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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.)

Premise: Because of its broad range in the neotropical rainforest and within tree canopies, the tank bromeliad Guzmania monostachia was investigated as a model of how varying leaf hydraulic conductance (K leaf ) could help plants resist and recover from episodic drought. The two pathways of K leaf , inside and outside the xylem, were also examined to determine the sites and causes of major hydraulic resistances within the leaf., Methods: We measured leaf hydraulic conductance for plants in the field and laboratory under wet, dry, and rewetted conditions and applied physiological, anatomical, and gene expression analysis with modeling to investigate changes in K leaf ., Results: After 7 d with no rain in the field or 14 days with no water in the glasshouse, K leaf decreased by 50% yet increased to hydrated values within 4 d of tank refilling. Staining to detect embolism combined with modeling indicated that changes outside the xylem were of greater importance to K leaf than were changes inside the xylem and were associated with changes in intercellular air spaces (aerenchyma), aquaporin expression and inhibition, and cuticular conductance., Conclusions: Low values for all conductances during drying, particularly in pathways outside the xylem, lead to hydraulic resilience for this species and may also contribute to its broad environmental tolerances., (© 2019 The Authors. American Journal of Botany is published by Wiley Periodicals, Inc. on behalf of the Botanical Society of America.)

Measuring and modeling the spatial distribution of chlorophyll within the leaf is critical for understanding the relationship between leaf structure and carbon assimilation, for defining the relative investments in leaf tissues from the perspective of leaf economics theory, and for the emerging application of in silico carbon assimilation models. Yet, spatially resolved leaf chlorophyll distribution data are limited. Here, we used epi-illumination fluorescence microscopy to estimate relative chlorophyll concentration as a function of mesophyll depth for 57 plant taxa. Despite interspecific variation due to differences in leaf thickness, mesophyll palisade fraction, and presence of large intercellular airspaces, the spatial distribution of chlorophyll in laminar leaves was remarkably well conserved across diverse lineages (ferns, cycads, conifers, ginkgo, basal angiosperms, magnoliids, monocots, and eudicots) and growth habits (tree, shrub, herbaceous, annual, perennial, evergreen, and deciduous). In the typical leaf, chlorophyll content increased gradually as a function of depth, peaking deep within the mesophyll. This chlorophyll distribution pattern is likely coupled to adaxial and abaxial intraleaf light gradients, including the relative enrichment of green light in the lower leaf. Chlorophyll distribution for the typical leaf from our dataset was well represented by a simple mathematical model (R 2 = 0.94). We present chlorophyll distribution data and model equations for many ecologically and commercially relevant species and plant functional types (defined according to chlorophyll profile similarity, clade, and leaf thickness). These findings represent an advancement toward more accurate photosynthesis modeling and increase our understanding of first principles in intraleaf physiology., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Maintaining water balance has been a critical constraint shaping the evolution of leaf form and function. However, flowers, which are heterotrophic and relatively short-lived, may not be constrained by the same physiological and developmental factors. We measured physiological parameters derived from pressure-volume curves for leaves and flowers of 22 species to characterize the diversity of hydraulic traits in flowers and to determine whether flowers are governed by the same constraints as leaves. Compared with leaves, flowers had high saturated water content, which was a strong predictor of hydraulic capacitance in both leaves and flowers. Principal component analysis revealed that flowers occupied a different region of multivariate trait space than leaves and that hydraulic traits are more diverse in flowers than in leaves. Without needing to maintain high rates of transpiration, flowers rely on other hydraulic traits, such as high hydraulic capacitance, to maintain turgor pressure. As a result, instead of employing a metabolically expensive but durable carbon (C)-based skeleton, flowers may rely predominantly on a metabolically cheaper, hydrostatic skeleton to keep their structures on display for pollinators, which has important implications for both the costs of reproduction and the biomechanical performance of flowers, particularly during drought., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Premise: Water deficit and drought conditions are increasing in intensity, frequency, and duration in the Iberian Peninsula. We observed natural variation in leaf traits across the range of Quercus suber L. (cork oak), an ecologically important species within this region. Stomatal traits (e.g., pore length, maximum aperture) and carbon isotope composition (δ 13 C) provide an opportunity to examine the integrative effects of drought and dry-season intensity on leaf development, maximum stomatal conductance, and adaptation to precipitation regimes., Methods: Gross leaf traits (e.g., area, thickness), stomatal traits (e.g., pore length, size, aperture), and carbon isotope discrimination were measured in Q. suber leaves, and maximum stomatal conductance to water vapor (g wmax ) was calculated. Trees were sampled from nine natural populations across a climate gradient in the Iberian Peninsula, including trees from two genetic lineages. Linear mixed models compared total water deficit to leaf traits, accounting for tree and site as random effects., Results: Quercus suber gross leaf morphology remained consistent across the climate gradient, but increasing water deficit was correlated with smaller stomata at the leaf level and low δ 13 C at the tree level. No traits were significantly different between the two genetic lineages., Conclusions: While there were no significant differences in gross leaf morphology across the climate gradient or between the genetic lineages, stomatal traits and δ 13 C responded to climate, suggesting that Q. suber can inhabit a range of environments in the Iberian Peninsula via micro-adjustments of the trait that controls water loss into the atmosphere., (© 2019 Botanical Society of America.)

