Author Correction: Increasing CO2 threatens human nutrition.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

New approaches to dissect leaf hydraulics reveal large gradients in living tissues of tomato leaves.

The water status of the living tissue in leaves is critical in determining plant function and global exchange of water and CO2. Despite significant advances in the past two decades, persistent questions remain about the tissue-specific origins of leaf hydraulic properties and their dependence on water status. We use a fluorescent nanoparticle reporter that provides water potential in the mesophyll apoplast adjacent to the epidermis of intact leaves to complement existing methods based on the Scholander Pressure Chamber (SPC). Working in tomato leaves, this approach provides access to the hydraulic conductance of the whole leaf, xylem, and outside-xylem tissues. These measurements show that, as stem water potential decreases, the water potential in the mesophyll apoplast can drop below that assessed with the SPC and can fall significantly below the turgor loss point of the leaf. We find that this drop in potential, dominated by the large loss (10-fold) of hydraulic conductance of the outside-xylem tissue, is not however strong enough to significantly limit transpiration. These observations highlight the need to reassess models of water transfer through the outside-xylem tissues, the potential importance of this tissue in regulating transpiration, and the power of new approaches for probing leaf hydraulics.

A minimally disruptive method for measuring water potential in planta using hydrogel nanoreporters.

Leaf water potential is a critical indicator of plant water status, integrating soil moisture status, plant physiology, and environmental conditions. There are few tools for measuring plant water status (water potential) in situ, presenting a critical barrier for developing appropriate phenotyping (measurement) methods for crop development and modeling efforts aimed at understanding water transport in plants. Here, we present the development of an in situ, minimally disruptive hydrogel nanoreporter (AquaDust) for measuring leaf water potential. The gel matrix responds to changes in water potential in its local environment by swelling; the distance between covalently linked dyes changes with the reconfiguration of the polymer, leading to changes in the emission spectrum via Förster Resonance Energy Transfer (FRET). Upon infiltration into leaves, the nanoparticles localize within the apoplastic space in the mesophyll; they do not enter the cytoplasm or the xylem. We characterize the physical basis for AquaDust's response and demonstrate its function in intact maize (Zea mays L.) leaves as a reporter of leaf water potential. We use AquaDust to measure gradients of water potential along intact, actively transpiring leaves as a function of water status; the localized nature of the reporters allows us to define a hydraulic model that distinguishes resistances inside and outside the xylem. We also present field measurements with AquaDust through a full diurnal cycle to confirm the robustness of the technique and of our model. We conclude that AquaDust offers potential opportunities for high-throughput field measurements and spatially resolved studies of water relations within plant tissues.

Xylem conduit deformation across vascular plants: an evolutionary spandrel or protective valve?

The hydraulic system of vascular plants and its integrity is essential for plant survival. To transport water under tension, the walls of xylem conduits must approximate rigid pipes. Against this expectation, conduit deformation has been reported in the leaves of a few species and hypothesized to function as a 'circuit breaker' against embolism. Experimental evidence is lacking, and its generality is unknown. We demonstrated the role of conduit deformation in protecting the upstream xylem from embolism through experiments on three species and surveyed a diverse selection of vascular plants for conduit deformation in leaves. Conduit deformation in minor veins occurred before embolism during slow dehydration. When leaves were exposed to transient increases in transpiration, conduit deformation was accompanied by large water potential differences from leaf to stem and minimal embolism in the upstream xylem. In the three species tested, collapsible vein endings provided clear protection of upstream xylem from embolism during transient increases in transpiration. We found conduit deformation in diverse vascular plants, including 11 eudicots, ginkgo, a cycad, a fern, a bamboo, and a grass species, but not in two bamboo and a palm species, demonstrating that the potential for 'circuit breaker' functionality may be widespread across vascular plants.

Changes in ploidy affect vascular allometry and hydraulic function in Mangifera indica trees.

