How and why do species break a developmental trade-off? Elucidating the association of trichomes and stomata across species.

PREMISE: Previous studies have suggested a trade-off between trichome density (Dt) and stomatal density (Ds) due to shared cell precursors. We clarified how, when, and why this developmental trade-off may be overcome across species. METHODS: We derived equations to determine the developmental basis for Dt and Ds in trichome and stomatal indices (it and is) and the sizes of epidermal pavement cells (e), trichome bases (t), and stomata (s) and quantified the importance of these determinants of Dt and Ds for 78 California species. We compiled 17 previous studies of Dt-Ds relationships to determine the commonness of Dt-Ds associations. We modeled the consequences of different Dt-Ds associations for plant carbon balance. RESULTS: Our analyses showed that higher Dt was determined by higher it and lower e, and higher Ds by higher is and lower e. Across California species, positive Dt-Ds coordination arose due to it-is coordination and impacts of the variation in e. A Dt-Ds trade-off was found in only 30% of studies. Heuristic modeling showed that species sets would have the highest carbon balance with a positive or negative relationship or decoupling of Dt and Ds, depending on environmental conditions. CONCLUSIONS: Shared precursor cells of trichomes and stomata do not limit higher numbers of both cell types or drive a general Dt-Ds trade-off across species. This developmental flexibility across diverse species enables different Dt-Ds associations according to environmental pressures. Developmental trait analysis can clarify how contrasting trait associations would arise within and across species.

The poorly-explored stomatal response to temperature at constant evaporative demand.

Changes in leaf temperature are known to drive stomatal responses, because the leaf-to-air water vapour gradient (Δw) increases with temperature if ambient vapour pressure is held constant, and stomata respond to changes in Δw. However, the direct response of stomata to temperature (DRST; the response when Δw is held constant by adjusting ambient humidity) has been examined far less extensively. Though the meagre available data suggest the response is usually positive, results differ widely and defy broad generalisation. As a result, little is known about the DRST. This review discusses the current state of knowledge about the DRST, including numerous hypothesised biophysical mechanisms, potential implications of the response for plant adaptation, and possible impacts of the DRST on plant-atmosphere carbon and water exchange in a changing climate.

Amphistomy increases leaf photosynthesis more in coastal than montane plants of Hawaiian 'ilima (Sida fallax).

PREMISE: The adaptive significance of amphistomy (stomata on both upper and lower leaf surfaces) is unresolved. A widespread association between amphistomy and open, sunny habitats suggests the adaptive benefit of amphistomy may be greatest in these contexts, but this hypothesis has not been tested experimentally. Understanding amphistomy informs its potential as a target for crop improvement and paleoenvironment reconstruction. METHODS: We developed a method to quantify "amphistomy advantage" ( AA $\text{AA}$ ) as the log-ratio of photosynthesis in an amphistomatous leaf to that of the same leaf but with gas exchange blocked through the upper surface (pseudohypostomy). Humidity modulated stomatal conductance and thus enabled comparing photosynthesis at the same total stomatal conductance. We estimated AA $\text{AA}$ and leaf traits in six coastal (open, sunny) and six montane (closed, shaded) populations of the indigenous Hawaiian species 'ilima (Sida fallax). RESULTS: Coastal 'ilima leaves benefit 4.04 times more from amphistomy than montane leaves. Evidence was equivocal with respect to two hypotheses: (1) that coastal leaves benefit more because they are thicker and have lower CO2 conductance through the internal airspace and (2) that they benefit more because they have similar conductance on each surface, as opposed to most conductance being through the lower surface. CONCLUSIONS: This is the first direct experimental evidence that amphistomy increases photosynthesis, consistent with the hypothesis that parallel pathways through upper and lower mesophyll increase CO2 supply to chloroplasts. The prevalence of amphistomatous leaves in open, sunny habitats can partially be explained by the increased benefit of amphistomy in "sun" leaves, but the mechanistic basis remains uncertain.

Leaf relative water content at 50% stomatal conductance measured by noninvasive NMR is linked to climate of origin in nine species of eucalypt.