Leaves are a nexus for the exchange of water, carbon, and energy between terrestrial plants and the atmosphere. Research in recent decades has highlighted the critical importance of the underlying biophysical and anatomical determinants of CO 2 and H 2 O transport, but a quantitative understanding of how detailed 3D leaf anatomy mediates within-leaf transport has been hindered by the lack of a consensus framework for analyzing or simulating transport and its spatial and temporal dynamics realistically, and by the difficulty of measuring within-leaf transport at the appropriate scales. We discuss how recent technological advancements now make a spatially explicit 3D leaf analysis possible, through new imaging and modeling tools that will allow us to address long-standing questions related to plant carbon-water exchange., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The influence of the dynamics of leaf hydraulic conductance ( K leaf ) diurnally and during dehydration on stomatal conductance and photosynthesis remains unclear. Using the model species Arabidopsis ( Arabidopsis thaliana ecotype Columbia-0), we applied a multitiered approach including physiological measurements, high-resolution x-ray microcomputed tomography, and modeling at a range of scales to characterize (1) K leaf decline during dehydration; (2) its basis in the hydraulic conductances of leaf xylem and outside-xylem pathways ( K ox ); (3) the dependence of its dynamics on irradiance; (4) its impact on diurnal patterns of stomatal conductance and photosynthetic rate; and (5) its influence on gas exchange and survival under simulated drought regimes. Arabidopsis leaves showed strong vulnerability to dehydration diurnally in both gas exchange and hydraulic conductance, despite lack of xylem embolism or conduit collapse above the turgor loss point, indicating a pronounced sensitivity of K ox to dehydration. K leaf increased under higher irradiance in well-hydrated leaves across the full range of water potential, but no shift in K leaf vulnerability was observed. Modeling indicated that responses to dehydration and irradiance are likely attributable to changes in membrane permeability and that a dynamic K ox would contribute strongly to stomatal closure, improving performance, survival, and efficient water use during drought. These findings for Columbia-0 provide a baseline for assessing variation across genotypes in hydraulic traits and their influence on gas exchange during dehydration., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

The leaf intercellular airspace (IAS) is generally considered to have high conductance to CO 2 diffusion relative to the liquid phase. While previous studies accounted for leaf-level variation in porosity and mesophyll thickness, they omitted 3D IAS traits that potentially influence IAS conductance (g IAS ). Here, we reevaluated the standard equation for g IAS by incorporating tortuosity, lateral path lengthening, and IAS connectivity. We measured and spatially mapped these geometric IAS traits for 19 Bromeliaceae species with Crassulacean acid metabolism (CAM) or C3 photosynthetic pathways using x-ray microcomputed tomography imaging and a novel computational approach. We found substantial variation in porosity (0.04-0.73 m 3 m -3 ), tortuosity (1.09-3.33 m 2 m -2 ), lateral path lengthening (1.12-3.19 m m -1 ), and IAS connectivity (0.81-0.97 m 2 m -2 ) across all bromeliad leaves. The revised g IAS model predicted significantly lower g IAS in CAM (0.01-0.19 mol m -2 s -1 bar -1 ) than in C3 (0.41-2.38 mol m -2 s -1 bar -1 ) plants due to a coordinated decline in these IAS traits. Our reevaluated equation also generally predicted lower g IAS values than the former one. Moreover, we observed high spatial heterogeneity in these IAS geometric traits throughout the mesophyll, especially within CAM leaves. Our data show that IAS traits that better capture the 3D complexity of leaves strongly influence g IAS and that the impact of the IAS on mesophyll conductance should be carefully considered with respect to leaf anatomy. We provide a simple function to estimate tortuosity and lateral path lengthening in the absence of access to imaging tools such as x-ray microcomputed tomography or other novel 3D image-processing techniques., (© 2018 American Society of Plant Biologists. All rights reserved.)

