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.

Localized measurements of water potential reveal large loss of conductance in living tissues of maize leaves.

The water status of the living tissue in leaves between the xylem and stomata (outside xylem zone (OXZ) plays a critical role in 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, nondestructive measurement of both ψ of xylem and highly localized ψ at the terminus of transpiration in the OXZ. Working in maize (Zea mays L.), these localized measurements reveal gradients in the OXZ that are several folds 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 the OXZ plays an active role in regulating the transpiration path, and our methods provide the means to study this phenomenon.

Trunk Water Potential Measured with Microtensiometers for Managing Water Stress in "Gala" Apple Trees.

The weather variations around the world are already having a profound impact on agricultural production. This impacts apple production and the quality of the product. Through agricultural precision, growers attempt to optimize both yield and fruit size and quality. Two experiments were conducted using field-grown "Gala" apple trees in Geneva, NY, USA, in 2021 and 2022. Mature apple trees (Malus × domestica Borkh. cv. Ultima "Gala") grafted onto G.11 rootstock planted in 2015 were used for the experiment. Our goal was to establish a relationship between stem water potential (Ψtrunk), which was continuously measured using microtensiometers, and the growth rate of apple fruits, measured continuously using dendrometers throughout the growing season. The second objective was to develop thresholds for Ψtrunk to determine when to irrigate apple trees. The economic impacts of different irrigation regimes were evaluated. Three different water regimes were compared (full irrigation, rainfed and rain exclusion to induce water stress). Trees subjected the rain-exclusion treatment were not irrigated during the whole season, except in the spring (April and May; 126 mm in 2021 and 100 mm in 2022); that is, these trees did not receive water during June, July, August and half of September. Trees subjected to the rainfed treatment received only rainwater (515 mm in 2021 and 382 mm in 2022). The fully irrigated trees received rain but were also irrigated by drip irrigation (515 mm in 2021 and 565 mm in 2022). Moreover, all trees received the same amount of water out of season in autumn and winter (245 mm in 2021 and 283 mm in 2022). The microtensiometer sensors detected differences in Ψtrunk among our treatments over the entire growing season. In both years, experimental trees with the same trunk cross-section area (TCSA) were selected (23-25 cm-2 TCSA), and crop load was adjusted to 7 fruits·cm-2 TCSA in 2021 and 8.5 fruits·cm-2 TCSA in 2022. However, the irrigated trees showed the highest fruit growth rates and final fruit weight (157 g and 70 mm), followed by the rainfed only treatment (132 g and 66 mm), while the rain-exclusion treatment had the lowest fruit growth rate and final fruit size (107 g and 61 mm). The hourly fruit shrinking and swelling rate (mm·h-1) measured with dendrometers and the hourly Ψtrunk (bar) measured with microtensiometers were correlated. We developed a logistic model to correlate Ψtrunk and fruit growth rate (g·h-1), which suggested a critical value of -9.7 bars for Ψtrunk, above which there were no negative effects on fruit growth rate due to water stress in the relatively humid conditions of New York State. A support vector machine model and a multiple regression model were developed to predict daytime hourly Ψtrunk with radiation and VPD as input variables. Yield and fruit size were converted to crop value, which showed that managing water stress with irrigation during dry periods improved crop value in the humid climate of New York State.

A signal-like role for floral humidity in a nocturnal pollination system.

Previous studies have considered floral humidity to be an inadvertent consequence of nectar evaporation, which could be exploited as a cue by nectar-seeking pollinators. By contrast, our interdisciplinary study of a night-blooming flower, Datura wrightii, and its hawkmoth pollinator, Manduca sexta, reveals that floral relative humidity acts as a mutually beneficial signal in this system. The distinction between cue- and signal-based functions is illustrated by three experimental findings. First, floral humidity gradients in Datura are nearly ten-fold greater than those reported for other species, and result from active (stomatal conductance) rather than passive (nectar evaporation) processes. These humidity gradients are sustained in the face of wind and are reconstituted within seconds of moth visitation, implying substantial physiological costs to these desert plants. Second, the water balance costs in Datura are compensated through increased visitation by Manduca moths, with concomitant increases in pollen export. We show that moths are innately attracted to humid flowers, even when floral humidity and nectar rewards are experimentally decoupled. Moreover, moths can track minute changes in humidity via antennal hygrosensory sensilla but fail to do so when these sensilla are experimentally occluded. Third, their preference for humid flowers benefits hawkmoths by reducing the energetic costs of flower handling during nectar foraging. Taken together, these findings suggest that floral humidity may function as a signal mediating the final stages of floral choice by hawkmoths, complementing the attractive functions of visual and olfactory signals beyond the floral threshold in this nocturnal plant-pollinator system.

