Rapid spatial assessment of leaf-absorbed irradiance.

Image-based high-throughput phenotyping promises the rapid determination of functional traits in large plant populations. However, interpretation of some traits - such as those related to photosynthesis or transpiration rates - is only meaningful if the irradiance absorbed by the measured leaves is known, which can differ greatly between different parts of the same plant and within canopies. No feasible method currently exists to rapidly measure absorbed irradiance in three-dimensional plants and canopies. We developed a method and protocols to derive absorbed irradiance at any visible part of a canopy with a thermal camera, by fitting a leaf energy balance model to transient changes in leaf temperature. Leaves were exposed to short light pulses (30 s) that were not long enough to trigger stomatal opening but strong enough to induce transient changes in leaf temperature that was proportional to the absorbed irradiance. The method was successfully validated against point measurements of absorbed irradiance in plant species with relatively simple architecture (sweet pepper, cucumber, tomato, and lettuce). Once calibrated, the model was used to produce absorbed irradiance maps from thermograms. Our method opens new avenues for the interpretation of plant responses derived from imaging techniques and can be adapted to existing high-throughput phenotyping platforms.

A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

Exploring natural genetic diversity in a bread wheat multi-founder population: Dual imaging of photosynthesis and stomatal kinetics.

A better understanding of crop phenotype under dynamic environmental conditions will help inform the development of new cultivars with superior adaptation to constantly changing field conditions. Recent research has shown that optimising photosynthetic and stomatal conductance traits holds promise for improved crop performance. However, standard phenotyping tools such as gas-exchange systems are limited by their throughput. In this work, a novel approach based on a bespoke gas-exchange chamber allowing combined measurement of the quantum yield of photosystem II (PSII) with an estimation of stomatal conductance via thermal imaging, was used to phenotype a range of bread wheat (Triticum aestivum L.) genotypes, that were a sub-set of a multi-founder experimental population. Datasets were further supplemented by measurement of photosynthetic capacity and stomatal density. First, we showed that measurement of stomatal traits using our dual imaging system compared to standard IRGA methods showed good agreement between the two methods (R2=0.86) for the rapidity of stomatal opening (Ki), with the dual-imager method resulting in less intra-genotype variation. Using the dual-imaging methods, and traditional approaches we found broad and significant variation in key traits, including photosynthetic CO2 uptake at saturating light and ambient CO2 concentration (Asat), photosynthetic CO2 uptake at saturating light and elevated CO2 concentration (Amax), the maximum velocity of Rubisco for carboxylation (Vcmax), time for stomatal opening (Ki), and leaf evaporative cooling. Anatomical analysis revealed significant variation in flag leaf adaxial stomatal density. Associations between traits highlighted significant relationships between leaf evaporative cooling, leaf stomatal conductance under low (gsmin) and high (gsmax) light intensity, and the operating efficiency of PSII (Fq'/Fm'), highlighting the importance of stomatal conductance and stomatal rapidity in maintaining optimal leaf temperature for photosynthesis in wheat. Additionally, gsmin and gsmax were positively associated, indicating that potential combination of preferable traits (i.e. inherently high gsmax, low Ki and maintained leaf evaporative cooling) are present in wheat. This work highlights for the first time the effectiveness of thermal imaging in screening dynamic stomatal conductance in a large panel of wheat genotypes. The wide phenotypic variation observed suggested the presence of exploitable genetic variability in bread wheat for dynamic stomatal conductance traits and photosynthetic capacity for targeted optimisation within future breeding programs.

The impact of growth at elevated [CO2] on stomatal anatomy and behavior differs between wheat species and cultivars.

