these adaptations provides valuable insights into the ability of plants to thrive under changing climate conditions.

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.

Unsaturation in the air spaces of leaves and its implications.

Modern plant physiological theory stipulates that the resistance to water movement from plants to the atmosphere is overwhelmingly dominated by stomata. This conception necessitates a corollary assumption-that the air spaces in leaves must be nearly saturated with water vapour; that is, with a relative humidity that does not decline materially below unity. As this idea became progressively engrained in scientific discourse and textbooks over the last century, observations inconsistent with this corollary assumption were occasionally reported. Yet, evidence of unsaturation gained little traction, with acceptance of the prevailing framework motivated by three considerations: (1) leaf water potentials measured by either thermocouple psychrometry or the Scholander pressure chamber are largely consistent with the framework; (2) being able to assume near saturation of intercellular air spaces was transformational to leaf gas exchange analysis; and (3) there has been no obvious mechanism to explain a variable, liquid-phase resistance in the leaf mesophyll. Here, we review the evidence that refutes the assumption of universal, near saturation of air spaces in leaves. Refining the prevailing paradigm with respect to this assumption provides opportunities for identifying and developing mechanisms for increased plant productivity in the face of increasing evaporative demand imposed by global climate change.

Variation in photosynthetic capacity of Salvia przewalskii along elevational gradients on the eastern Qinghai-Tibetan Plateau, China

Elevational variation in plant growing environment drives diversification of photosynthetic capacity, however, the mechanism behind this reaction is poorly understood. We measured leaf gas exchange, chlorophyll fluorescence, anatomical characteristics, and biochemical traits of Salvia przewalskii at elevations ranging from 2400 m to 3400 m above sea level (a.s.l) on the eastern Qinghai-Tibetan Plateau, China. We found that photosynthetic capacity showed an initial increase and then a decrease with rising elevation, and the best state observed at 2800 m a.s.l. Environmental factors indirectly regulated photosynthetic capacity by affecting stomatal conductance (gs), mesophyll conductance (gm), maximum velocity of carboxylation (Vc max), and maximum capacity for photosynthetic electron transport (Jmax). The average temperature (T) and total precipitation (P) during the growing season had the highest contribution to the variation of photosynthetic capacity of S. przewalskii in subalpine areas, which were 25% and 24%, respectively. Photosynthetic capacity was mainly affected by diffusional limitations (71%–89%), and mesophyll limitation (lm) played a leading role. The variation of gm was attributed to the effects of environmental factors on the volume fraction of intercellular air space (fias), the thickness of cell wall (Tcw), the surface of mesophyll cells and chloroplasts exposed to intercellular airspace (Sm, Sc), and plasma membrane intrinsic protein (PIPs, PIP1, PIP2), independent of carbonic anhydrase (CA). Optimization of leaf tissue structure and adaptive physiological responses enabled plants to efficiently cope with variable climate conditions of high-elevation areas, and the while maintaining high levels of carbon assimilation.

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.

Variation of mesophyll conductance mediated by nitrogen form is related to changes in cell wall property and chloroplast number.

Plants primarily incorporate nitrate (NO3 -) and ammonium (NH4 +) as the primary source of inorganic nitrogen (N); the physiological mechanisms of photosynthesis (A) dropdown under NH4 + nutrition has been investigated in many studies. Leaf anatomy is a major determinant to mesophyll conductance (g m) and photosynthesis; however, it remains unclear whether the photosynthesis variations of plants exposed to different N forms is related to leaf anatomical variation. In this work, a common shrub, Lonicera japonica was hydroponically grown under NH4 +, NO3 - and 50% NH4 +/NO3 -. We found that leaf N significantly accumulated under NH4 +, whereas the photosynthesis was significantly decreased, which was mainly caused by a reduced g m. The reduced g m under NH4 + was related to the decreased intercellular air space, the reduced chloroplast number and especially the thicker cell walls. Among the cell wall components, lignin and hemicellulose contents under NH4 + nutrition were significantly higher than those in the other two N forms and were scaled negatively correlated with g m; while pectin content was independent from N forms. Pathway analysis further revealed that the cell wall components might indirectly regulate g m by influencing the thickness of the cell wall. These results highlight the importance of leaf anatomical variation characterized by modifications of chloroplasts number and cell wall thickness and compositions, in the regulation of photosynthesis in response to varied N sources.