Stomata are the gatekeepers of plant water use and must quickly respond to changes in plant water status to ensure plant survival under fluctuating environmental conditions. The mechanism for their closure is highly sensitive to disturbances in leaf water status, which makes isolating their response to declining water content difficult to characterise and to compare responses among species. Using a small-scale non-destructive nuclear magnetic resonance spectrometer as a leaf water content sensor, we measure the stomatal response to rapid induction of water deficit in the leaves of nine species of eucalypt from contrasting climates. We found a strong linear correlation between relative water content at 50% stomatal conductance (RWCgs50 ) and mean annual temperature at the climate of origin of each species. We also show evidence for stomata to maintain control over water loss well below turgor loss point in species adapted to warmer climates and secondary increases in stomatal conductance despite declining water content. We propose that RWCgs50 is a promising trait to guide future investigations comparing stomatal responses to water deficit. It may provide a useful phenotyping trait to delineate tolerance and adaption to hot temperatures and high leaf-to-air vapour pressure deficits.

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

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

Hyperspectral Remote Sensing for Phenotyping the Physiological Drought Response of Common and Tepary Bean.

Proximal remote sensing offers a powerful tool for high-throughput phenotyping of plants for assessing stress response. Bean plants, an important legume for human consumption, are often grown in regions with limited rainfall and irrigation and are therefore bred to further enhance drought tolerance. We assessed physiological (stomatal conductance and predawn and midday leaf water potential) and ground- and tower-based hyperspectral remote sensing (400 to 2,400 nm and 400 to 900 nm, respectively) measurements to evaluate drought response in 12 common bean and 4 tepary bean genotypes across 3 field campaigns (1 predrought and 2 post-drought). Hyperspectral data in partial least squares regression models predicted these physiological traits (R 2 = 0.20 to 0.55; root mean square percent error 16% to 31%). Furthermore, ground-based partial least squares regression models successfully ranked genotypic drought responses similar to the physiologically based ranks. This study demonstrates applications of high-resolution hyperspectral remote sensing for predicting plant traits and phenotyping drought response across genotypes for vegetation monitoring and breeding population screening.

TSWIFT: Tower Spectrometer on Wheels for Investigating Frequent Timeseries for high-throughput phenotyping of vegetation physiology.

BACKGROUND: Remote sensing instruments enable high-throughput phenotyping of plant traits and stress resilience across scale. Spatial (handheld devices, towers, drones, airborne, and satellites) and temporal (continuous or intermittent) tradeoffs can enable or constrain plant science applications. Here, we describe the technical details of TSWIFT (Tower Spectrometer on Wheels for Investigating Frequent Timeseries), a mobile tower-based hyperspectral remote sensing system for continuous monitoring of spectral reflectance across visible-near infrared regions with the capacity to resolve solar-induced fluorescence (SIF). RESULTS: We demonstrate potential applications for monitoring short-term (diurnal) and long-term (seasonal) variation of vegetation for high-throughput phenotyping applications. We deployed TSWIFT in a field experiment of 300 common bean genotypes in two treatments: control (irrigated) and drought (terminal drought). We evaluated the normalized difference vegetation index (NDVI), photochemical reflectance index (PRI), and SIF, as well as the coefficient of variation (CV) across the visible-near infrared spectral range (400 to 900 nm). NDVI tracked structural variation early in the growing season, following initial plant growth and development. PRI and SIF were more dynamic, exhibiting variation diurnally and seasonally, enabling quantification of genotypic variation in physiological response to drought conditions. Beyond vegetation indices, CV of hyperspectral reflectance showed the most variability across genotypes, treatment, and time in the visible and red-edge spectral regions. CONCLUSIONS: TSWIFT enables continuous and automated monitoring of hyperspectral reflectance for assessing variation in plant structure and function at high spatial and temporal resolutions for high-throughput phenotyping. Mobile, tower-based systems like this can provide short- and long-term datasets to assess genotypic and/or management responses to the environment, and ultimately enable the spectral prediction of resource-use efficiency, stress resilience, productivity and yield.

Kinetic factors of physiology and the dynamic light environment influence the economic landscape of short-term hydraulic risk.

Optimality-based models of stomatal conductance unify biophysical and evolutionary constraints and can improve predictions of land-atmosphere carbon and water exchange. Recent models incorporate hydraulic constraints by penalizing excessive stomatal opening in relation to hydraulic damage caused by low water potentials. We used simulation models to test whether penalties based solely on vulnerability curves adequately represent the optimality hypothesis, given that they exclude the effects of kinetic factors on stomatal behavior and integrated carbon balance. To quantify the effects of nonsteady-state phenomena on the landscape of short-term hydraulic risk, we simulated diurnal dynamics of leaf physiology for 10 000 patches of leaf in a canopy and used a ray-tracing model, Helios, to simulate realistic variation in sunfleck dynamics. Our simulations demonstrated that kinetic parameters of leaf physiology and sunfleck properties influence the economic landscape of short-term hydraulic risk, as characterized by the effect of stomatal strategy (gauged by the water potential causing a 50% hydraulic penalty) on both aggregated carbon gain and the aggregated carbon cost of short-term hydraulic risk. Hydraulic penalties in optimization models should be generalized to allow their parameters to account for kinetic factors, in addition to parameters of hydraulic vulnerability.