The ability of plants to respond to changes in the environment is crucial to their survival and reproductive success. The impact of increasing the atmospheric CO2 concentration (a[CO2]), mediated by behavioral and developmental responses of stomata, on crop performance remains a concern under all climate change scenarios, with potential impacts on future food security. To identify possible beneficial traits that could be exploited for future breeding, phenotypic variation in morphological traits including stomatal size and density, as well as physiological responses and, critically, the effect of growth [CO2] on these traits, was assessed in six wheat relative accessions (including Aegilops tauschii, Triticum turgidum ssp. Dicoccoides, and T. turgidum ssp. dicoccon) and five elite bread wheat T. aestivum cultivars. Exploiting a range of different species and ploidy, we identified key differences in photosynthetic capacity between elite hexaploid wheat and wheat relatives. We also report differences in the speed of stomatal responses which were found to be faster in wheat relatives than in elite cultivars, a trait that could be useful for enhanced photosynthetic carbon gain and water use efficiency. Furthermore, these traits do not all appear to be influenced by elevated [CO2], and determining the underlying genetics will be critical for future breeding programmes.

Guard Cell Starch Degradation Yields Glucose for Rapid Stomatal Opening in Arabidopsis.

Starch in Arabidopsis (Arabidopsis thaliana) guard cells is rapidly degraded at the start of the day by the glucan hydrolases α-AMYLASE3 (AMY3) and ß-AMYLASE1 (BAM1) to promote stomatal opening. This process is activated via phototropin-mediated blue light signaling downstream of the plasma membrane H+-ATPase. It remains unknown how guard cell starch degradation integrates with light-regulated membrane transport processes in the fine control of stomatal opening kinetics. We report that H+, K+, and Cl- transport across the guard cell plasma membrane is unaltered in the amy3 bam1 mutant, suggesting that starch degradation products do not directly affect the capacity to transport ions. Enzymatic quantification revealed that after 30 min of blue light illumination, amy3 bam1 guard cells had similar malate levels as the wild type, but had dramatically altered sugar homeostasis, with almost undetectable amounts of Glc. Thus, Glc, not malate, is the major starch-derived metabolite in Arabidopsis guard cells. We further show that impaired starch degradation in the amy3 bam1 mutant resulted in an increase in the time constant for opening of 40 min. We conclude that rapid starch degradation at dawn is required to maintain the cytoplasmic sugar pool, clearly needed for fast stomatal opening. The conversion and exchange of metabolites between subcellular compartments therefore coordinates the energetic and metabolic status of the cell with membrane ion transport.

Stomata on the abaxial and adaxial leaf surfaces contribute differently to leaf gas exchange and photosynthesis in wheat.

Although stomata are typically found in greater numbers on the abaxial surface, wheat flag leaves have greater densities on the adaxial surface. We determine the impact of this less common stomatal patterning on gaseous fluxes using a novel chamber that simultaneously measures both leaf surfaces. Using a combination of differential illuminations and CO2 concentrations at each leaf surface, we found that mesophyll cells associated with the adaxial leaf surface have a higher photosynthetic capacity than those associated with the abaxial leaf surface, which is supported by an increased stomatal conductance (driven by differences in stomatal density). When vertical gas flux at the abaxial leaf surface was blocked, no compensation by adaxial stomata was observed, suggesting each surface operates independently. Similar stomatal kinetics suggested some co-ordination between the two surfaces, but factors other than light intensity played a role in these responses. Higher photosynthetic capacity on the adaxial surface facilitates greater carbon assimilation, along with higher adaxial stomatal conductance, which would also support greater evaporative leaf cooling to maintain optimal leaf temperatures for photosynthesis. Furthermore, abaxial gas exchange contributed c. 50% to leaf photosynthesis and therefore represents an important contributor to overall leaf gas exchange.

Light, power, action! Interaction of respiratory energy- and blue light-induced stomatal movements.