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.

Re-entrant transition as a bridge of broken ergodicity in confined monolayers of hexagonal prisms and cylinders.

The entropy-driven monolayer assembly of hexagonal prisms and cylinders was studied under hard slit confinement. At the conditions investigated, the particles have two distinct and dynamically disconnected rotational states: unflipped and flipped, depending on whether their circular/hexagonal face is parallel or perpendicular to the wall plane. Importantly, these two rotational states cast distinct projection areas over the wall plane that favor either hexagonal or tetragonal packing. Monte Carlo simulations revealed a re-entrant melting transition where an intervening disordered Flipped-Unflipped (FUN) phase is sandwiched between a fourfold tetratic phase at high concentrations and a sixfold triangular solid at intermediate concentrations. The FUN phase contains a mixture of flipped and unflipped particles and is translationally and orientationally disordered. Complementary experiments were conducted with photolithographically fabricated cylindrical microparticles confined in a wedge cell. Both simulations and experiments show the formation of phases with comparable fraction of flipped particles and structure, i.e., the FUN phase, triangular solid, and tetratic phase, indicating that both approaches sample analogous basins of particle-orientation phase-space. The phase behavior of hexagonal prisms in a soft-repulsive wall model was also investigated to exemplify how tunable particle-wall interactions can provide an experimentally viable strategy to dynamically bridge the flipped and unflipped states.

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.

Ex Situ and In Situ Measurement of Water Activity with a MEMS Tensiometer.

Water acts as the solvent for natural biotic and abiotic processes and in many technological contexts. The availability of water to participate in chemical and physical processes is captured by thermodynamic variables which track the energetic state of water such as water activity and water potential. Our understanding of the energetic state of water in relevant processes is limited by a lack of sensors capable of providing accurate and reliable ex situ and in situ measurements of water activity. To address this technology gap, we present applications of a microtensiometer (µTM): a biomimetic microelectromechanical system (MEMS) sensor capable of measuring water activity in liquid, vapor, and semisolid (e.g., hydrogels, cheese) phases. We developed packaging, measurement systems, and methodology to enable us to make water activity measurements previously inaccessible to tensiometry. We present measurements in two contexts: (1) a small benchtop unit for ex situ measurements and (2) a probe format for in situ measurements. We demonstrate that the µTM can accurately measure water activity in a diversity of complex samples and agrees with chilled mirror hygrometry, an industry standard for water activity measurement.

Adsorption, Desorption, and Crystallization of Aqueous Solutions in Nanopores.

Probing nanoconfined solutions in tortuous, mesoporous media is challenging because of pore size, complex pore connectivity, and the coexistence of multiple components and phases. Here, we use optical reflectance to experimentally investigate the wetting and drying of a mesoporous medium with ∼3-nm-diameter pores containing aqueous solutions of sodium chloride and lithium chloride. We show that the vapor activities (i.e., relative humidities) that correspond to optical features in the isotherms for solutions can be used to deduce the thermodynamic state of a nanoscopic solution that undergoes evaporation and crystallization upon drying and condensation and deliquescence when increasing the relative humidity. We emphasize specific equilibrium states of the system: the onset of draining during desorption and the end of filling during adsorption as well as percolation-induced scattering and crystallization. We find that theoretical arguments involving classical thermodynamics (a modified Kelvin-Laplace equation and classical nucleation theory) explain quantitatively the evolution of the optical features and thereby the state of the solution as a function of imposed vapor activity and solute concentration.

How Solutes Modify the Thermodynamics and Dynamics of Filling and Emptying in Extreme Ink-Bottle Pores.

