Stomatal function results from structural movements of guard cells in which an increase in their volume opens the pore, while a decrease in volume closes the pore. In angiosperms, neighboring epidermal cells are described as having a mechanical advantage over the guard cells, yet we have limited information on how epidermal cells contribute to stomatal pore formation. Here we visualize the structural interactions between guard cells and neighboring epidermal cells in 2 angiosperms, Vicia faba and Tradescantia virginiana, using cryo-SEM to capture guard cell and epidermal cell cross-sectional conformations in both open (light adapted and transpiring) and closed (dark adapted) stomatal states, and 3D confocal imaging in intact leaves to track the shape and movement of guard cells and epidermal cells during light-induced pore opening. We find that the change in guard cell shape as the cells increase in volume does not result in pore formation; only lateral movement of the guard-epidermal cell wall boundary results in the formation of a pore. Although it is often assumed that decreases in epidermal cell volume are required to create space in the epidermis for pore opening to occur, we found no evidence of a decrease in epidermal cell volume in T. virginiana. Instead, guard cell intrusion forces a change in epidermal cell shape, moving epidermal cell volume out of plane and creating space for pore formation to occur.

Xylem pit membranes are discrete regions of primary cell wall that are permeable to water but prevent the spread of air embolism. Here, we investigate how pectin, a cell wall hydrogel with a kPa-scale modulus, affects the functioning of intervessel pit membranes in Acer rubrum (red maple). We show that enzymatically digesting pectin significantly lowers the resistance against embolism spread, confirming earlier reports in other species and providing evidence that pectin is present in at least some portions of A. rubrum's intervessel pit membranes. Removing calcium with a chelating agent had a smaller effect that became nonsignificant after accounting for radial flows. Vulnerability curves of control and calcium removal stems measured with and without the addition of a surfactant (0.1% w/v Triton-X) exhibited large differences in P50 as expected for the change in surface tension and implying an invariant effective Laplace radius. In contrast, pectin removal stems did not exhibit a significant change in P50 when vulnerability curves were measured with and without surfactant. Instead, the difference in surface tension implied large changes in the effective Laplace radius with increasing xylem tension, suggesting that pectin removal increases the local compliance of pit membranes. We hypothesize that pectin stabilizes the spacing of cellulose microfibrils, thereby contributing to the transport of water under tension.

Leaves maintain a pool of non-structural carbohydrates (NSC) whose size can vary over hourly and longer timescales. We tested two long-standing hypotheses regarding potential physiological roles of changes in foliar NSC levels. The first is that soluble NSC plays a critical role in osmotic adjustment, with their increase enabling stomatal opening despite daily and seasonal reductions in leaf water potential (Ψleaf). The second is that increases in NSC are a sign of excess assimilation relative to sink demand and serve as a signal to downregulate gas exchange. To explore these questions, we monitored the diurnal and seasonal dynamics of gas exchange, Ψleaf, osmotic potential, and NSC of irrigated and dehydrated grapevines (Vitis vinifera) through two consecutive growing seasons. We found that the daily accumulation of soluble sugars constitutes approximately 50% of the daily osmotic adjustment (0.2 MPa), enabling the vines to maintain turgor under low Ψleaf. At the same time, the importance of NSC as osmolytes decreased as the season progressed, and they did not contribute to osmotic adjustments when water was withheld. Additionally, there was no negative correlation between NSC and gas exchange, implying that bulk NSC concentration is not the signal for photosynthetic feedback inhibition.

Plants responding to excessive soil salinity by discharging brine onto their leaf surface risk dehydration through the osmotic continuity between the living tissue and the surface brine, which further enriches with evaporation. Cuticle cracks have long been identified as essential for salt to reach the leaf surface but enable a potentially desiccating continuity between the brine and the gland interior. Using the secreting salt gland of Nolana mollis as a model system, we integrate mathematical modeling, imaging, and physiological measurements to examine the mechanical and biochemical processes required for efficient salt removal. We find that the subcuticular space between the concentrated surface brine and the more dilute secreting cell eases the energetic limits of active salt secretion by reducing the concentration gradient of salt across the cell membrane. We show that crack size plays a critical role in balancing the osmotic and pressure gradients required for salt removal without runaway foliar desiccation.