Although the signalling pathway of blue light (BL)-dependent stomatal opening is well characterized, little is known about the interspecific diversity, the role it plays in the regulation of gas exchange and the source of energy used to drive the commonly observed increase in pore aperture. Using a combination of red and BL under ambient and low [O2 ] (to inhibit respiration), the interaction between BL, photosynthesis and respiration in determining stomatal conductance was investigated. These findings were used to develop a novel model to predict the feedback between photosynthesis and stomatal conductance under these conditions. Here we demonstrate that BL-induced stomatal responses are far from universal, and that significant species-specific differences exist in terms of both rapidity and magnitude. Increased stomatal conductance under BL reduced photosynthetic limitation, at the expense of water loss. Moreover, we stress the importance of the synergistic effect of BL and respiration in driving rapid stomatal movements, especially when photosynthesis is limited. These observations will help reshape our understanding of diurnal gas exchange in order to exploit the dynamic coordination between the rate of carbon assimilation (A) and stomatal conductance (gs ), as a target for enhancing crop performance and water use efficiency.

Variation in key leaf photosynthetic traits across wheat wild relatives is accession dependent not species dependent.

The wild relatives of modern wheat represent an underutilized source of genetic and phenotypic diversity and are of interest in breeding owing to their wide adaptation to diverse environments. Leaf photosynthetic traits underpin the rate of production of biomass and yield and have not been systematically explored in the wheat relatives. This paper identifies and quantifies the phenotypic variation in photosynthetic, stomatal, and morphological traits in up to 88 wheat wild relative accessions across five genera. Both steady-state measurements and dynamic responses to step changes in light intensity are assessed. A 2.3-fold variation for flag leaf light and CO2 -saturated rates of photosynthesis Amax was observed. Many accessions showing higher and more variable Amax , maximum rates of carboxylation, electron transport, and Rubisco activity when compared with modern genotypes. Variation in dynamic traits was also significant; with distinct genus-specific trends in rates of induction of nonphotochemical quenching and rate of stomatal opening. We conclude that utilization of wild relatives for improvement of photosynthesis is supported by the existence of a high degree of natural variation in key traits and should consider not only genus-level properties but variation between individual accessions.

Thermography methods to assess stomatal behaviour in a dynamic environment.

Although thermography allows rapid, non-invasive measurements of large numbers of plants, it has not been used extensively due to the difficulty in deriving biologically relevant information such as leaf transpiration (E) and stomatal conductance (gsw) from thermograms. Methods normalizing leaf temperature using temperatures from reference materials (e.g. with and without evaporative flux) to generate stress indices are generally preferred due to their ease of use to assess plant water status. Here, a simplified method to solve dynamic energy balance equations is presented, which enables the calculation of 'wet' and 'dry' leaf temperatures in order to derive stress indices, whilst providing accurate estimates of E and gsw. Comparing stress indices and gas exchange parameters highlights the limitation of stress indices in a dynamic environment and how this problem can be overcome using artificial leaf references with known conductance. Additionally, applying the equations for each pixel of a thermogram to derive the rapidity of stomatal response over the leaf lamina in wheat revealed the spatial heterogeneity of stomatal behaviour. Rapidity of stomatal movements is an important determinant of water use efficiency, and our results showed 'patchy' responses that were linked to both the spatial and temporal response of gsw.

Role of blue and red light in stomatal dynamic behaviour.

Plants experience changes in light intensity and quality due to variations in solar angle and shading from clouds and overlapping leaves. Stomatal opening to increasing irradiance is often an order of magnitude slower than photosynthetic responses, which can result in CO2 diffusional limitations on leaf photosynthesis, as well as unnecessary water loss when stomata continue to open after photosynthesis has reached saturation. Stomatal opening to light is driven by two distinct pathways; the 'red' or photosynthetic response that occurs at high fluence rates and saturates with photosynthesis, and is thought to be the main mechanism that coordinates stomatal behaviour with photosynthesis; and the guard cell-specific 'blue' light response that saturates at low fluence rates, and is often considered independent of photosynthesis, and important for early morning stomatal opening. Here we review the literature on these complicated signal transduction pathways and osmoregulatory processes in guard cells that are influenced by the light environment. We discuss the possibility of tuning the sensitivity and magnitude of stomatal response to blue light which potentially represents a novel target to develop ideotypes with the 'ideal' balance between carbon gain, evaporative cooling, and maintenance of hydraulic status that is crucial for maximizing crop performance and productivity.