Structural changes of mesophyll cells in the rice leaf tissue in response to salinity stress based on the three-dimensional analysis.

Rice leaf blades have intricate-shaped mesophyll cells (MCs) with a large volume of chloroplasts enhancing gas exchange between stroma and intercellular airspace (IAS). Since the rice MCs do not form palisade or spongy tissue cells and are considered monotypic cells, the structural analysis of MCs in the middle part of the leaf tissue has been done, neglecting the various shapes of MCs can be observed on the cross-section of rice leaves. Moreover, the middle MC layer is sandwiched between the upper and lower layers and is more restricted in its demand for light and CO2 entering from the outside. Therefore, the different layers of MCs may differ in their sensitivity to salt stress that causes structural changes in cells. This study aims to elucidate the intra- and extra-cellular structures of MC in different layers of leaf tissue and determine how salinity affects the MC structure in each layer. The mesophyll tissue was divided into adaxial, middle and abaxial layers, and eight MCs and chloroplast regions were selected from each layer and reconstructed into three-dimensional (3D) representations. The whole leaf anatomical and physiological parameters were measured to find the effects of salinity stress on the MC structures. As a result,the 3D analysis of rice leaf tissue revealed the different structures of MCs with greater diversity in the adaxial and abaxial layers than in the middle layer. Salinity stress reduced the size and height of the MCs and coverage of the chloroplast on the cytoplasm periphery of the adaxial and abaxial layers, as well as the chloroplast size of adaxial MCs. Overall,these results reveal the variation of rice MC in leaf tissue and suggest the higher sensitivity to salt stress in the adaxial mesophyll among the layers, which may partly account for the decrease in photosynthetic capacity.

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.

Magnetized Seeds and Structured Water: Effects on Resilience of Velvet Bean Seedlings (Mucuna pruriens) under Deficit Irrigation

A custom-built water generator supplied structured water (SW) for applying the deficit irrigation treatments to velvet bean plants (Mucuns pruriens). The objectives of the study were to 1) determine the effects of magnetized seed treatment on velvet bean plants, 2) determine the effects of magnetized and hydroxylated water treatments on velvet bean plants, and 4) determine the effects of deficit irrigation, using three soil moisture levels, on velvet bean plants. The optimal water-saving treatment was magnetized seeds plus 10 MT + HWT. This treatment had a 226% increase in transpiration and a 22% increase in water vapor concentration in the intercellular airspace for the low soil moisture watering schedule. The three study factors in the optimal seed and water treatment had a synchronistic effect for enhancing metabolic efficiency by increasing whole plant WUE by 87% and carbon assimilation efficiency by 66% in the low soil moisture schedule. Plants irrigated with SW water and grown from magnetized seeds had enhanced resilience to high water stress conditions by maintaining adequate levels of biologically structured water. The rapid deactivation of a suite of highly interconnected defense activities in the optimal seed and water treatments implies that the plants exhibit macroscopic coherence properties. Coherence at the macroscopic level resulted in complex synchronization between metabolic efficiency, plant health, and deactivation of a suite of regulatory defenses in plants exposed to high water stress.

Photosynthetic plasticity aggravates the susceptibility of magnesium-deficient leaf to high light in rapeseed plants: the importance of Rubisco and mesophyll conductance.

