Developmental and biophysical determinants of grass leaf size worldwide.

One of the most notable ecological trends-described more than 2,300  years ago by Theophrastus-is the association of small leaves with dry and cold climates, which has recently been recognized for eudicotyledonous plants at a global scale1-3. For eudicotyledons, this pattern has been attributed to the fact that small leaves have a thinner boundary layer that helps to avoid extreme leaf temperatures4 and their leaf development results in vein traits that improve water transport under cold or dry climates5,6. However, the global distribution of leaf size and its adaptive basis have not been tested in the grasses, which represent a diverse lineage that is distinct in leaf morphology and that contributes 33% of terrestrial primary productivity (including the bulk of crop production)7. Here we demonstrate that grasses have shorter and narrower leaves under colder and drier climates worldwide. We show that small grass leaves have thermal advantages and vein development that contrast with those of eudicotyledons, but that also explain the abundance of small leaves in cold and dry climates. The worldwide distribution of leaf size in grasses exemplifies how biophysical and developmental processes result in convergence across major lineages in adaptation to climate globally, and highlights the importance of leaf size and venation architecture for grass performance in past, present and future ecosystems.

Pinpointing the causal influences of stomatal anatomy and behavior on minimum, operational, and maximum leaf surface conductance.

Leaf surface conductance to water vapor and CO2 across the epidermis (gleaf) strongly determines rates of gas exchange. Thus, clarifying the drivers of gleaf has important implications for resolving mechanisms of photosynthetic productivity and leaf and plant responses and tolerance to drought. It is well recognized that gleaf is a function of the conductances of the stomata (gs) and of the epidermis + cuticle (gec). Yet, controversies have arisen around the relative roles of stomatal density (d) and size (s), fractional stomatal opening (α; aperture relative to maximum) and gec in determining gleaf. Resolving the importance of these drivers is critical across the range of leaf surface conductances, from strong stomatal closure under drought (gleaf, min), to typical opening for photosynthesis (gleaf, op), to maximum achievable opening (gleaf, max). We derived equations and analyzed a compiled database of published and measured data for approximately 200 species and genotypes. On average, within and across species, higher gleaf, min was determined ten times more strongly by α and gec than by d, and negligibly by s; higher gleaf, op was determined approximately equally by α (47%) than by stomatal anatomy (45% by d, and 8% by s), and negligibly by gec; and higher gleaf, max was determined entirely by d. These findings clarify how diversity in stomatal functioning arises from multiple structural and physiological causes with importance shifting with context. The rising importance of d relative to α, from gleaf, min to gleaf, op, enables even species with low gleaf, min, which can retain leaves through drought, to possess high d and thereby achieve rapid gas exchange in periods of high water availability.

The dynamic multi-functionality of leaf water transport outside the xylem.

A surge of papers have reported low leaf vulnerability to xylem embolism during drought. Here, we focus on the less studied, and more sensitive, outside-xylem leaf hydraulic responses to multiple internal and external conditions. Studies of 34 species have resolved substantial vulnerability to dehydration of the outside-xylem pathways, and studies of leaf hydraulic responses to light also implicate dynamic outside-xylem responses. Detailed experiments suggest these dynamic responses arise at least in part from strong control of radial water movement across the vein bundle sheath. While leaf xylem vulnerability may influence leaf and plant survival during extreme drought, outside-xylem dynamic responses are important for the control and resilience of water transport and leaf water status for gas exchange and growth.

Thresholds for persistent leaf photochemical damage predict plant drought resilience in a tropical rainforest.

Water stress can cause declines in plant function that persist after rehydration. Recent work has defined 'resilience' traits characterizing leaf resistance to persistent damage from drought, but whether these traits predict resilience in whole-plant function is unknown. It is also unknown whether the coordination between resilience and 'resistance' - the ability to maintain function during drought - observed globally occurs within ecosystems. For eight rainforest species, we dehydrated and subsequently rehydrated leaves, and measured water stress thresholds for declines in rehydration capacity and maximum quantum yield of photosystem II (Fv /Fm ). We tested correlations with embolism resistance and dry season water potentials (ΨMD ), and calculated safety margins for damage (ΨMD - thresholds) and tested correlations with drought resilience in sap flow and growth. Ψ thresholds for persistent declines in Fv /Fm , indicating resilience, were positively correlated with ΨMD and thresholds for leaf vein embolism. Safety margins for persistent declines in Fv /Fm , but not rehydration capacity, were positively correlated with drought resilience in sap flow. Correlations between resistance and resilience suggest that species' differences in performance during drought are perpetuated after drought, potentially accelerating shifts in forest composition. Resilience to photochemical damage emerged as a promising functional trait to characterize whole-plant drought resilience.