The enucleated vascular elements of the xylem and the phloem offer an excellent system to test the effect of ploidy on plant function because variation in vascular geometry has a direct influence on transport efficiency. However, evaluations of conduit sizes in polyploid plants have remained elusive, most remarkably in woody species. We used a combination of molecular, physiological and microscopy techniques to model the hydraulic resistance between source and sinks in tetraploid and diploid mango trees. Tetraploids exhibited larger chloroplasts, mesophyll cells and stomatal guard cells, resulting in higher leaf elastic modulus and lower dehydration rates, despite the high water potentials of both ploidies in the field. Both the xylem and the phloem displayed a scaling of conduits with ploidy, revealing attenuated hydraulic resistance in tetraploids. Conspicuous wall hygroscopic moieties in the cells involved in transpiration and transport indicate a role in volumetric adjustments as a result of turgor change in both ploidies. In autotetraploids, the enlargement of organelles, cells and tissues, which are critical for water and photoassimilate transport at long distances, point to major physiological novelties associated with whole-genome duplication.

Sieve tube structural variation in Austrobaileya scandens and its significance for lianescence.

Lianas combine large leaf areas with slender stems, features that require an efficient vascular system. The only extant member of the Austrobaileyaceae is an endemic twining liana of the tropical Australian forests with well-known xylem hydraulics, but the vascular phloem continuum aboveground remains understudied. Microscopy analysis across leaf vein orders and stems of Austrobaileya scandens revealed a low foliar xylem:phloem ratio, with isodiametric vascular elements along the midrib, but tapered across vein orders. Sieve plate pore radii increased from 0.08 µm in minor veins to 0.12 µm in the petiole, but only to 0.20 µm at the stem base, tens of metres away. In easily bent searcher branches, phloem conduits have pectin-rich walls and simple plates, whereas in twining stems, conduits were connected through highly angled and densely porated sieve plates. The hydraulic resistance of phloem conduits in the twisted and elongated stems of A. scandens is large compared with trees of similar stature; phloem hydraulic resistance decreases from leaves to stems, consistent with the efficient delivery of photoassimilates from sources under Münch predictions. Sink strength of a continuously growing canopy might be stronger than in self-supporting understory plants, favoring resource allocation to aerial organs and the attainment of vertical stature.

Extreme undersaturation in the intercellular airspace of leaves: a failure of Gaastra or Ohm?

BACKGROUND: Recent reports of extreme levels of undersaturation in internal leaf air spaces have called into question one of the foundational assumptions of leaf gas exchange analysis, that leaf air spaces are effectively saturated with water vapour at leaf surface temperature. Historically, inferring the biophysical states controlling assimilation and transpiration from the fluxes directly measured by gas exchange systems has presented a number of challenges, including: (1) a mismatch in scales between the area of flux measurement, the biochemical cellular scale and the meso-scale introduced by the localization of the fluxes to stomatal pores; (2) the inaccessibility of the internal states of CO2 and water vapour required to define conductances; and (3) uncertainties about the pathways these internal fluxes travel. In response, plant physiologists have adopted a set of simplifying assumptions that define phenomenological concepts such as stomatal and mesophyll conductances. SCOPE: Investigators have long been concerned that a failure of basic assumptions could be distorting our understanding of these phenomenological conductances, and the biophysical states inside leaves. Here we review these assumptions and historical efforts to test them. We then explore whether artefacts in analysis arising from the averaging of fluxes over macroscopic leaf areas could provide alternative explanations for some part, if not all, of reported extreme states of undersaturation. CONCLUSIONS: Spatial heterogeneities can, in some cases, create the appearance of undersaturation in the internal air spaces of leaves. Further refinement of experimental approaches will be required to separate undersaturation from the effects of spatial variations in fluxes or conductances. Novel combinations of current and emerging technologies hold promise for meeting this challenge.

Raman spectroscopy reveals high phloem sugar content in leaves of canopy red oak trees.