Drought-induced tree mortality is associated with hydraulic failure and subsequent cell death. Leaf vitality is a critical indicator of tree water stress; however, current methods for assessing cell death are destructive and unsuitable for large-scale measurements. The optical method, which detects changes in leaf light transmittance, has been traditionally used to monitor vein cavitation. However, emerging studies have reported transmittance changes extending beyond veins, suggesting a potential link with mesophyll cell death. In this study, we investigated whether the loss of light transmittance in leaves reflects underlying cellular mortality. We subjected seedlings from six tree species with contrasting drought resistance to progressive water stress. Changes in leaf light transmittance were continuously monitored using the optical method while cell mortality was quantified using classical methods. Cryo-SEM imaging provided anatomical insights into cell structural degradation and its influence on transmittance. Our results revealed a strong synchronicity between transmittance loss and cell mortality increases across species, supporting the use of light transmittance as a nondestructive proxy for cellular damage. Overall, our results highlight the potential of the optical method for early detection of drought-induced stress and mortality in trees and offer a promising, scalable, and noninvasive tool for monitoring tree health under drought.

Background and Aims Nolana mollis is a dominant plant species in the hyper-arid Atacama Desert. A previous hypothesis states that N. mollis owes its success to the condensation of atmospheric water from undersaturated air onto its leaf surfaces by exuded salts, and absorption of this water by its leaves, or by shallow roots following drip onto the soil surface; living roots of N. mollis were suggested to only exist near the soil surface.Methods We conducted a field experiment with three treatments to establish the source of water for N. mollis: control, root cutting to block uptake of all soil moisture, and plastic skirting at the soil surface to block leaf drip of atmospheric water.Key Results Xylem tensions monotonically increased after root cutting until the plants wilted irreversibly, diverging clearly from the skirted and control treatments showing diurnal patterns of increasing tension in the day followed by recovery overnight.Conclusions Hydration in N. mollis requires access to deep soil water, motivating an alternative hypothesis: imperfect salt exclusion at the root surface and salt exudation by the leaf results in less root fouling and lower xylem tensions, while during the day evaporation of the surface brine, condensed overnight, increases the water use efficiency of carbon gain.

The conventional assumption is that stomatal conductance (gs) dominates the regulation of water and carbon dioxide fluxes between leaves and the atmosphere. Here, a nanoreporter of water status at the mesophyll cell surface and local xylem within intact maize leaves documents significant undersaturation of water vapor in the outside-xylem zone (OXZ) and a large loss of conductance of this zone (goxz) at moderate xylem water stress (no turgor loss). The ratio of the resistances (1/goxz)/(1/gs) serves as a predictive phenotype of undersaturation, non-stomatal regulation of transpiration, errors in standard gas exchange analysis, and an increase of intrinsic water use efficiency (iWUE). Cell scale access to water status reveals symplasmic-apoplasmic disequilibrium and informs a biophysical model that can explain experimental observations quantitatively based on localization of variable conductance to the plasma membrane. This work opens new paths of inquiry into the molecular basis and functional consequences of non-stomatal regulation of transpiration. ### Competing Interest Statement Piyush Jain and Abraham D. Stroock are listed as inventors on US Patent 11,536,660 titled, 'In situ sensing of water potential' owned by Cornell University.

Diversification of plant hydraulic architecture and stomatal function coincides with radical changes in the Earth's atmosphere over the past 400 my. Due to shared stomatal anatomy with the earliest land plants, bryophyte stomatal behavior may provide insights into the evolution of stomatal function, but significant uncertainty remains due to technical limitations of measuring guard cell turgor pressure in situ. Here, we introduce a method for monitoring cell turgor pressure by nucleating microbubbles within the guard cells of intact plant tissue and then examining microbubble growth and dissolution dynamics. First, we show that maximum microbubble radius decreases with increasing pressure as the pressure of the surrounding fluid constrains its growth according to a modified version of the Epstein-Plesset equation. We then apply this method to monitor turgor pressure in dark- vs. light-acclimated guard cells across bryophyte taxa with stomata, where their role in gas-exchange remains ambiguous, and in vascular plants with well-documented light-dependent turgor modulation. Our findings show no light-activated change in turgor in bryophyte guard cells, with pressures not significantly different than neighboring epidermal cells. In contrast, vascular plants show distinct pressure modulation in response to light that drives reversible changes in stomatal aperture. Complete guard cell turgor loss had no effect on bryophyte stomatal aperture but resulted in partial or complete closure in vascular plants. These results suggest that despite conserved stomatal morphology, the sampled bryophytes lack dynamic control over guard cell turgor that is critical for sustaining photosynthesis and inhibiting desiccation.

Droughts of increasing severity and frequency are a primary cause of forest mortality associated with climate change. Yet, fundamental knowledge gaps regarding the complex physiology of trees limit the development of more effective management strategies to mitigate drought effects on forests. Here, we highlight some of the basic research needed to better understand tree drought physiology and how new technologies and interdisciplinary approaches can be used to address them. Our discussion focuses on how trees change wood development to mitigate water stress, hormonal responses to drought, genetic variation underlying adaptive drought phenotypes, how trees 'remember' prior stress exposure, and how symbiotic soil microbes affect drought response. Next, we identify opportunities for using research findings to enhance or develop new strategies for managing drought effects on forests, ranging from matching genotypes to environments, to enhancing seedling resilience through nursery treatments, to landscape-scale monitoring and predictions. We conclude with a discussion of the need for co-producing research with land managers and extending research to forests in critical ecological regions beyond the temperate zone.