Rapid drought-recovery of gas exchange in Caragana species adapted to low mean annual precipitation.

While variation in mean annual precipitation (MAP) of the native habitat of a species has been shown to determine the ability of a species to resist a hydraulic decrease during drought, it remains unknown whether these variations in MAP also influence the ability of a species to recover and survive drought. Leaf hydraulic and gas exchange recovery following drought and the underlying mechanisms of these responses in six Caragana species from habitats along a large precipitation gradient were investigated during rehydration in a common garden. The gas exchange of species from arid habitats recovered more rapidly during rehydration after mild, moderate and severe drought stress treatments than species from humid habitats. The recovery of gas exchange was not associated with foliar abscisic acid concentration, but tightly related to the recovery of leaf hydraulic conductance (Kleaf ). The recovery of Kleaf was associated with the loss of Kleaf during dehydration under mild and moderate drought stress, and to leaf xylem embolism formation under severe drought stress. Results pointed to the different ability to recover in gas exchange in six Caragana species post-drought is associated with the MAP of the species in its native habitat.

Low baseline intraspecific variation in leaf pressure-volume traits: Biophysical basis and implications for spectroscopic sensing.

Intra-specific trait variation (ITV) plays a role in processes at a wide range of scales from organs to ecosystems across climate gradients. Yet, ITV remains rarely quantified for many ecophysiological traits typically assessed for species means, such as pressure volume (PV) curve parameters including osmotic potential at full turgor and modulus of elasticity, which are important in plant water relations. We defined a baseline "reference ITV" (ITVref ) as the variation among fully exposed, mature sun leaves of replicate individuals of a given species grown in similar, well-watered conditions, representing the conservative sampling design commonly used for species-level ecophysiological traits. We hypothesized that PV parameters would show low ITVref relative to other leaf morphological traits, and that their intraspecific relationships would be similar to those previously established across species and proposed to arise from biophysical constraints. In a database of novel and published PV curves and additional leaf structural traits for 50 diverse species, we found low ITVref for PV parameters relative to other morphological traits, and strong intraspecific relationships among PV traits. Simulation modeling showed that conservative ITVref enables the use of species-mean PV parameters for scaling up from spectroscopic measurements of leaf water content to enable sensing of leaf water potential.

Testing the association of relative growth rate and adaptation to climate across natural ecotypes of Arabidopsis.

Ecophysiologists have reported a range of relationships, including intrinsic trade-offs across and within species between plant relative growth rate in high resource conditions (RGR) vs adaptation to tolerate cold or arid climates, arising from trait-based mechanisms. Few studies have considered ecotypes within a species, in which the lack of a trade-off would contribute to a wide species range and resilience to climate change. For 15 ecotypes of Arabidopsis thaliana in a common garden we tested for associations between RGR vs adaptation to cold or dry native climates and assessed hypotheses for its mediation by 15 functional traits. Ecotypes native to warmer, drier climates had higher leaf density, leaf mass per area, root mass fraction, nitrogen per leaf area and carbon isotope ratio, and lower osmotic potential at full turgor. Relative growth rate was statistically independent of the climate of the ecotype native range and of individual functional traits. The decoupling of RGR and cold or drought adaptation in Arabidopsis is consistent with multiple stress resistance and avoidance mechanisms for ecotypic climate adaptation and would contribute to the species' wide geographic range and resilience as the climate changes.

Combined high leaf hydraulic safety and efficiency provides drought tolerance in Caragana species adapted to low mean annual precipitation.

Clarifying the coordination of leaf hydraulic traits with gas exchange across closely-related species adapted to varying rainfall can provide insights into plant habitat distribution and drought adaptation. The leaf hydraulic conductance (Kleaf ), stomatal conductance (gs ), net assimilation (A), vein embolism and abscisic acid (ABA) concentration during dehydration were quantified, as well as pressure-volume curve traits and vein anatomy in 10 Caragana species adapted to a range of mean annual precipitation (MAP) conditions and growing in a common garden. We found a positive correlation between Ψleaf at 50% loss of Kleaf (Kleaf P50 ) and maximum Kleaf (Kleaf-max ) across species. Species from low-MAP environments exhibited more negative Kleaf P50 and turgor loss point, and higher Kleaf-max and leaf-specific capacity at full turgor, along with higher vein density and midrib xylem per leaf area, and a higher ratio of Kleaf-max : maximum gs . Tighter stomatal control mediated by higher ABA accumulation during dehydration in these species resulted in an increase in hydraulic safety and intrinsic water use efficiency (WUEi ) during drought. Our results suggest that high hydraulic safety and efficiency combined with greater stomatal sensitivity triggered by ABA production and leading to greater WUEi provides drought tolerance in Caragana species adapted to low-MAP environments.