Unexpected Connections between Humidity and Ion Transport Discovered Using a Model to Bridge Guard Cell-to-Leaf Scales.

Stomatal movements depend on the transport and metabolism of osmotic solutes that drive reversible changes in guard cell volume and turgor. These processes are defined by a deep knowledge of the identities of the key transporters and of their biophysical and regulatory properties, and have been modeled successfully with quantitative kinetic detail at the cellular level. Transpiration of the leaf and canopy, by contrast, is described by quasilinear, empirical relations for the inputs of atmospheric humidity, CO2, and light, but without connection to guard cell mechanics. Until now, no framework has been available to bridge this gap and provide an understanding of their connections. Here, we introduce OnGuard2, a quantitative systems platform that utilizes the molecular mechanics of ion transport, metabolism, and signaling of the guard cell to define the water relations and transpiration of the leaf. We show that OnGuard2 faithfully reproduces the kinetics of stomatal conductance in Arabidopsis thaliana and its dependence on vapor pressure difference (VPD) and on water feed to the leaf. OnGuard2 also predicted with VPD unexpected alterations in K+ channel activities and changes in stomatal conductance of the slac1 Cl- channel and ost2 H+-ATPase mutants, which we verified experimentally. OnGuard2 thus bridges the micro-macro divide, offering a powerful tool with which to explore the links between guard cell homeostasis, stomatal dynamics, and foliar transpiration.

Speedy stomata, photosynthesis and plant water use efficiency.

Contents Summary 93 I. Introduction 93 II. Influence of the speed of gs responses on A and Wi 93 III. Determinants of the rapidity of gs responses 95 IV. Conclusion 97 Acknowledgements 97 References 97 SUMMARY: Stomatal movements control CO2 uptake for photosynthesis and water loss through transpiration, and therefore play a key role in plant productivity and water use efficiency. The predicted doubling of global water usage by 2030 mean that stomatal behaviour is central to current efforts to increase photosynthesis and crop yields, particularly under conditions of reduced water availability. In the field, slow stomatal responses to dynamic environmental conditions add a temporal dimension to gaseous fluxes between the leaf and atmosphere. Here, we review recent work on the rapidity of stomatal responses and present some of the possible anatomical and biochemical mechanisms that influence the rapidity of stomatal movements.

Acclimation to Fluctuating Light Impacts the Rapidity of Response and Diurnal Rhythm of Stomatal Conductance.

Plant acclimation to growth light environment has been studied extensively; however, the majority of these studies have focused on light intensity and photo-acclimation, with few studies exploring the impact of dynamic growth light on stomatal acclimation and behavior. To assess the impact of growth light regime on stomatal acclimation, we grew Arabidopsis (Arabidopsis thaliana) plants in three different lighting regimes (with the same average daily intensity), fluctuating with a fixed pattern of light, fluctuating with a randomized pattern of light (sinusoidal), and nonfluctuating (square wave), to assess the effect of light regime dynamics on gas exchange. We demonstrated that gs (stomatal conductance to water vapor) acclimation is influenced by both intensity and light pattern, modifying the stomatal kinetics at different times of the day and resulting in differences in the rapidity and magnitude of the gs response. We also describe and quantify the response to an internal signal that uncouples variation in A and gs over the majority of the diurnal period and represents 25% of the total diurnal gs This gs response can be characterized by a Gaussian element and when incorporated into the widely used Ball-Berry model greatly improved the prediction of gs in a dynamic environment. From these findings, we conclude that acclimation of gs to growth light could be an important strategy for maintaining carbon fixation and overall plant water status and should be considered when inferring responses in the field from laboratory-based experiments.

Dynamic leaf energy balance: deriving stomatal conductance from thermal imaging in a dynamic environment.