Thermal sensitivity across forest vertical profiles: patterns, mechanisms, and ecological implications.

Rising temperatures are influencing forests on many scales, with potentially strong variation vertically across forest strata. Using published research and new analyses, we evaluate how microclimate and leaf temperatures, traits, and gas exchange vary vertically in forests, shaping tree, and ecosystem ecology. In closed-canopy forests, upper canopy leaves are exposed to the highest solar radiation and evaporative demand, which can elevate leaf temperature (Tleaf ), particularly when transpirational cooling is curtailed by limited stomatal conductance. However, foliar traits also vary across height or light gradients, partially mitigating and protecting against the elevation of upper canopy Tleaf . Leaf metabolism generally increases with height across the vertical gradient, yet differences in thermal sensitivity across the gradient appear modest. Scaling from leaves to trees, canopy trees have higher absolute metabolic capacity and growth, yet are more vulnerable to drought and damaging Tleaf than their smaller counterparts, particularly under climate change. By contrast, understory trees experience fewer extreme high Tleaf 's but have fewer cooling mechanisms and thus may be strongly impacted by warming under some conditions, particularly when exposed to a harsher microenvironment through canopy disturbance. As the climate changes, integrating the patterns and mechanisms reviewed here into models will be critical to forecasting forest-climate feedback.

Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

Predicting ecosystem productivity based on plant community traits.

With the rapid accumulation of plant trait data, major opportunities have arisen for the integration of these data into predicting ecosystem primary productivity across a range of spatial extents. Traditionally, traits have been used to explain physiological productivity at cell, organ, or plant scales, but scaling up to the ecosystem scale has remained challenging. Here, we show the need to combine measures of community-level traits and environmental factors to predict ecosystem productivity at landscape or biogeographic scales. We show how theory can extend the production ecology equation to enormous potential for integrating traits into ecological models that estimate productivity-related ecosystem functions across ecological scales and to anticipate the response of terrestrial ecosystems to global change.

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

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

The three-dimensional construction of leaves is coordinated with water use efficiency in conifers.

Conifers prevail in the canopies of many terrestrial biomes, holding a great ecological and economic importance globally. Current increases in temperature and aridity are imposing high transpirational demands and resulting in conifer mortality. Therefore, identifying leaf structural determinants of water use efficiency is essential for predicting physiological impacts due to environmental variation. Using synchrotron-generated microtomography imaging, we extracted leaf volumetric anatomy and stomatal traits in 34 species across conifers with a special focus on Pinus, the richest conifer genus. We show that intrinsic water use efficiency (WUEi ) is positively driven by leaf vein volume. Needle-like leaves of Pinus, as opposed to flat leaves or flattened needles of other genera, showed lower mesophyll porosity, decreasing the relative mesophyll volume. This led to increased ratios of stomatal pore number per mesophyll or intercellular airspace volume, which emerged as powerful explanatory variables, predicting both stomatal conductance and WUEi . Our results clarify how the three-dimensional organisation of tissues within the leaf has a direct impact on plant water use and carbon uptake. By identifying a suite of structural traits that influence important physiological functions, our findings can help to understand how conifers may respond to the pressures exerted by climate change.

Genome-wide association identifies candidate genes for drought tolerance in coast redwood and giant sequoia.

Drought is a major limitation for survival and growth in plants. With more frequent and severe drought episodes occurring due to climate change, it is imperative to understand the genomic and physiological basis of drought tolerance to be able to predict how species will respond in the future. In this study, univariate and multitrait multivariate genome-wide association study methods were used to identify candidate genes in two iconic and ecosystem-dominating species of the western USA, coast redwood and giant sequoia, using 10 drought-related physiological and anatomical traits and genome-wide sequence-capture single nucleotide polymorphisms. Population-level phenotypic variation was found in carbon isotope discrimination, osmotic pressure at full turgor, xylem hydraulic diameter, and total area of transporting fibers in both species. Our study identified new 78 new marker × trait associations in coast redwood and six in giant sequoia, with genes involved in a range of metabolic, stress, and signaling pathways, among other functions. This study contributes to a better understanding of the genomic basis of drought tolerance in long-generation conifers and helps guide current and future conservation efforts in the species.

