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.

Leaves are arguably the most complex and important physicobiological systems in the ecosphere. Yet, water transport outside the leaf xylem remains poorly understood, despite its impacts on stomatal function and photosynthesis. We applied anatomical measurements from 14 diverse species to a novel model of water flow in an areole (the smallest region bounded by minor veins) to predict the impact of anatomical variation across species on outside-xylem hydraulic conductance (K ox ). Several predictions verified previous correlational studies: (1) vein length per unit area is the strongest anatomical determinant of K ox , due to effects on hydraulic pathlength and bundle sheath (BS) surface area; (2) palisade mesophyll remains well hydrated in hypostomatous species, which may benefit photosynthesis, (3) BS extensions enhance K ox ; and (4) the upper and lower epidermis are hydraulically sequestered from one another despite their proximity. Our findings also provided novel insights: (5) the BS contributes a minority of outside-xylem resistance; (6) vapor transport contributes up to two-thirds of K ox ; (7) K ox is strongly enhanced by the proximity of veins to lower epidermis; and (8) K ox is strongly influenced by spongy mesophyll anatomy, decreasing with protoplast size and increasing with airspace fraction and cell wall thickness. Correlations between anatomy and K ox across species sometimes diverged from predicted causal effects, demonstrating the need for integrative models to resolve causation. For example, (9) K ox was enhanced far more in heterobaric species than predicted by their having BS extensions. Our approach provides detailed insights into the role of anatomical variation in leaf function.

Leaf vein traits are implicated in the determination of gas exchange rates and plant performance. These traits are increasingly considered as causal factors affecting the 'leaf economic spectrum' (LES), which includes the light-saturated rate of photosynthesis, dark respiration, foliar nitrogen concentration, leaf dry mass per area (LMA) and leaf longevity. This article reviews the support for two contrasting hypotheses regarding a key vein trait, vein length per unit leaf area (VLA). Recently, Blonder et al. (2011, 2013) proposed that vein traits, including VLA, can be described as the 'origin' of the LES by structurally determining LMA and leaf thickness, and thereby vein traits would predict LES traits according to specific equations. Careful re-examination of leaf anatomy, published datasets, and a newly compiled global database for diverse species did not support the 'vein origin' hypothesis, and moreover showed that the apparent power of those equations to predict LES traits arose from circularity. This review provides a 'flux trait network' hypothesis for the effects of vein traits on the LES and on plant performance, based on a synthesis of the previous literature. According to this hypothesis, VLA, while virtually independent of LMA, strongly influences hydraulic conductance, and thus stomatal conductance and photosynthetic rate. We also review (i) the specific physiological roles of VLA; (ii) the role of leaf major veins in influencing LES traits; and (iii) the role of VLA in determining photosynthetic rate per leaf dry mass and plant relative growth rate. A clear understanding of leaf vein traits provides a new perspective on plant function independently of the LES and can enhance the ability to explain and predict whole plant performance under dynamic conditions, with applications towards breeding improved crop varieties.

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 ) during dehydration was driven by embolism initiating in petioles and midribs across all species, and K vulnerability was strongly correlated with petiole and midrib conduit dimensions. Our simulations showed no significant impact of conduit collapse on K decline. We found xylem conduit dimensions play a major role in determining the susceptibility of the leaf water transport system during strong leaf dehydration.

Leaf dry mass per unit leaf area (LMA) is a central trait in ecology, but its anatomical and compositional basis has been unclear. An explicit mathematical and physical framework for quantifying the cell and tissue determinants of LMA will enable tests of their influence on species, communities and ecosystems. We present an approach to explaining LMA from the numbers, dimensions and mass densities of leaf cells and tissues, which provided unprecedented explanatory power for 11 broadleaved woody angiosperm species diverse in LMA (33-262 g m ; R = 0.94; P < 0.001). Across these diverse species, and in a larger comparison of evergreen vs. deciduous angiosperms, high LMA resulted principally from larger cell sizes, greater major vein allocation, greater numbers of mesophyll cell layers and higher cell mass densities. This explicit approach enables relating leaf anatomy and composition to a wide range of processes in physiological, evolutionary, community and macroecology.

Stomata, the microvalves on leaf surfaces, exert major influences across scales, from plant growth and productivity to global carbon and water cycling. Stomatal opening enables leaf photosynthesis, and plant growth and water use, whereas plant survival of drought depends on stomatal closure. Here we report that stomatal function is constrained by a safety-efficiency trade-off, such that species with greater stomatal conductance under high water availability ( g max ) show greater sensitivity to closure during leaf dehydration, i.e., a higher leaf water potential at which stomatal conductance is reduced by 50% (Ψ gs50 ). The g max - Ψ gs50 trade-off and its mechanistic basis is supported by experiments on leaves of California woody species, and in analyses of previous studies of the responses of diverse flowering plant species around the world. Linking the two fundamental key roles of stomata—the enabling of gas exchange, and the first defense against drought—this trade-off constrains the rates of water use and the drought sensitivity of leaves, with potential impacts on ecosystems.

The sites of evaporation within leaves are unknown, but they have drawn attention for decades due to their perceived implications for many factors, including patterns of leaf isotopic enrichment, the maintenance of mesophyll water status, stomatal regulation, and the interpretation of measured stomatal and leaf hydraulic conductances. We used a spatially explicit model of coupled water and heat transport outside the xylem, MOFLO 2.0, to map the distribution of net evaporation across leaf tissues in relation to anatomy and environmental parameters. Our results corroborate earlier predictions that most evaporation occurs from the epidermis at low light and moderate humidity but that the mesophyll contributes substantially when the leaf center is warmed by light absorption, and more so under high humidity. We also found that the bundle sheath provides a significant minority of evaporation (15% in darkness and 18% in high light), that the vertical center of amphistomatous leaves supports net condensation, and that vertical temperature gradients caused by light absorption vary over 10-fold across species, reaching 0.3°C. We show that several hypotheses that depend on the evaporating sites require revision in light of our findings, including that experimental measurements of stomatal and hydraulic conductances should be affected directly by changes in the location of the evaporating sites. We propose a new conceptual model that accounts for mixed-phase water transport outside the xylem. These conclusions have far-reaching implications for inferences in leaf hydraulics, gas exchange, water use, and isotope physiology.The pathways for water transport through a plant are often perceived to end at the sites of evaporation outside the xylem within leaves (Holmgren et al

Summary Given increasing water deficits across numerous ecosystems world‐wide, it is urgent to understand the sequence of failure of leaf function during dehydration. We assessed dehydration‐induced losses of rehydration capacity and maximum quantum yield of the photosystem II (Fv/Fm) in the leaves of 10 diverse angiosperm species, and tested when these occurred relative to turgor loss, declines of stomatal conductance gs, and hydraulic conductance Kleaf, including xylem and outside xylem pathways for the same study plants. We resolved the sequences of relative water content and leaf water potential Ψleaf thresholds of functional impairment. On average, losses of leaf rehydration capacity occurred at dehydration beyond 50% declines of gs, Kleaf and turgor loss point. Losses of Fv/Fm occurred after much stronger dehydration and were not recovered with leaf rehydration. Across species, tissue dehydration thresholds were intercorrelated, suggesting trait co‐selection. Thresholds for each type of functional decline were much less variable across species in terms of relative water content than Ψleaf. The stomatal and leaf hydraulic systems show early functional declines before cell integrity is lost. Substantial damage to the photochemical apparatus occurs at extreme dehydration, after complete stomatal closure, and seems to be irreversible.