In spite of the significant progress made in recent years, the use of thermography to derive biologically relevant traits remains a challenge under fluctuating conditions. The aim of this study was to rethink the current method to process thermograms and derive temporal responses of stomatal conductance (gsw) using dynamic energy balance equations. Time-series thermograms provided the basis for a spatial and temporal characterization of gsw responses in wheat (Triticum aestivum). A leaf replica with a known conductance was used to validate the approach and to test the ability of our model to be used with any material and under any environmental conditions. The results highlighted the importance of the co-ordinated stomatal responses that run parallel to the leaf blade despite their patchy distribution. The diversity and asymmetry of the temporal response of gsw observed after a step increase and step decrease in light intensity can be interpreted as a strategy to maximize photosynthesis per unit of water loss and avoid heat stress in response to light flecks in a natural environment. This study removes a major bottleneck for plant phenotyping platforms and will pave the way to further developments in our understanding of stomatal behaviour.

Time-Series Transcriptomics Reveals That AGAMOUS-LIKE22 Affects Primary Metabolism and Developmental Processes in Drought-Stressed Arabidopsis.

In Arabidopsis thaliana, changes in metabolism and gene expression drive increased drought tolerance and initiate diverse drought avoidance and escape responses. To address regulatory processes that link these responses, we set out to identify genes that govern early responses to drought. To do this, a high-resolution time series transcriptomics data set was produced, coupled with detailed physiological and metabolic analyses of plants subjected to a slow transition from well-watered to drought conditions. A total of 1815 drought-responsive differentially expressed genes were identified. The early changes in gene expression coincided with a drop in carbon assimilation, and only in the late stages with an increase in foliar abscisic acid content. To identify gene regulatory networks (GRNs) mediating the transition between the early and late stages of drought, we used Bayesian network modeling of differentially expressed transcription factor (TF) genes. This approach identified AGAMOUS-LIKE22 (AGL22), as key hub gene in a TF GRN. It has previously been shown that AGL22 is involved in the transition from vegetative state to flowering but here we show that AGL22 expression influences steady state photosynthetic rates and lifetime water use. This suggests that AGL22 uniquely regulates a transcriptional network during drought stress, linking changes in primary metabolism and the initiation of stress responses.

Diurnal Variation in Gas Exchange: The Balance between Carbon Fixation and Water Loss.

Stomatal control of transpiration is critical for maintaining important processes, such as plant water status, leaf temperature, as well as permitting sufficient CO2 diffusion into the leaf to maintain photosynthetic rates (A). Stomatal conductance often closely correlates with A and is thought to control the balance between water loss and carbon gain. It has been suggested that a mesophyll-driven signal coordinates A and stomatal conductance responses to maintain this relationship; however, the signal has yet to be fully elucidated. Despite this correlation under stable environmental conditions, the responses of both parameters vary spatially and temporally and are dependent on species, environment, and plant water status. Most current models neglect these aspects of gas exchange, although it is clear that they play a vital role in the balance of carbon fixation and water loss. Future efforts should consider the dynamic nature of whole-plant gas exchange and how it represents much more than the sum of its individual leaf-level components, and they should take into consideration the long-term effect on gas exchange over time.

Importance of Fluctuations in Light on Plant Photosynthetic Acclimation.

The acclimation of plants to light has been studied extensively, yet little is known about the effect of dynamic fluctuations in light on plant phenotype and acclimatory responses. We mimicked natural fluctuations in light over a diurnal period to examine the effect on the photosynthetic processes and growth of Arabidopsis (Arabidopsis thaliana). High and low light intensities, delivered via a realistic dynamic fluctuating or square wave pattern, were used to grow and assess plants. Plants subjected to square wave light had thicker leaves and greater photosynthetic capacity compared with fluctuating light-grown plants. This, together with elevated levels of proteins associated with electron transport, indicates greater investment in leaf structural components and photosynthetic processes. In contrast, plants grown under fluctuating light had thinner leaves, lower leaf light absorption, but maintained similar photosynthetic rates per unit leaf area to square wave-grown plants. Despite high light use efficiency, plants grown under fluctuating light had a slow growth rate early in development, likely due to the fact that plants grown under fluctuating conditions were not able to fully utilize the light energy absorbed for carbon fixation. Diurnal leaf-level measurements revealed a negative feedback control of photosynthesis, resulting in a decrease in total diurnal carbon assimilated of at least 20%. These findings highlight that growing plants under square wave growth conditions ultimately fails to predict plant performance under realistic light regimes and stress the importance of considering fluctuations in incident light in future experiments that aim to infer plant productivity under natural conditions in the field.