Deciduous and evergreen oaks show contrasting adaptive responses in leaf mass per area across environments.

Increases in leaf mass per area (LMA) are commonly observed in response to environmental stresses and are achieved through increases in leaf thickness and/or leaf density. Here, we investigated how the two underlying components of LMA differ in relation to species native climates and phylogeny, across deciduous and evergreen species. Using a phylogenetic approach, we quantified anatomical, compositional and climatic variables from 40 deciduous and 45 evergreen Quercus species from across the Northern Hemisphere growing in a common garden. Deciduous species from shorter growing seasons tended to have leaves with lower LMA and leaf thickness than those from longer growing seasons, while the opposite pattern was found for evergreens. For both habits, LMA and thickness increased in arid environments. However, this shift was associated with increased leaf density in evergreens but reduced density in deciduous species. Deciduous and evergreen oaks showed fundamental leaf morphological differences that revealed a diverse adaptive response. While LMA in deciduous species may have diversified in tight coordination with thickness mainly modulated by aridity, diversification of LMA within evergreens appears to be dependent on the infrageneric group, with diversification in leaf thickness modulated by both aridity and cold, while diversification in leaf density is only modulated by aridity.

Coordinated decline of leaf hydraulic and stomatal conductances under drought is not linked to leaf xylem embolism for different grapevine cultivars.

Drought decreases water transport capacity of leaves and limits gas exchange, which involves reduced leaf leaf hydraulic conductance (Kleaf) in both the xylem and outside-xylem pathways. Some literature suggests that grapevines are hyper-susceptible to drought-induced xylem embolism. We combined Kleaf and gas exchange measurements, micro-computed tomography of intact leaves, and spatially explicit modeling of the outside-xylem pathways to evaluate the role of vein embolism and Kleaf in the responses of two different grapevine cultivars to drought. Cabernet Sauvignon and Chardonnay exhibited similar vulnerabilities of Kleaf and gs to dehydration, decreasing substantially prior to leaf xylem embolism. Kleaf and gs decreased by 80% for both cultivars by Ψ leaf approximately -0.7 MPa and -1.2 MPa, respectively, while leaf xylem embolism initiated around Ψ leaf = -1.25 MPa in the midribs and little to no embolism was detected in minor veins even under severe dehydration for both cultivars. Modeling results indicated that reduced membrane permeability associated with a Casparian-like band in the leaf vein bundle sheath would explain declines in Kleaf of both cultivars. We conclude that during moderate water stress, changes in the outside-xylem pathways, rather than xylem embolism, are responsible for reduced Kleaf and gs. Understanding this mechanism could help to ensure adequate carbon capture and crop performance under drought.

Thresholds for leaf damage due to dehydration: declines of hydraulic function, stomatal conductance and cellular integrity precede those for photochemistry.

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.

The Causes of Leaf Hydraulic Vulnerability and Its Influence on Gas Exchange in Arabidopsis thaliana.

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.

The Sites of Evaporation within Leaves.

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.

Outside-Xylem Vulnerability, Not Xylem Embolism, Controls Leaf Hydraulic Decline during Dehydration.

Leaf hydraulic supply is crucial to maintaining open stomata for CO2 capture and plant growth. During drought-induced dehydration, the leaf hydraulic conductance (Kleaf) declines, which contributes to stomatal closure and, eventually, to leaf death. Previous studies have tended to attribute the decline of Kleaf 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 Kleaf 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 Kleaf 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.

Is xylem of angiosperm leaves less resistant to embolism than branches? Insights from microCT, hydraulics, and anatomy.