We investigate the filling and emptying of extreme ink-bottle porous media-micrometer-scale pores connected by nanometer-scale pores-when changing the pressure of the external vapor, in a case where the pore liquid contains solutes. These phenomena are relevant in diverse contexts, such as the weathering of building materials and artwork, aerosol formation in the atmosphere, and the hydration of soils and plants. Using model systems made of vein-shaped microcavities interconnected by a mesoporous matrix, we show experimentally that the presence of a nonvolatile solute shifts the condensation and evaporation transitions and in a way that is consistent with a modified Kelvin-Laplace equation that takes into account the osmotic pressure of the solution. Emptying occurs far below saturation, when the Kelvin stress, mediated by the large curvature of the liquid-vapor interfaces in the nanopores, is negative enough to induce spontaneous bubble nucleation in the microveins. Filling, on the other hand, occurs close to equilibrium (i.e., at saturation, psat for pure water and ps < psat for a solution), driven by the weak capillary pressure of the liquid-vapor interface in the microveins. Interestingly, solutes allow the system to reach situations where the vapor is supersaturated with respect to the solution ( ps < p < psat). We show that in that latter situation, a condensation layer covers the outer surface of the porous system, preventing the generation of Kelvin stresses but inducing osmotic stresses and flows that are vapor pressure-dependent. The timescales and dynamics reflect these different driving forces: emptying proceeds through discrete, stochastic nucleation events with very fast, unsteady bubble growth associated with a poroelastic relaxation process, while filling occurs collectively in all veins of the sample through a slower steady-state process driven by a combination of osmosis and capillarity. The dynamics can however be rendered symmetrical between filling and emptying if bubbles pre-exist during emptying, a case that we explore using cycling of the vapor pressure around equilibrium.

Multi-scale computational study of the Warburg effect, reverse Warburg effect and glutamine addiction in solid tumors.

Cancer metabolism has received renewed interest as a potential target for cancer therapy. In this study, we use a multi-scale modeling approach to interrogate the implications of three metabolic scenarios of potential clinical relevance: the Warburg effect, the reverse Warburg effect and glutamine addiction. At the intracellular level, we construct a network of central metabolism and perform flux balance analysis (FBA) to estimate metabolic fluxes; at the cellular level, we exploit this metabolic network to calculate parameters for a coarse-grained description of cellular growth kinetics; and at the multicellular level, we incorporate these kinetic schemes into the cellular automata of an agent-based model (ABM), iDynoMiCS. This ABM evaluates the reaction-diffusion of the metabolites, cellular division and motion over a simulation domain. Our multi-scale simulations suggest that the Warburg effect provides a growth advantage to the tumor cells under resource limitation. However, we identify a non-monotonic dependence of growth rate on the strength of glycolytic pathway. On the other hand, the reverse Warburg scenario provides an initial growth advantage in tumors that originate deeper in the tissue. The metabolic profile of stromal cells considered in this scenario allows more oxygen to reach the tumor cells in the deeper tissue and thus promotes tumor growth at earlier stages. Lastly, we suggest that glutamine addiction does not confer a selective advantage to tumor growth with glutamine acting as a carbon source in the tricarboxylic acid (TCA) cycle, any advantage of glutamine uptake must come through other pathways not included in our model (e.g., as a nitrogen donor). Our analysis illustrates the importance of accounting explicitly for spatial and temporal evolution of tumor microenvironment in the interpretation of metabolic scenarios and hence provides a basis for further studies, including evaluation of specific therapeutic strategies that target metabolism.

Enhanced Oxygen Solubility in Metastable Water under Tension.

Despite its relevance in numerous natural and industrial processes, the solubility of molecular oxygen has never been directly measured in capillary-condensed liquid water. In this article, we measure oxygen solubility in liquid water trapped within nanoporous samples, in metastable equilibrium with a subsaturated vapor. We show that solubility increases two fold at moderate subsaturations (relative humidity ∼0.55). This evolution with relative humidity is in good agreement with a simple thermodynamic prediction using properties of bulk water, previously verified experimentally at positive pressure. Our measurement thus verifies the validity of this macroscopic thermodynamic theory to strong confinement and large negative pressures, where significant nonidealities are expected. This effect has strong implications for important oxygen-dependent chemistries in natural and technological contexts.

Phloem Loading through Plasmodesmata: A Biophysical Analysis.