Plants grown under low magnesium (Mg) soils are highly susceptible to encountering light intensities that exceed the capacity of photosynthesis (A), leading to a depression of photosynthetic efficiency and eventually to photooxidation (i.e., leaf chlorosis). Yet, it remains unclear which processes play a key role in limiting the photosynthetic energy utilization of Mg-deficient leaves, and whether the plasticity of A in acclimation to irradiance could have cross-talk with Mg, hence accelerating or mitigating the photodamage. We investigated the light acclimation responses of rapeseed (Brassica napus) grown under low- and adequate-Mg conditions. Magnesium deficiency considerably decreased rapeseed growth and leaf A, to a greater extent under high than under low light, which is associated with higher level of superoxide anion radical and more severe leaf chlorosis. This difference was mainly attributable to a greater depression in dark reaction under high light, with a higher Rubisco fallover and a more limited mesophyll conductance to CO2 (gm ). Plants grown under high irradiance enhanced the content and activity of Rubisco and gm to optimally utilize more light energy absorbed. However, Mg deficiency could not fulfill the need to activate the higher level of Rubisco and Rubisco activase in leaves of high-light-grown plants, leading to lower Rubisco activation and carboxylation rate. Additionally, Mg-deficient leaves under high light invested more carbon per leaf area to construct a compact leaf structure with smaller intercellular airspaces, lower surface area of chloroplast exposed to intercellular airspaces, and CO2 diffusion conductance through cytosol. These caused a more severe decrease in within-leaf CO2 diffusion rate and substrate availability. Taken together, plant plasticity helps to improve photosynthetic energy utilization under high light but aggravates the photooxidative damage once the Mg nutrition becomes insufficient.

Effects of leaf age during drought and recovery on photosynthesis, mesophyll conductance and leaf anatomy in wheat leaves.

statement: Mesophyll conductance (g m) was negatively correlated with wheat leaf age but was positively correlated with the surface area of chloroplasts exposed to intercellular airspaces (S c). The rate of decline in photosynthetic rate and g m as leaves aged was slower for water-stressed than well-watered plants. Upon rewatering, the degree of recovery from water-stress depended on the age of the leaves, with the strongest recovery for mature leaves, rather than young or old leaves. Diffusion of CO2 from the intercellular airspaces to the site of Rubisco within C3 plant chloroplasts (gm) governs photosynthetic CO2 assimilation (A). However, variation in g m in response to environmental stress during leaf development remains poorly understood. Age-dependent changes in leaf ultrastructure and potential impacts on g m, A, and stomatal conductance to CO2 (g sc) were investigated for wheat (Triticum aestivum L.) in well-watered and water-stressed plants, and after recovery by re-watering of droughted plants. Significant reductions in A and g m were found as leaves aged. The oldest plants (15 days and 22 days) in water-stressed conditions showed higher A and gm compared to irrigated plants. The rate of decline in A and g m as leaves aged was slower for water-stressed compared to well-watered plants. When droughted plants were rewatered, the degree of recovery depended on the age of the leaves, but only for g m. The surface area of chloroplasts exposed to intercellular airspaces (S c) and the size of individual chloroplasts declined as leaves aged, resulting in a positive correlation between g m and S c. Leaf age significantly affected cell wall thickness (t cw), which was higher in old leaves compared to mature/young leaves. Greater knowledge of leaf anatomical traits associated with g m partially explained changes in physiology with leaf age and plant water status, which in turn should create more possibilities for improving photosynthesis using breeding/biotechnological strategies.

Exploring 3D leaf anatomical traits for C4 photosynthesis: chloroplast and plasmodesmata pit field size in maize and sugarcane.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C4 species, maize and sugarcane, with respect to cell surface area and cell volume. Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM. Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells. This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C4 photosynthesis.

Anatomical drivers of stomatal conductance in sorghum lines with different leaf widths grown under different temperatures.

Sustaining crop productivity and resilience in water-limited environments and under rising temperatures are matters of concern worldwide. We investigated the leaf anatomical traits that underpin our recently identified link between leaf width (LW) and intrinsic water use efficiency (iWUE), as traits of interest in plant breeding. Ten sorghum lines with varying LW were grown under three temperatures to expand the range of variation of both LW and gas exchange rates. Leaf gas exchange, surface morphology and cross-sectional anatomy were measured and analysed using structural equations modelling. Narrower leaves had lower stomatal conductance (gs ) and higher iWUE across growth temperatures. They also had smaller intercellular airspaces, stomatal size, percentage of open stomatal aperture relative to maximum, hydraulic pathway, mesophyll thickness, and leaf mass per area. Structural modelling revealed a developmental association among leaf anatomical traits that underpinned gs variation in sorghum. Growing temperature and LW both impacted leaf gas exchange rates, but only LW directly impacted leaf anatomy. Wider leaves may be more productive under well-watered conditions, but consume more water for growth and development, which is detrimental under water stress.