A robust understanding of phloem functioning in tall trees evades us because current methods for collecting phloem sap do not lend themselves to measuring actively photosynthesizing canopy leaves. We show that Raman spectroscopy can be used as a quantitative tool to assess sucrose concentration in leaf samples. Specifically, we found that Raman spectroscopy can predict physiologically relevant sucrose concentrations (adjusted R2 of 0.9) in frozen leaf extract spiked with sucrose. We then apply this method to estimate sieve element sucrose concentration in rapidly frozen petioles of canopy red oak (Quercus rubra) trees and found that sucrose concentrations are > 1100 mM at midday and midnight. This concentration is predicted to generate a sieve element turgor pressure high enough to generate bulk flow through the phloem, but is potentially too high to allow for sucrose diffusion from photosynthetic cells. Our findings support the Münch hypothesis for phloem transport once the carbon is in the phloem and challenge the passive-loading hypothesis for carbon movement into the phloem for red oak. This study provides the first ˜in-situ (frozen in the functioning state) source sieve element sucrose concentration characterization in any plant, opening a new avenue for investigation of phloem functioning.

Leaf Carbon Export and Nonstructural Carbohydrates in Relation to Diurnal Water Dynamics in Mature Oak Trees.

Trees typically experience large diurnal depressions in water potential, which may impede carbon export from leaves during the day because the xylem is the source of water for the phloem. As water potential becomes more negative, higher phloem osmotic concentrations are needed to draw water in from the xylem. Generating this high concentration of sugar in the phloem is particularly an issue for the ∼50% of trees that exhibit passive loading. These ideas motivate the hypothesis that carbon export in woody plants occurs predominantly at night, with sugars that accumulate during the day assisting in mesophyll turgor maintenance or being converted to starch. To test this, diurnal and seasonal patterns of leaf nonstructural carbohydrates, photosynthesis, solute, and water potential were measured, and carbon export was estimated in leaves of five mature (>20 m tall) red oak (Quercus rubra) trees, a species characterized as a passive loader. Export occurred throughout the day at equal or higher rates than at night despite a decrease in water potential to -1.8 MPa at midday. Suc and starch accumulated over the course of the day, with Suc contributing ∼50% of the 0.4 MPa diurnal osmotic adjustment. As a result of this diurnal osmotic adjustment, estimates of midday turgor were always >0.7 MPa. These findings illustrate the robustness of phloem functioning despite diurnal fluctuations in leaf water potential and the role of nonstructural carbohydrates in leaf turgor maintenance.

Idioblasts and peltate hairs as distribution networks for water absorbed by xerophilous leaves.

Capparis odoratissima is a tree species native to semi-arid environments of South America where low soil water availability coexists with frequent night-time fog. A previous study showed that water applied to leaf surfaces enhanced leaf hydration, photosynthesis and growth, but the mechanisms of foliar water uptake are unknown. Here, we combine detailed anatomical evaluations with water and dye uptake experiments in the laboratory, and use immunolocalization of pectin and arabinogalactan protein epitopes to characterize water uptake pathways in leaves. Abaxially, the leaves of C. odoratissima are covered with peltate hairs, while the adaxial surfaces are glabrous. Both surfaces are able to absorb condensed water, but the abaxial surface has higher rates of water uptake. Thousands of idioblasts per cm2 , a higher density than stomata, connect the adaxial leaf surface and the abaxial peltate hairs, both of which contain hygroscopic substances such as arabinogalactan proteins and pectins. The highly specialized anatomy of the leaves of C odoratissima fulfils the dual function of minimizing water loss when stomata are closed, while maintaining the ability to absorb liquid water. Cell-wall related hygroscopic compounds in the peltate hairs and idioblasts create a network of microchannels that maintain leaf hydration and promote water uptake.

Ecophysiological differentiation between life stages in filmy ferns (Hymenophyllaceae).

Desiccation tolerance was a key trait that allowed plants to colonize land. However, little is known about the transition from desiccation tolerant non-vascular plants to desiccation sensitive vascular ones. Filmy ferns (Hymenophyllaceae) represent a useful system to investigate how water-stress strategies differ between non-vascular and vascular stages within a single organism because they have vascularized sporophytes and nonvascular gametophytes that are each capable of varying degrees of desiccation tolerance. To explore this, we surveyed sporophytes and gametophytes of 19 species (22 taxa including varieties) of filmy ferns on Moorea (French Polynesia) and used chlorophyll fluorescence to measure desiccation tolerance and light responses. We conducted phylogenetically informed analyses to identify differences in physiology between life stages and growth habits. Gametophytes had similar or less desiccation tolerance (ability to recover from 2 days desiccation at - 86 MPa) and lower photosynthetic optima (maximum electron transport rate of photosystem II and light level at 95% of that rate) than sporophytes. Epiphytes were more tolerant of desiccation than terrestrial species in both life stages. Despite their lack of greater physiological tolerances, gametophytes of several species occurred over a wider elevational range than conspecific sporophytes. Our results demonstrate that filmy fern gametophytes and sporophytes differ in their physiology and niche requirements, and point to the importance of microhabitat in shaping the evolution of water-use strategies in vascular plants.