In many species, Suc en route out of the leaf migrates from photosynthetically active mesophyll cells into the phloem down its concentration gradient via plasmodesmata, i.e. symplastically. In some of these plants, the process is entirely passive, but in others phloem Suc is actively converted into larger sugars, raffinose and stachyose, and segregated (trapped), thus raising total phloem sugar concentration to a level higher than in the mesophyll. Questions remain regarding the mechanisms and selective advantages conferred by both of these symplastic-loading processes. Here, we present an integrated model-including local and global transport and kinetics of polymerization-for passive and active symplastic loading. We also propose a physical model of transport through the plasmodesmata. With these models, we predict that (1) relative to passive loading, polymerization of Suc in the phloem, even in the absence of segregation, lowers the sugar content in the leaf required to achieve a given export rate and accelerates export for a given concentration of Suc in the mesophyll and (2) segregation of oligomers and the inverted gradient of total sugar content can be achieved for physiologically reasonable parameter values, but even higher export rates can be accessed in scenarios in which polymers are allowed to diffuse back into the mesophyll. We discuss these predictions in relation to further studies aimed at the clarification of loading mechanisms, fitness of active and passive symplastic loading, and potential targets for engineering improved rates of export.

Passive phloem loading and long-distance transport in a synthetic tree-on-a-chip.

Vascular plants rely on differences in osmotic pressure to export sugars from regions of synthesis (mature leaves) to sugar sinks (roots, fruits). In this process, known as Münch pressure flow, the loading of sugars from photosynthetic cells to the export conduit (the phloem) is crucial, as it sets the pressure head necessary to power long-distance transport. Whereas most herbaceous plants use active mechanisms to increase phloem sugar concentration above that of the photosynthetic cells, in most tree species, for which transport distances are largest, loading seems, counterintuitively, to occur by means of passive symplastic diffusion from the mesophyll to the phloem. Here, we use a synthetic microfluidic model of a passive loader to explore the non-linear dynamics that arise during export and determine the ability of passive loading to drive long-distance transport. We first demonstrate that in our device, the phloem concentration is set by the balance between the resistances to diffusive loading from the source and convective export through the phloem. Convection-limited export corresponds to classical models of Münch transport, where the phloem concentration is close to that of the source; in contrast, diffusion-limited export leads to small phloem concentrations and weak scaling of flow rates with hydraulic resistance. We then show that the effective regime of convection-limited export is predominant in plants with large transport resistances and low xylem pressures. Moreover, hydrostatic pressures developed in our synthetic passive loader can reach botanically relevant values as high as 10 bars. We conclude that passive loading is sufficient to drive long-distance transport in large plants, and that trees are well suited to take full advantage of passive phloem loading strategies.

Imbibition Triggered by Capillary Condensation in Nanopores.

We study the spatiotemporal dynamics of water uptake by capillary condensation from unsaturated vapor in mesoporous silicon layers (pore radius rp ≃ 2 nm), taking advantage of the local changes in optical reflectance as a function of water saturation. Our experiments elucidate two qualitatively different regimes as a function of the imposed external vapor pressure: at low vapor pressures, equilibration occurs via a diffusion-like process; at high vapor pressures, an imbibition-like wetting front results in fast equilibration toward a fully saturated sample. We show that the imbibition dynamics can be described by a modified Lucas-Washburn equation that takes into account the liquid stresses implied by Kelvin equation.

Capillarity-driven flows at the continuum limit.

We experimentally investigate the dynamics of capillary-driven flows at the nanoscale, using an original platform that combines nanoscale pores (⋍3 nm in diameter) and microfluidic features. In particular, we show that drying involves a fine coupling between thermodynamics and fluid mechanics that can be used to generate precisely controlled nanoflows driven by extreme stresses - up to 100 MPa of tension. We exploit these tunable flows to provide quantitative tests of continuum theories (e.g. Kelvin-Laplace equation and Poiseuille flow) across an unprecedented range and we isolate the breakdown of continuum as a negative slip length of molecular dimension. Our results show a coherent picture across multiple experiments including drying-induced permeation flows, imbibition and poroelastic transients.

Stability Limit of Water by Metastable Vapor-Liquid Equilibrium with Nanoporous Silicon Membranes.