Ecosystems worldwide are facing increasingly severe and prolonged droughts during which hydraulic failure from drought-induced embolism can lead to organ or whole plant death. Understanding the determinants of xylem failure across species is especially critical in leaves, the engines of plant growth. If the vulnerability segmentation hypothesis holds within leaves, higher order veins that are most terminal in the plant hydraulic system should be more susceptible to embolism to protect the rest of the water transport system. Increased vulnerability in the higher order veins would also be consistent with these experiencing the greatest tensions in the plant xylem network. To test this hypothesis, we combined X-ray micro-computed tomography imaging, hydraulic experiments, cross-sectional anatomy and 3D physiological modelling to investigate how embolisms spread throughout petioles and vein orders during leaf dehydration in relation to conduit dimensions. Decline of leaf xylem hydraulic conductance (K x ) during dehydration was driven by embolism initiating in petioles and midribs across all species, and K x vulnerability was strongly correlated with petiole and midrib conduit dimensions. Our simulations showed no significant impact of conduit collapse on K x decline. We found xylem conduit dimensions play a major role in determining the susceptibility of the leaf water transport system during strong leaf dehydration., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Leaf hydraulic supply is crucial to maintaining open stomata for CO 2 capture and plant growth. During drought-induced dehydration, the leaf hydraulic conductance (K leaf ) declines, which contributes to stomatal closure and, eventually, to leaf death. Previous studies have tended to attribute the decline of K leaf to embolism in the leaf vein xylem. We visualized at high resolution and quantified experimentally the hydraulic vulnerability of xylem and outside-xylem pathways and modeled their respective influences on plant water transport. Evidence from all approaches indicated that the decline of K leaf during dehydration arose first and foremost due to the vulnerability of outside-xylem tissues. In vivo x-ray microcomputed tomography of dehydrating leaves of four diverse angiosperm species showed that, at the turgor loss point, only small fractions of leaf vein xylem conduits were embolized, and substantial xylem embolism arose only under severe dehydration. Experiments on an expanded set of eight angiosperm species showed that outside-xylem hydraulic vulnerability explained 75% to 100% of K leaf decline across the range of dehydration from mild water stress to beyond turgor loss point. Spatially explicit modeling of leaf water transport pointed to a role for reduced membrane conductivity consistent with published data for cells and tissues. Plant-scale modeling suggested that outside-xylem hydraulic vulnerability can protect the xylem from tensions that would induce embolism and disruption of water transport under mild to moderate soil and atmospheric droughts. These findings pinpoint outside-xylem tissues as a central locus for the control of leaf and plant water transport during progressive drought., (© 2017 The author(s). All Rights Reserved.)

Coastal redwood (Sequoia sempervirens), the world's tallest tree species, rehydrates leaves via foliar water uptake during fog/rain events. Here we examine if bark also permits water uptake in redwood branches, exploring potential flow mechanisms and biological significance. Using isotopic labelling and microCT imaging, we observed that water entered the xylem via bark and reduced tracheid embolization. Moreover, prolonged bark wetting (16 h) partially restored xylem hydraulic conductivity in isolated branch segments and whole branches. Partial hydraulic recovery coincided with an increase in branch water potential from about -5.5 ± 0.4 to -4.2 ± 0.3 MPa, suggesting localized recovery and possibly hydraulic isolation. As bark water uptake rate correlated with xylem osmotic potential (R(2)  = 0.88), we suspect a symplastic role in transferring water from bark to xylem. Using historical weather data from typical redwood habitat, we estimated that bark and leaves are wet more than 1000 h per year on average, with over 30 events being sufficiently long (>24 h) to allow for bark-assisted hydraulic recovery. The capacity to uptake biologically meaningful volumes of water via bark and leaves for localized hydraulic recovery throughout the crown during rain/fog events might be physiologically advantageous, allowing for relatively constant transpiration., (© 2015 John Wiley & Sons Ltd.)

Measuring leaf light absorptance is central to many areas of plant biology including photosynthesis and energy balance. Absorptance is calculated from measured values of transmittance and reflectance, and most such measurements have used direct beam light. However, photosynthesis and other processes can differ under direct and diffuse light. Optical properties under diffuse light may be different, but there have been technical difficulties involved in measuring total reflectance of diffuse light. We developed instrumentation to measure this reflectance using a chopped measuring beam delivered alternately to sample and reference integrating spheres, and lock-in detection. We also built instrumentation for measuring transmittance of diffuse light. We developed standards to calibrate our instruments and correct for substitution error, a known systematic error with integrating sphere-based measurements. Helianthus annuus leaves measured under diffuse light reflected 5-10% more and transmitted a few percent less 400-700 nm light than under direct light. Overall absorptance was only a few percent higher under direct light, but leaves may utilize absorbed direct and diffuse light differently. For example, of the light entering the leaf, significantly more direct light than diffuse light is transmitted through the leaf, suggesting differences in localization of absorption within the leaf., (© 2010 The Authors. Journal Compilation. The American Society of Photobiology.)

Global-change scenarios suggest a trend of increasing diffuse light due to expected increases in cloud cover. Canopy-level measurements of plant-community photosynthesis under diffuse light show increased productivity attributed to more uniform distribution of light within the forest canopy, yet the effect of the directional quality of light at the leaf level is unknown. Here we show that leaf-level photosynthesis in sun leaves of both C(3) and C(4) plants can be 10-15% higher under direct light compared to equivalent absorbed irradiances of diffuse light. High-light-grown leaves showed significant photosynthetic enhancement in direct light, while shade-adapted leaves showed no preference for direct or diffuse light at any irradiance. High-light-grown leaves with multiple palisade layers may be adapted to better utilize direct than diffuse light, while shade leaf structure does not appear to discriminate light based on its directionality. Based upon our measurements, it appears that leaf-level and canopy-level photosynthetic processes react differently to the directionality of light, and previously observed increases in canopy-level photosynthesis occur even though leaf-level photosynthesis decreases under diffuse light.