Visualizing Embolism Propagation in Gas-Injected Leaves.

Because the xylem in leaves is thought to be at the greatest risk of cavitation, reliable and efficient methods to characterize leaf xylem vulnerability are of interest. We report a method to generate leaf xylem vulnerability curves (VCs) by gas injection. Using optical light transmission, we visualized embolism propagation in grapevine (Vitis vinifera) and red oak (Quercus rubra) leaves injected with positive gas pressure. This resulted in a rapid, stepwise reduction of transmitted light, identical to that observed during leaf dehydration, confirming that the optical method detects gas bubbles and provides insights into the air-seeding hypothesis. In red oak, xylem VCs generated using gas injection were similar to those generated using bench dehydration, but indicated 50% loss of conductivity at lower tension (∼0.4 MPa) in grapevine. In determining VC, this method eliminates the need to ascertain xylem tension, thus avoiding potential errors in water potential estimations. It is also much faster (1 h per VC). However, severing the petiole and applying high-pressure gas could affect air-seeding and the generated VC. We discuss potential artifacts arising from gas injection and recommend comparison of this method with a more standard procedure before it is assumed to be suitable for a given species.

Wood day capacitance is related to water content, wood density, and anatomy across 30 temperate tree species.

Water released from wood during transpiration (capacitance) can meaningfully affect daily water use and drought response. To provide context for better understanding of capacitance mechanisms, we investigated links between capacitance and wood anatomy. On twigs of 30 temperate angiosperm tree species, we measured day capacitance (between predawn and midday), water content, wood density, and anatomical traits, that is, vessel dimensions, tissue fractions, and vessel-tissue contact fractions (fraction of vessel circumference in contact with other tissues). Across all species, wood density (WD) and predawn lumen volumetric water content (VWCL-pd ) together were the strongest predictors of day capacitance (r2adj = .44). Vessel-tissue contact fractions explained an additional ~10% of the variation in day capacitance. Regression models were not improved by including tissue lumen fractions. Among diffuse-porous species, VWCL-pd and vessel-ray contact fraction together were the best predictors of day capacitance, whereas among semi/ring-porous species, VWCL-pd , WD and vessel-fibre contact fraction were the best predictors. At predawn, wood was less than fully saturated for all species (lumen relative water content = 0.52 ± 0.17). Our findings imply that day capacitance depends on the amount of stored water, tissue connectivity and the bulk wood properties arising from WD (e.g., elasticity), rather than the fraction of any particular tissue.

Ontogenetic scaling of phloem sieve tube anatomy and hydraulic resistance with tree height in Quercus rubra.

PREMISE: The dimensions of phloem sieve elements have been shown to vary as a function of tree height, decreasing hydraulic resistance as the transport pathway lengthens. However, little is known about ontogenetic patterns of sieve element scaling. Here we examine within a single species (Quercus rubra) how decreases in hydraulic resistance with distance from the plant apex are mediated by overall plant size. METHODS: We sampled and imaged phloem tissue at multiple heights along the main stem and in the live crown of four size classes of trees using fluorescence and scanning electron microscopy. Sieve element length and radius, the number of sieve areas per compound plate, pore number, and pore radius were used to calculate total hydraulic resistance at each sampling location. RESULTS: Sieve element length varied with tree size, while sieve element radius, sieve pore radius, and the number of sieve areas per compound plate varied with sampling position. When data from all size classes were aggregated, all four variables followed a power-law trend with distance from the top of the tree. The net effect of these ontogenetic scalings was to make total hydraulic sieve tube resistance independent of tree height from 0.5 to over 20 m. CONCLUSIONS: Sieve element development responded to two pieces of information, tree size and distance from the apex, in a manner that conserved total sieve tube resistance across size classes. A further differentiated response between the phloem in the live crown and in the main stem is also suggested.