Functional consequences of light intensity on soybean leaf hydraulic conductance: Coordinated variations in leaf venation architecture and mesophyll structure

Leaf hydraulic conductance (Kleaf) is a critical determinant of photosynthesis. Prior research has assessed the influence of light intensity on Kleaf by affecting leaf veins. However, whether the mesophyll structure is associated with leaf hydraulic acclimation to different light intensities remains unclear. We investigated the responses of leaf gas exchange and Kleaf to different growth light intensities and checked for the correlation of Kleaf with leaf structural traits in soybean. Low light significantly reduced Kleaf, but Kleaf and stomatal conductance remain coordinated. Low light decreased the stomatal density to match the balance between in water supply and carbon gain capacity. The decline in Kleaf under low growth light condition was partly due to for the reduction of the water movement through leaf veins because of reduced leaf vein thickness and density and xylem conduit size. In addition, low light reduces the mesophyll cell‐to‐cell connectivity and increased the volume fraction of intercellular air space. As a result, the area available for liquid‐phase flow decreases, and subsequently decreased Kleaf. The present study highlights the coordination role between leaf venation architecture and mesophyll structure influencing Kleaf in response to light intensity.

Exploring the potential of Δ17O in CO2 for determining mesophyll conductance.

Mesophyll conductance to CO2 from the intercellular air space to the CO2-H2O exchange site has been estimated using δ18O measurements (gm18). However, the gm18 estimates are affected by the uncertainties in the δ18O of leaf water where the CO2-H2O exchange takes place and the degree of equilibration between CO2 and H2O. We show that measurements of Δ17O (i.e.Δ17O=δ17O-0.528×δ18O) can provide independent constraints on gm (gmΔ17) and that these gm estimates are less affected by fractionation processes during gas exchange. The gm calculations are applied to combined measurements of δ18O and Δ17O, and gas exchange in two C3 species, sunflower (Helianthus annuus L. cv. 'sunny') and ivy (Hedera hibernica L.), and the C4 species maize (Zea mays). The gm18 and gmΔ17 estimates agree within the combined errors (P-value, 0.876). Both approaches are associated with large errors when the isotopic composition in the intercellular air space becomes close to the CO2-H2O exchange site. Although variations in Δ17O are low, it can be measured with much higher precision compared with δ18O. Measuring gmΔ17 has a few advantages compared with gm18: (i) it is less sensitive to uncertainty in the isotopic composition of leaf water at the isotope exchange site and (ii) the relative change in the gm due to an assumed error in the equilibration fraction θeq is lower for gmΔ17 compared with gm18. Thus, using Δ17O can complement and improve the gm estimates in settings where the δ18O of leaf water varies strongly, affecting the δ18O (CO2) difference between the intercellular air space and the CO2-H2O exchange site.

Leaf optical properties and photosynthesis of fern species with a wide range of divergence time in relation to mesophyll anatomy.

For a comprehensive understanding of the mechanisms of changing plant photosynthetic capacity during plant evolutionary history, knowledge of leaf gas exchange and optical properties are essential, both of which relate strongly to mesophyll anatomy. Although ferns are suitable for investigating the evolutionary history of photosynthetic capacity, comprehensive research of fern species has yet to be undertaken in this regard. We investigated leaf optical properties, gas exchange and mesophyll anatomy of fern species with a wide range of divergence time, using 66 ferns from natural habitats and eight glasshouse-grown ferns. We used a spectroradiometer and an integrating sphere to measure light absorptance and reflectance by the leaves. The more newly divergent fern species had a thicker mesophyll, a larger surface area of chloroplasts facing the intercellular airspaces (Sc), thicker cell walls and large light absorptance. Although no trend with divergence time was obtained in leaf photosynthetic capacity on a leaf-area basis, when the traits were expressed on a mesophyll-thickness basis, trends in leaf photosynthetic capacity became apparent. On a mesophyll-thickness basis, the more newly divergent species had a low maximum photosynthesis rate, accompanied by a low Sc. We found a strong link between light capture, mesophyll anatomy and photosynthesis rate in fern species for the first time. The thick mesophyll of the more newly divergent ferns does not necessarily relate to the high photosynthetic capacity on a leaf-area basis. Rather, the thick mesophyll accompanied by thick cell walls allowed the ferns to adapt to a wider range of environments through increasing leaf toughness, which would contribute to the diversification of fern species.