Liquid can sustain mechanical tension as its pressure drops below the vapor-liquid coexistence line and becomes less than zero, until it reaches the stability limit-the pressure at which cavitation inevitably occurs. For liquid water, its stability limit is still a subject of debate: the results obtained by researchers using a variety of techniques show discrepancies between the values of the stability limit and its temperature dependence as temperature approaches 0 °C. In this work, we present a study of the stability limit of water by the metastable vapor-liquid equilibrium (MVLE) method with nanoporous silicon membranes. We also report on an experimental system which enables tests of the temperature dependence of the stability limit with MVLE. The stability limit we found increases monotonically (larger tension) as temperature approaches 0 °C; this trend contradicts the centrifugal result of Briggs but agrees with the experiments by acoustic cavitation. This result confirms that a quasi-static method can reach stability values similar to that from the dynamic stretching technique, even close to 0 °C. Nevertheless, our results fall in the range of ∼ -20 to -30 MPa, a range that is consistent with the majority of experiments but is far less negative than the limit obtained in experiments involving quartz inclusions and that predicted for homogeneous nucleation.

Adipose-derived stem cells increase angiogenesis through matrix metalloproteinase-dependent collagen remodeling.

Adipose-derived stem cells (ASCs) are key regulators of new blood vessel formation and widely investigated for their role in tissue regeneration and tumorigenesis. However, the cellular and molecular mechanisms through which ASCs regulate angiogenesis are not well understood. Here, it was our goal to test the functional contribution of ASC-mediated extracellular matrix (ECM) remodeling on endothelial cell invasion. To isolate the effect of ECM-remodeling, ASCs were embedded within 3-D collagen type I hydrogels and pre-cultured for 7 days; controls were not pre-cultured. A confluent monolayer of human umbilical vein endothelial cells (HUVECs) was seeded on top and its invasion into the underlying hydrogel was analyzed. Without pre-culture, ASCs inhibited vascular sprouting by stabilizing the endothelium. In contrast, 7 day pre-culture of ASCs drastically increased invasion by HUVECs. This effect was largely mediated by proteolytic ECM degradation by ASC-derived matrix metalloproteinases (MMPs) rather than vascular endothelial growth factor (VEGF), as our results indicated that blockade of MMPs, but not VEGF, inhibited endothelial sprouting. Collectively, these data suggest that the angiogenic capability of ASCs is modulated by their proteolytic remodeling of the ECM, opening new avenues for pro- and anti-angiogenic therapies.

Endothelial cell dynamics during anastomosis in vitro.

Vascular anastomosis - the fusion of vessels from two distinct branches of the vascular system - represents a critical step in vascular growth under both healthy and pathological conditions, in vivo, and presents an important target for engineering of vascularized tissues, in vitro. Recent works in animal models have advanced our understanding of the molecular and cellular players in vascular anastomosis, but questions remain related to cellular dynamics and control of this process, in vitro. In this study, we exploited a three-dimensional (3-D) culture platform to examine the dynamics of endothelial cell (EC) during and after vascular anastomosis by allowing angiogenesis and vasculogenesis to proceed in parallel. We show that anastomosis occurs between sprouts formed by angiogenesis from an endothelium and tubes formed by vasculogenesis in the bulk of a 3-D matrix. This fusion leads to highly connected vessels that span from the surface of the matrix into the bulk in a manner that depends on cell density and identity. Further, we observe and analyze intermixing of endothelial cells of distinct origin (surface versus bulk) within the vessels structures that are formed; we provide evidence that the cells migrate along pre-existing vessels segments as part of this intermixing process. We conclude that anastomosis can occur between vessels emerging by angiogenesis and vasculogenesis and that this process may play an important role in contexts such as wound healing.

Drying by cavitation and poroelastic relaxations in porous media with macroscopic pores connected by nanoscale throats.

We investigate the drying dynamics of porous media with two pore diameters separated by several orders of magnitude. Nanometer-sized pores at the edge of our samples prevent air entry, while drying proceeds by heterogeneous nucleation of vapor bubbles--cavitation--in the liquid in micrometer-sized voids within the sample. We show that the dynamics of cavitation and drying are set by the interplay of the deterministic poroelastic mass transport in the porous medium and the stochastic nucleation process. Spatiotemporal patterns emerge in this unusual reaction-diffusion system, with temporal oscillations in the drying rate and variable roughness of the drying front.