Elevated atmospheric carbon dioxide (eCO2) can affect plant growth and physiology, which can, in turn, impact herbivorous insects, including by altering pollen or plant tissue nutrition. Previous research suggests that eCO2 can reduce pollen nutrition in some species, but it is unknown whether this effect is consistent across flowering plant species. We experimentally quantified the effects of eCO2 across multiple flowering plant species on plant growth in 9 species and pollen chemistry (%N an estimate for protein content and nutrition in 12 species; secondary chemistry in 5 species) in greenhouses. For pollen nutrition, only buckwheat significantly responded to eCO2, with %N increasing in eCO2; CO2 treatment did not affect pollen amino acid composition but altered secondary metabolites in buckwheat and sunflower. Plant growth under eCO2 exhibited two trends across species: plant height was taller in 44% of species and flower number was affected for 63% of species (3 species with fewer and 2 species with more flowers). The remaining growth metrics (leaf number, above-ground biomass, flower size, and flowering initiation) showed divergent, species-specific responses, if any. Our results indicate that future eCO2 is unlikely to uniformly change pollen chemistry or plant growth across flowering species but may have the potential to alter ecological interactions, or have particularly important effects on specialized pollinators.

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.

Understanding the relationship between wind speed and gas exchange in plants is a longstanding challenge. Our aim was to investigate the impact of wind speed on maximum rates of gas exchange and the kinetics of stomatal responses. We conducted experiments in different angiosperm and fern species using an infrared gas analyzer equipped with a controlled leaf fan, enabling precise control of the boundary layer conductance. We first showed that the chamber was adequately mixed even at extremely low wind speed (<0.005 m s-1) and evaluated the link between fan speed, wind speed, and boundary layer conductance. We observed that higher wind speeds led to increased gas exchange of both water vapor and CO₂, primarily due to the increase in boundary layer conductance. This increase in transpiration subsequently reduced epidermal pressure, leading to stomatal opening. We documented that stomatal opening in response to light was 2.5 times faster at a wind speed of 2 m s-1 compared to minimal wind speed in Vicia faba, while epidermal peels in a buffer with no transpiration exhibited a similar opening rate. The increase in stomatal conductance under high wind was also observed in four angiosperm species under field conditions, but it was not observed in Boston fern (Nephrolepis exaltata), which lacks epidermal mechanical advantage. Our findings highlight the significant impact of boundary layer conductance on determining gas exchange rates and the kinetics of gas exchange responses to environmental changes.

Characteristic of all conifer needles, the transfusion tissue mediates the radial transport of water and sugar between the endodermis and axial vasculature. Physical constraints imposed by the needle's linear geometry introduce two potential extravascular bottlenecks where the opposition of sugar and water flows may frustrate sugar export: one at the vascular access point and the other at the endodermis. We developed a network model of the transfusion tissue to explore how its structure and composition affect the delivery of sugars to the axial phloem. To describe extravascular transport with cellular resolution, we construct networks from images of Pinus pinea needles obtained through tomographic microscopy, as well as fluorescence and electron microscopy. The transfusion tissue provides physically distinct pathways for sugar and water, reducing resistance between the vasculature and endodermis and mitigating flow constriction at the vascular flank. Dissipation of flow velocities through the transfusion tissue's branched structure allows for bidirectional transport of an inbound diffusive sugar flux against an outbound advective water flux across the endodermis. Our results clarify the structure-function relationships of the transfusion tissue under conditions free of physiological stress. The presented model framework is also applicable to different transfusion tissue morphologies in other gymnosperms.

The water status of the living tissue in leaves between the xylem and stomata (outside xylem zone - OXZ) play a critical role for plant function and global mass and energy balance but has remained largely inaccessible. We resolve the local water relations of OXZ tissue using a nanogel reporter of water potential ( ψ ), AquaDust, that enables an in-situ, non-destructive measurement of both ψ of xylem and highly localized ψ at the terminus of transpiration in the OXZ. Working in maize, these localized measurements reveal gradients in the OXZ that are several fold larger than those based on conventional methods, and values of ψ in the mesophyll apoplast well below the macroscopic turgor loss potential. We find a strong loss of hydraulic conductance in both the bundle sheath and the mesophyll with decreasing xylem potential but not with evaporative demand. Our measurements suggest an active role played by the OXZ in regulating the transpiration path and our methods provide novel means to study this phenomenon.