According to the hydraulic vulnerability segmentation hypothesis, leaves are more vulnerable to decline of hydraulic conductivity than branches, but whether stem xylem is more embolism resistant than leaves remains unclear. Drought-induced embolism resistance of leaf xylem was investigated based on X-ray microcomputed tomography (microCT) for Betula pendula, Laurus nobilis, and Liriodendron tulipifera, excluding outside-xylem, and compared with hydraulic vulnerability curves for branch xylem. Moreover, bordered pit characters related to embolism resistance were investigated for both organs. Theoretical P50 values (i.e. the xylem pressure corresponding to 50% loss of hydraulic conductance) of leaves were generally within the same range as hydraulic P50 values of branches. P50 values of leaves were similar to branches for L. tulipifera (-2.01 versus -2.10 MPa, respectively), more negative for B. pendula (-2.87 versus -1.80 MPa), and less negative for L. nobilis (-6.4 versus -9.2 MPa). Despite more narrow conduits in leaves than branches, mean interconduit pit membrane thickness was similar in both organs, but significantly higher in leaves of B. pendula than in branches. This case study indicates that xylem shows a largely similar embolism resistance across leaves and branches, although differences both within and across organs may occur, suggesting interspecific variation with regard to the hydraulic vulnerability segmentation hypothesis.

Evolution of leaf structure and drought tolerance in species of Californian Ceanothus.

PREMISE OF THE STUDY: Studies across diverse species have established theory for the contribution of leaf traits to plant drought tolerance. For example, species in more arid climates tend to have smaller leaves of higher vein density, higher leaf mass per area, and more negative osmotic potential at turgor loss point (πTLP ). However, few studies have tested these associations for species within a given lineage that have diversified across an aridity gradient. METHODS: We analyzed the anatomy and physiology of 10 Ceanothus (Rhamnaceae) species grown in a common garden for variation between and within "wet" and "dry" subgenera (Ceanothus and Cerastes, respectively) and analyzed a database for 35 species for leaf size and leaf mass per area (LMA). We used a phylogenetic generalized least squares approach to test hypothesized relationships among traits, and of traits with climatic aridity in the native range. We also tested for allometric relationships among anatomical traits. KEY RESULTS: Leaf form, anatomy, and drought tolerance varied strongly among species within and between subgenera. Cerastes species had specialized anatomy including hypodermis and encrypted stomata that may confer superior water storage and retention. The osmotic potentials at turgor loss point (πTLP ) and full turgor (πo ) showed evolutionary correlations with the aridity index (AI) and precipitation of the 10 species' native distributions, and LMA with potential evapotranspiration for the 35 species in the larger database. We found an allometric correlation between upper and lower epidermal cell wall thicknesses, but other anatomical traits diversified independently. CONCLUSIONS: Leaf traits and drought tolerance evolved within and across lineages of Ceanothus consistently with climatic distributions. The πTLP has signal to indicate the evolution of drought tolerance within small clades.

The anatomical and compositional basis of leaf mass per area.

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-2 ; R2  = 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.

Leaf vein xylem conduit diameter influences susceptibility to embolism and hydraulic decline.

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

The causes and consequences of leaf hydraulic decline with dehydration.

Resolving the drivers of hydraulic decline during drought is crucial for understanding drought tolerance in crops and natural ecosystems. In the past 15 years, studies of the decline of leaf hydraulic conductance (Kleaf) have supported a major role in controlling plant drought responses. We analyzed the variation in Kleaf decline with dehydration in a global database of 310 species, providing novel insights into its underlying mechanisms, its co-ordination with stem hydraulics, its influence on gas exchange and drought tolerance, and its linkage with species ecological distributions. Kleaf vulnerability varied strongly within and across lineages, growth forms, and biomes. A critical literature review indicates that changes in hydraulic conductance outside the xylem with dehydration drive the overall decline of Kleaf. We demonstrate a significant leaf hydraulic safety-efficiency trade-off across angiosperm species and discuss the importance of the large variation around this trend. Leaves tend to be more vulnerable than stems, with their vulnerabilities co-ordinated across species, and importantly linked with adaptation across biomes. We hypothesize a novel framework to explain diversity across species in the co-ordination of Kleaf and gas exchange during dehydration. These findings reflect considerable recent progress, yet new tools for measurement, visualization, and modeling will result in ongoing discoveries important across fields in plant biology.

How Does Leaf Anatomy Influence Water Transport outside the Xylem?

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 (Kox). Several predictions verified previous correlational studies: (1) vein length per unit area is the strongest anatomical determinant of Kox, 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 Kox; 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 Kox; (7) Kox is strongly enhanced by the proximity of veins to lower epidermis; and (8) Kox is strongly influenced by spongy mesophyll anatomy, decreasing with protoplast size and increasing with airspace fraction and cell wall thickness. Correlations between anatomy and Kox across species sometimes diverged from predicted causal effects, demonstrating the need for integrative models to resolve causation. For example, (9) Kox 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.