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.

Probing nano-confined solutions in tortuous, mesoporous media is challenging due to pore size, complex pore connectivity, and 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 1 Page 1 of 41 ACS Paragon Plus Environment Langmuir of the system: the onset of draining during desorption and the end of filling during adsorption, percolation-induced scattering and crystallization. We find that theoretical arguments involving classical thermodynamics (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.

Background Recent reports of extreme levels of undersaturation in the 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 vapor 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 vapor 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 artifacts 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.

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.

Although visual and olfactory floral signals attract pollinators from a distance, at the flower’s threshold, pollinators can use floral humidity as an index cue for nectar presence. We evaluate the role of floral humidity in the Datura wrightii-Manduca sexta nocturnal pollination system. In addition to our finding that M. sexta shows strong innate attraction toward humid flowers, we identify the hygrosensing sensillum on their antennae, demonstrate its extreme sensitivity to minute changes in RH, and observe the elimination of moths’ behavioral preference towards humid flowers following experimental occlusion of the sensilla. Despite Manduca’s attraction toward humid flowers, we find that floral humidity is not a reliable cue for nectar presence in this system. While Datura floral headspace sustains an enormous humidity gradient, it is not a consequence of nectar evaporation, but an outcome of gas exchange through floral stomata and is decoupled from nectar presence. Using interdisciplinary tools, we demonstrate the function of floral humidity as an attractive signal, not a cue, in this pollination system, thus showcasing an underappreciated modality by which flowers may manipulate their visitors.

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 the development of 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 Fluorescence Resonance Energy Transfer (FRET). Upon infiltration into leaves, the nanoparticles localize within the apoplastic space in the mesophyll; they do not enter the cytoplast 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 highthroughput, field measurements and spatially resolved studies of water relations within plant tissues.

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 bothψof xylem and highly localizedψat 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 ofψin 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.