Increasing CO2 threatens human nutrition.

Dietary deficiencies of zinc and iron are a substantial global public health problem. An estimated two billion people suffer these deficiencies, causing a loss of 63 million life-years annually. Most of these people depend on C3 grains and legumes as their primary dietary source of zinc and iron. Here we report that C3 grains and legumes have lower concentrations of zinc and iron when grown under field conditions at the elevated atmospheric CO2 concentration predicted for the middle of this century. C3 crops other than legumes also have lower concentrations of protein, whereas C4 crops seem to be less affected. Differences between cultivars of a single crop suggest that breeding for decreased sensitivity to atmospheric CO2 concentration could partly address these new challenges to global health.

Scaling of phloem hydraulic resistance in stems and leaves of the understory angiosperm shrub Illicium parviflorum.

PREMISE OF THE STUDY: Recent studies in canopy-dominant trees revealed axial scaling of phloem structure. However, whether this pattern is found in woody plants of the understory, the environment of most angiosperms from the ANA grade (Amborellales-Nymphaeales-Austrobaileyales), is unknown. METHODS: We used seedlings and adult plants of the understory tropical shrub Illicium parviflorum, a member of the lineage Austrobaileyales, to explore the anatomy and physiology of the phloem in their aerial parts, including changes through ontogeny. KEY RESULTS: Adult plants maintain a similar proportion of phloem tissue across stem diameters, but larger conduit dimensions and number cause the hydraulic resistance of the phloem to decrease toward the base of the plant. Small sieve plate pores resulted in an overall higher sieve tube hydraulic resistance than has been reported in other woody angiosperms. Sieve elements increase in size from minor to major leaf veins, but were shorter and narrower in petioles. The low carbon assimilation rates of seedlings and mature plants contrasted with a 3-fold higher phloem sap velocity in seedlings, suggesting that phloem transport velocity is modulated through ontogeny. CONCLUSIONS: The overall architecture of the phloem tissue in this understory angiosperm shrub scales in a manner consistent with taller trees that make up the forest canopy. Thus, the evolution of larger sieve plate pores in canopy-dominant trees may have played a key role in allowing woody angiosperms to extend beyond their understory origins.

Stomatal Closure, Basal Leaf Embolism, and Shedding Protect the Hydraulic Integrity of Grape Stems.

The time scale of stomatal closure and xylem cavitation during plant dehydration, as well as the fate of embolized organs, are under debate, largely due to methodological limitations in the evaluation of embolism. While some argue that complete stomatal closure precedes the occurrence of embolism, others believe that the two are contemporaneous processes that are accompanied by daily xylem refilling. Here, we utilize an optical light transmission method to continuously monitor xylem cavitation in leaves of dehydrating grapevine (Vitis vinifera) in concert with stomatal conductance and stem and petiole hydraulic measurements. Magnetic resonance imaging was used to continuously monitor xylem cavitation and flow rates in the stem of an intact vine during 10 d of dehydration. The results showed that complete stomatal closure preceded the appearance of embolism in the leaves and the stem by several days. Basal leaves were more vulnerable to xylem embolism than apical leaves and, once embolized, were shed, thereby preventing further water loss and protecting the hydraulic integrity of younger leaves and the stem. As a result, embolism in the stem was minimal even when drought led to complete leaf shedding. These findings suggest that grapevine avoids xylem embolism rather than tolerates it.

Comparing optimal and empirical stomatal conductance models for application in Earth system models.

