Increasing drought is one of the most critical challenges facing species and ecosystems worldwide, and improved theory and practices are needed for quantification of species tolerances. Leaf water potential at turgor loss, or wilting (π(tlp) ), is classically recognised as a major physiological determinant of plant water stress response. However, the cellular basis of π(tlp) and its importance for predicting ecological drought tolerance have been controversial. A meta-analysis of 317 species from 72 studies showed that π(tlp) was strongly correlated with water availability within and across biomes, indicating power for anticipating drought responses. We derived new equations giving both π(tlp) and relative water content at turgor loss point (RWC(tlp) ) as explicit functions of osmotic potential at full turgor (π(o) ) and bulk modulus of elasticity (ε). Sensitivity analyses and meta-analyses showed that π(o) is the major driver of π(tlp) . In contrast, ε plays no direct role in driving drought tolerance within or across species, but sclerophylly and elastic adjustments act to maintain RWC(tlp,) preventing cell dehydration, and additionally protect against nutrient, mechanical and herbivory stresses independent of drought tolerance. These findings clarify biogeographic trends and the underlying basis of drought tolerance parameters with applications in comparative assessments of species and ecosystems worldwide.

983I.983II.983III.984IV.990V.992VI.992VII.993VIII.995IX.996996References996 Summary The design and function of leaf venation are important to plant performance, with key implications for the distribution and productivity of ecosystems, and applications in paleobiology, agriculture and technology. We synthesize classical concepts and the recent literature on a wide range of aspects of leaf venation. We describe 10 major structural features that contribute to multiple key functions, and scale up to leaf and plant performance. We describe the development and plasticity of leaf venation and its adaptation across environments globally, and a new global data compilation indicating trends relating vein length per unit area to climate, growth form and habitat worldwide. We synthesize the evolution of vein traits in the major plant lineages throughout paleohistory, highlighting the multiple origins of individual traits. We summarize the strikingly diverse current applications of leaf vein research in multiple fields of science and industry. A unified core understanding will enable an increasing range of plant biologists to incorporate leaf venation into their research.

Leaf size and venation show remarkable diversity across dicotyledons, and are key determinants of plant adaptation in ecosystems past and present. Here we present global scaling relationships of venation traits with leaf size. Across a new database for 485 globally distributed species, larger leaves had major veins of larger diameter, but lower length per leaf area, whereas minor vein traits were independent of leaf size. These scaling relationships allow estimation of intact leaf size from fragments, to improve hindcasting of past climate and biodiversity from fossil remains. The vein scaling relationships can be explained by a uniquely synthetic model for leaf anatomy and development derived from published data for numerous species. Vein scaling relationships can explain the global biogeographical trend for smaller leaves in drier areas, the greater construction cost of larger leaves and the ability of angiosperms to develop larger and more densely vascularised lamina to outcompete earlier-evolved plant lineages.

Across plant species, leaves vary enormously in their size and their venation architecture, of which one major function is to replace water lost to transpiration. The leaf hydraulic conductance (K leaf ) represents the capacity of the transport system to deliver water, allowing stomata to remain open for photosynthesis. Previous studies showed that K leaf relates to vein density (vein length per area). Additionally, venation architecture determines the sensitivity of K leaf to damage; severing the midrib caused K leaf and gas exchange to decline, with lesser impacts in leaves with higher major vein density that provided more numerous water flow pathways around the damaged vein. Because xylem embolism during dehydration also reduces K leaf , we hypothesized that higher major vein density would also reduce hydraulic vulnerability. Smaller leaves, which generally have higher major vein density, would thus have lower hydraulic vulnerability. Tests using simulations with a spatially explicit model confirmed that smaller leaves with higher major vein density were more tolerant of major vein embolism. Additionally, for 10 species ranging strongly in drought tolerance, hydraulic vulnerability, determined as the leaf water potential at 50% and 80% loss of K leaf , was lower with greater major vein density and smaller leaf size (|r| = 0.85-0.90; P , 0.01). These relationships were independent of other aspects of physiological and morphological drought tolerance. These findings point to a new functional role of venation architecture and small leaf size in drought tolerance, potentially contributing to well-known biogeographic trends in leaf size.

Summary1. Across plant species, drought tolerance and distributions with respect to water availability are strongly correlated with two physiological traits, the leaf water potential at wilting, that is, turgor loss point (p tlp ), and the cell solute potential at full hydration, that is, osmotic potential (p o ). We present methods to determine these parameters 30 times more rapidly than the standard pressurevolume (p-v) curve approach, making feasible community-scale studies of plant drought tolerance. 2. We optimized existing methods for measurements of p o using vapour-pressure osmometry of freeze-thawed leaf discs from 30 species growing in two precipitation regimes, and developed the first regression relationships to accurately estimate pressure-volume curve values of both p o and p tlp from osmometer values . 3. The p o determined with the osmometer (p osm ) was an excellent predictor of the p o determined from the p-v curve (p pv, r 2 = 0AE80). Although the correlation of p osm and p pv enabled prediction, the relationship departed from the 1 : 1 line. The discrepancy between the methods could be quantitatively accounted for by known sources of error in osmometer measurements, that is, dilution by the apoplastic water, and solute dissolution from destroyed cell walls. An even stronger prediction of p pv could be made using p osm, leaf density (q), and their interaction (r 2 = 0AE85, all P < 2 · 10 )10). 4. The p osm could also be used to predict p tlp (r 2 = 0AE86). Indeed, p osm was a better predictor of p tlp than leaf mass per unit area (LMA; r 2 = 0AE54), leaf thickness (T; r 2 = 0AE12), q (r 2 = 0AE63), and leaf dry matter content (LDMC; r 2 = 0AE60), which have been previously proposed as drought tolerance indicators. Models combining p osm with LMA, T, q, or LDMC or other p-v curve parameters (i.e. elasticity and apoplastic fraction) did not significantly improve prediction of p tlp . 5. This osmometer method enables accurate measurements of drought tolerance traits across a wide range of leaf types and for plants with diverse habitat preferences, with a fraction of effort of previous methods. We expect it to have wide application for predicting species responses to climate variability and for assessing ecological and evolutionary variation in drought tolerance in natural populations and agricultural cultivars.

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.

Two highly contrasting variables summarizing the efficiency of transport of materials within the leaf are recognized as playing central roles in determining gas exchange and plant performance. This paper summarizes current approaches for the measurement of mesophyll conductance to CO2 (g m) and leaf hydraulic conductance (K leaf) and addresses the physiological integration of these parameters. First, the most common methods to determine g m and K leaf are summarized. Next, novel data compilation is analysed, which indicates that, across diverse species, g m is strongly linked with gas exchange parameters such as net CO2 assimilation (A area) and stomatal conductance (g s), and with K leaf, independently of leaf vein length per leaf area. Based on their parallel responses to a number of environmental variables, this review proposes that g m is linked to the outside-xylem but not to the xylem component of K leaf. Further, a mechanistic hypothesis is proposed to explain the interactions among all these and other physiological parameters. Finally, the possibility of estimating g m based on this hypothesis was tested using a regression analysis and a neurofuzzy logic approach. These approaches enabled the estimation of g m of given species from K leaf and leaf mass per area, providing a higher predictive power than from either parameter alone. The possibility of estimating g m from measured K leaf or vice-versa would result in a rapid increase in available data. Studies in which g m, K leaf, and leaf mass per area are simultaneously determined are needed in order to confirm and strengthen predictive and explanatory models for these parameters and importantly improve resolution of the integrated hydraulic-stomatal-photosynthetic system.