Analyzing anatomy over three dimensions unpacks the differences in mesophyll diffusive area between sun and shade Vitis vinifera leaves.

Leaves grown at different light intensities exhibit considerable differences in physiology, morphology and anatomy. Because plant leaves develop over three dimensions, analyses of the leaf structure should account for differences in lengths, surfaces, as well as volumes. In this manuscript, we set out to disentangle the mesophyll surface area available for diffusion per leaf area (S m,LA) into underlying one-, two- and three-dimensional components. This allowed us to estimate the contribution of each component to S m,LA, a whole-leaf trait known to link structure and function. We introduce the novel concept of a 'stomatal vaporshed,' i.e. the intercellular airspace unit most closely connected to a single stoma, and use it to describe the stomata-to-diffusive-surface pathway. To illustrate our new theoretical framework, we grew two cultivars of Vitis vinifera L. under high and low light, imaged 3D leaf anatomy using microcomputed tomography (microCT) and measured leaf gas exchange. Leaves grown under high light were less porous and thicker. Our analysis showed that these two traits and the lower S m per mesophyll cell volume (S m,Vcl) in sun leaves could almost completely explain the difference in S m,LA. Further, the studied cultivars exhibited different responses in carbon assimilation per photosynthesizing cell volume (A Vcl). While Cabernet Sauvignon maintained A Vcl constant between sun and shade leaves, it was lower in Blaufränkisch sun leaves. This difference may be related to genotype-specific strategies in building the stomata-to-diffusive-surface pathway.

Photosynthesis and leaf structure of F1 hybrids between Cymbidium ensifolium (C3) and C. bicolor subsp. pubescens (CAM).

The introduction of crassulacean acid metabolism (CAM) into C3 crops has been considered as a means of improving water-use efficiency. In this study, we investigated photosynthetic and leaf structural traits in F1 hybrids between Cymbidium ensifolium (female C3 parent) and C. bicolor subsp. pubescens (male CAM parent) of the Orchidaceae. Seven F1 hybrids produced through artificial pollination and in vitro culture were grown in a greenhouse with the parent plants. Structural, biochemical and physiological traits involved in CAM in their leaves were investigated. Cymbidium ensifolium accumulated very low levels of malate without diel fluctuation, whereas C. bicolor subsp. pubescens showed nocturnal accumulation and diurnal consumption of malate. The F1s also accumulated malate at night, but much less than C. bicolor subsp. pubescens. This feature was consistent with low nocturnal fixation of atmospheric CO2 in the F1s. The δ13C values of the F1s were intermediate between those of the parents. Leaf thickness was thicker in C. bicolor subsp. pubescens than in C. ensifolium, and those of the F1s were more similar to that of C. ensifolium. This was due to the difference in mesophyll cell size. The chloroplast coverage of mesophyll cell perimeter adjacent to intercellular air spaces of C. bicolor subsp. pubescens was lower than that of C. ensifolium, and that of the F1s was intermediate between them. Interestingly, one F1 had structural and physiological traits more similar to those of C. bicolor subsp. pubescens than the other F1s. Nevertheless, all F1s contained intermediate levels of phosphoenolpyruvate carboxylase but as much pyruvate, Pi dikinase as C. bicolor subsp. pubescens. CAM traits were intricately inherited in the F1 hybrids, the level of CAM expression varied widely among F1 plants, and the CAM traits examined were not necessarily co-ordinately transmitted to the F1s.