Global Sensitivity Analysis of OnGuard Models Identifies Key Hubs for Transport Interaction in Stomatal Dynamics.

The physical requirement for charge to balance across biological membranes means that the transmembrane transport of each ionic species is interrelated, and manipulating solute flux through any one transporter will affect other transporters at the same membrane, often with unforeseen consequences. The OnGuard systems modeling platform has helped to resolve the mechanics of stomatal movements, uncovering previously unexpected behaviors of stomata. To date, however, the manual approach to exploring model parameter space has captured little formal information about the emergent connections between parameters that define the most interesting properties of the system as a whole. Here, we introduce global sensitivity analysis to identify interacting parameters affecting a number of outputs commonly accessed in experiments in Arabidopsis (Arabidopsis thaliana). The analysis highlights synergies between transporters affecting the balance between Ca2+ sequestration and Ca2+ release pathways, notably those associated with internal Ca2+ stores and their turnover. Other, unexpected synergies appear, including with the plasma membrane anion channels and H+-ATPase and with the tonoplast TPK K+ channel. These emergent synergies, and the core hubs of interaction that they define, identify subsets of transporters associated with free cytosolic Ca2+ concentration that represent key targets to enhance plant performance in the future. They also highlight the importance of interactions between the voltage regulation of the plasma membrane and tonoplast in coordinating transport between the different cellular compartments.

An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells.

Oscillations in cytosolic-free Ca(2+) concentration ([Ca(2+)]i) have been proposed to encode information that controls stomatal closure. [Ca(2+)]i oscillations with a period near 10 min were previously shown to be optimal for stomatal closure in Arabidopsis (Arabidopsis thaliana), but the studies offered no insight into their origins or mechanisms of encoding to validate a role in signaling. We have used a proven systems modeling platform to investigate these [Ca(2+)]i oscillations and analyze their origins in guard cell homeostasis and membrane transport. The model faithfully reproduced differences in stomatal closure as a function of oscillation frequency with an optimum period near 10 min under standard conditions. Analysis showed that this optimum was one of a range of frequencies that accelerated closure, each arising from a balance of transport and the prevailing ion gradients across the plasma membrane and tonoplast. These interactions emerge from the experimentally derived kinetics encoded in the model for each of the relevant transporters, without the need of any additional signaling component. The resulting frequencies are of sufficient duration to permit substantial changes in [Ca(2+)]i and, with the accompanying oscillations in voltage, drive the K(+) and anion efflux for stomatal closure. Thus, the frequency optima arise from emergent interactions of transport across the membrane system of the guard cell. Rather than encoding information for ion flux, these oscillations are a by-product of the transport activities that determine stomatal aperture.

Effects of kinetics of light-induced stomatal responses on photosynthesis and water-use efficiency.

Both photosynthesis (A) and stomatal conductance (gs ) respond to changing irradiance, yet stomatal responses are an order of magnitude slower than photosynthesis, resulting in noncoordination between A and gs in dynamic light environments. Infrared gas exchange analysis was used to examine the temporal responses and coordination of A and gs to a step increase and decrease in light in a range of different species, and the impact on intrinsic water use efficiency was evaluated. The temporal responses revealed a large range of strategies to save water or maximize photosynthesis in the different species used in this study but also displayed an uncoupling of A and gs in most of the species. The shape of the guard cells influenced the rapidity of response and the overall gs values achieved, with different impacts on A and Wi . The rapidity of gs in dumbbell-shaped guard cells could be attributed to size, whilst in elliptical-shaped guard cells features other than anatomy were more important for kinetics. Our findings suggest significant variation in the rapidity of stomatal responses amongst species, providing a novel target for improving photosynthesis and water use.