Earth system models (ESMs) rely on the calculation of canopy conductance in land surface models (LSMs) to quantify the partitioning of land surface energy, water, and CO2 fluxes. This is achieved by scaling stomatal conductance, gw , determined from physiological models developed for leaves. Traditionally, models for gw have been semi-empirical, combining physiological functions with empirically determined calibration constants. More recently, optimization theory has been applied to model gw in LSMs under the premise that it has a stronger grounding in physiological theory and might ultimately lead to improved predictive accuracy. However, this premise has not been thoroughly tested. Using original field data from contrasting forest systems, we compare a widely used empirical type and a more recently developed optimization-type gw model, termed BB and MED, respectively. Overall, we find no difference between the two models when used to simulate gw from photosynthesis data, or leaf gas exchange from a coupled photosynthesis-conductance model, or gross primary productivity and evapotranspiration for a FLUXNET tower site with the CLM5 community LSM. Field measurements reveal that the key fitted parameters for BB and MED, g1B and g1M, exhibit strong species specificity in magnitude and sensitivity to CO2 , and CLM5 simulations reveal that failure to include this sensitivity can result in significant overestimates of evapotranspiration for high-CO2 scenarios. Further, we show that g1B and g1M can be determined from mean ci /ca (ratio of leaf intercellular to ambient CO2 concentration). Applying this relationship with ci /ca values derived from a leaf δ13 C database, we obtain a global distribution of g1B and g1M , and these values correlate significantly with mean annual precipitation. This provides a new methodology for global parameterization of the BB and MED models in LSMs, tied directly to leaf physiology but unconstrained by spatial boundaries separating designated biomes or plant functional types.

Reversible Leaf Xylem Collapse: A Potential "Circuit Breaker" against Cavitation.

We report a novel form of xylem dysfunction in angiosperms: reversible collapse of the xylem conduits of the smallest vein orders that demarcate and intrusively irrigate the areoles of red oak (Quercus rubra) leaves. Cryo-scanning electron microscopy revealed gradual increases in collapse from approximately -2 MPa down to -3 MPa, saturating thereafter (to -4 MPa). Over this range, cavitation remained negligible in these veins. Imaging of rehydration experiments showed spatially variable recovery from collapse within 20 s and complete recovery after 2 min. More broadly, the patterns of deformation induced by desiccation in both mesophyll and xylem suggest that cell wall collapse is unlikely to depend solely on individual wall properties, as mechanical constraints imposed by neighbors appear to be important. From the perspective of equilibrium leaf water potentials, petioles, whose vessels extend into the major veins, showed a vulnerability to cavitation that overlapped in the water potential domain with both minor vein collapse and buckling (turgor loss) of the living cells. However, models of transpiration transients showed that minor vein collapse and mesophyll capacitance could effectively buffer major veins from cavitation over time scales relevant to the rectification of stomatal wrong-way responses. We suggest that, for angiosperms, whose subsidiary cells give up large volumes to allow large stomatal apertures at the cost of potentially large wrong-way responses, vein collapse could make an important contribution to these plants' ability to transpire near the brink of cavitation-inducing water potentials.

The tomato plastidic fructokinase SlFRK3 plays a role in xylem development.

Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylation, cytosolic and plastidic. The major cytosolic tomato FRK, SlFRK2, is essential for the development of xylem vessels. In order to study the role of SlFRK3, which encodes the only plastidic FRK, we generated transgenic tomato (Solanum lycopersicon) plants with RNAi suppression of SlFRK3 as well as plants expressing beta-glucoronidase (GUS) under the SlFRK3 promoter. GUS staining indicated SlFRK3 expression in vascular tissues of the leaves and stems, including cambium, differentiating xylem, young xylem fibers and phloem companion cells. Suppression of SlFRK3 reduced the stem xylem area, stem and root water conductance, and whole-plant transpiration, with minor effects on plant development. However, suppression of SlFRK3 accompanied by partial suppression of SlFRK2 induced significant growth-inhibition effects, including the wilting of mature leaves. Grafting experiments revealed that these growth effects are imposed primarily by the leaves, whose petioles had unlignified, thin-walled xylem fibers with collapsed parenchyma cells around the vessels. A cross between the SlFRK2-antisense and SlFRK3-RNAi lines exhibited similar wilting and anatomical effects, confirming that these effects are the result of the combined suppression of SlFRK3 and SlFRK2. These results demonstrate a role of the plastidic SlFRK3 in xylem development and hydraulic conductance.