CO2, nitrogen deposition and a discontinuous climate response drive water use efficiency in global forests.

Reduced stomatal conductance is a common plant response to rising atmospheric CO2 and increases water use efficiency (W). At the leaf-scale, W depends on water and nitrogen availability in addition to atmospheric CO2. In hydroclimate models W is a key driver of rainfall, droughts, and streamflow extremes. We used global climate data to derive Aridity Indices (AI) for forests over the period 1965-2015 and synthesised those with data for nitrogen deposition and W derived from stable isotopes in tree rings. AI and atmospheric CO2 account for most of the variance in W of trees across the globe, while cumulative nitrogen deposition has a significant effect only in regions without strong legacies of atmospheric pollution. The relation of aridity and W displays a clear discontinuity. W and AI are strongly related below a threshold value of AI ≈ 1 but are not related where AI > 1. Tree ring data emphasise that effective demarcation of water-limited from non-water-limited behaviour of stomata is critical to improving hydrological models that operate at regional to global scales.

A double-ratio method to measure fast, slow and reverse sap flows.

Sap velocity measurements are useful in fields ranging from plant water relations to hydrology at a variety of scales. Techniques based on pulses of heat are among the most common methods to measure sap velocity, but most lack ability to measure velocities across a wide range, including very high, very low and negative velocities (reverse flow). We propose a new method, the double-ratio method (DRM), which is robust across an unprecedented range of sap velocities and provides real-time estimates of the thermal diffusivity of wood. The DRM employs one temperature sensor upstream (proximal) and two sensors downstream (distal) to the source of heat. This facilitates several theoretical, heat-based approaches to quantifying sap velocity. We tested the DRM using whole-tree lysimetry in Eucalyptus cypellocarpa L.A.S. Johnson and found strong agreement across a wide range of velocities.

Optimal carbon partitioning helps reconcile the apparent divergence between optimal and observed canopy profiles of photosynthetic capacity.

Photosynthetic capacity per unit irradiance is greater, and the marginal carbon revenue of water (∂A/∂E) is smaller, in shaded leaves than sunlit leaves, apparently contradicting optimization theory. I tested the hypothesis that these patterns arise from optimal carbon partitioning subject to biophysical constraints on leaf water potential. In a whole plant model with two canopy modules, I adjusted carbon partitioning, nitrogen partitioning and leaf water potential to maximize carbon profit or canopy photosynthesis, and recorded how gas exchange parameters compared between shaded and sunlit modules in the optimum. The model predicted that photosynthetic capacity per unit irradiance should be larger, and ∂A/∂E smaller, in shaded modules compared to sunlit modules. This was attributable partly to radiation-driven differences in evaporative demand, and partly to differences in hydraulic conductance arising from the need to balance marginal returns on stem carbon investment between modules. The model verified, however, that invariance in the marginal carbon revenue of N (∂A/∂N) is in fact optimal. The Cowan-Farquhar optimality solution (invariance of ∂A/∂E) does not apply to spatial variation within a canopy. The resulting variation in carbon-water economy explains differences in capacity per unit irradiance, reconciling optimization theory with observations.

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

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

Wide variation in the suboptimal distribution of photosynthetic capacity in relation to light across genotypes of wheat.

Suboptimal distribution of photosynthetic capacity in relation to light among leaves reduces potential whole-canopy photosynthesis. We quantified the degree of suboptimality in 160 genotypes of wheat by directly measuring photosynthetic capacity and daily irradiance in flag and penultimate leaves. Capacity per unit daily irradiance was systematically lower in flag than penultimate leaves in most genotypes, but the ratio (γ) of capacity per unit irradiance between flag and penultimate leaves varied widely across genotypes, from less than 0.5 to over 1.2. Variation in γ was most strongly associated with differences in photosynthetic capacity in penultimate leaves, rather than with flag leaf photosynthesis or canopy light penetration. Preliminary genome-wide association analysis identified nine strong marker-trait associations with this trait, which should be validated in future work in other environments and/or materials. Our modelling suggests canopy photosynthesis could be increased by up to 5 % under sunny conditions by harnessing this variation through selective breeding for increased γ.