"Carlquist's Law" isa striking pattern of association between anatomical features in the wood of vessel-bearing plants. It derives from Sherwin Carlquist's observation that xylem vessels tend to be solitary when embedded in a matrix of imperforate tracheary elements that appear to be conductive, whereas xylem vessels tend to be grouped when surrounded by seemingly non-conductive cells. Vessel-vessel contacts (vessel grouping) allow water to travel between conduits, but also provide pathways for air to propagate from embolized (air-filled) vessels into functional vessels. If the background matrix is conductive, it is conceivable that water could bypass embolized vessels, providing an alternative transport route in species with conductive backgrounds and solitary vessels. Much remains to be tested in this hypothesis, including the topology of the vessel networks in species with solitary versus grouped vessels and how conductive the different imperforate tracheary element types are. Exploring Carlquist's Law promises to provide key insight into the causes of embolism in plant conduits, the modes of embolism passage between conduits, and how vessels and the cells in which they are imbedded may interact to govern the pathways of water flow through plants.

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.

A growing literature argues that ecosystem-scale evapotranspiration is more sensitive to drying of the atmosphere because of stomatal regulation by plants than to reductions in surface soil moisture. Past studies analysed observations, for which it is difficult to conclusively control for potential relations among plant physiology, measurable state variables such as vapour pressure deficit (VPD) or soil moisture, and ecosystem-scale water flux. Here we analyse natural mechanism-denial experiments at non-vegetated but hydrologically active salt flats. At these sites, any apparent sensitivity of the ecosystem-scale surface conductance ( g s , a bulk measure of how the land surface influences evapotranspiration) to VPD cannot be due to stomatal closure. Over the salt flats we find a VPD– g s relation similar to that commonly attributed to stomatal closure, and reproduce similar relations using a parsimonious boundary layer model that excludes plants. We conclude that observational studies probably overstate the sensitivity of ecosystem-scale surface conductance to atmospheric drying and understate the importance of variations in surface soil moisture. This finding has broad implications for future ecosystems, because anthropogenic trends in soil moisture are uncertain and spatially heterogeneous whereas ubiquitous atmospheric drying is expected due to global warming.

A frequently expressed viewpoint across the Earth science community is that global soil moisture estimates from satellite L-band (1.4 GHz) measurements represent moisture only in a shallow surface layer (0-5 cm) and consequently are of limited value for studying global terrestrial ecosystems because plants use water from deeper rootzones. Using this argumentation, many observation-based land surface studies avoid satellite-observed soil moisture. Here, based on peer-reviewed literature across several fields, we argue that such a viewpoint is overly limiting for two reasons. First, microwave soil emission depth considerations and statistical considerations of vertically correlated soil moisture information together indicate that L-band measurements carry information about soil moisture extending below the commonly referenced 5 cm in many conditions. However, spatial variations of effective depths of representation remain uncertain. Second, in reviewing isotopic tracer field studies of plant water uptake, we find a prevalence of vegetation that primarily draws moisture from these upper soil layers. This is especially true for grasslands and croplands covering more than a third of global vegetated surfaces. Even some deeper-rooted species (i.e., shrubs and trees) preferentially or seasonally draw water from the upper soil layers. Therefore, L-band satellite soil moisture estimates are more relevant to global vegetation water uptake than commonly appreciated (i.e., relevant beyond only shallow soil processes like soil evaporation). Our commentary encourages the application of satellite soil moisture across a broader range of terrestrial hydrosphere and biosphere studies while urging more rigorous estimates of its effective depth of representation.

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.

Summary Trade‐offs among carbon sinks constrain how trees physiologically, ecologically, and evolutionarily respond to their environments. These trade‐offs typically fall along a productive growth to conservative, bet‐hedging continuum. How nonstructural carbohydrates (NSCs) stored in living tree cells (known as carbon stores) fit in this trade‐off framework is not well understood. We examined relationships between growth and storage using both within species genetic variation from a common garden, and across species phenotypic variation from a global database. We demonstrate that storage is actively accumulated, as part of a conservative, bet‐hedging life history strategy. Storage accumulates at the expense of growth both within and across species. Within the species Populus trichocarpa, genetic trade‐offs show that for each additional unit of wood area growth (in cm2 yr−1) that genotypes invest in, they lose 1.2 to 1.7 units (mg g−1 NSC) of storage. Across species, for each additional unit of area growth (in cm2 yr−1), trees, on average, reduce their storage by 9.5% in stems and 10.4% in roots. Our findings impact our understanding of basic plant biology, fit storage into a widely used growth‐survival trade‐off spectrum describing life history strategy, and challenges the assumptions of passive storage made in ecosystem models today.