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

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

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

Acclimation to fluctuating light impacts the rapidity 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. In order to assess the impact of growth light regime on stomatal acclimation, we grew 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 non-fluctuating (square wave), to assess the effect of light regime dynamics on gas exchange. We demonstrated that gs acclimation is influenced by both intensity and light pattern, modifying the stomatal kinetics at different times of the day resulting in differences in the rapidity and magnitude of the gs response. We also describe and quantify 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

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

Development of an accurate low cost NDVI imaging system for assessing plant health.

BACKGROUND: Spectral imaging is a key method for high throughput phenotyping that can be related to a large variety of biological parameters. The Normalised Difference Vegetation Index (NDVI), uses specific wavelengths to compare crop health and performance. Increasing the accessibility of spectral imaging systems through the development of small, low cost, and easy to use platforms will generalise its use for precision agriculture. We describe a method for using a dual camera system connected to a Raspberry Pi to produce NDVI imagery, referred to as NDVIpi. Spectral reference targets were used to calibrate images into values of reflectance, that are then used to calculated NDVI with improved accuracy compared with systems that use single references/standards. RESULTS: NDVIpi imagery showed strong performance against standard spectrometry, as an accurate measurement of leaf NDVI. The NDVIpi was also compared to a relatively more expensive commercial camera (Micasense RedEdge), with both cameras having a comparable performance in measuring NDVI. There were differences between the NDVI values of the NDVIpi and the RedEdge, which could be attributed to the measurement of different wavelengths for use in the NDVI calculation by each camera. Subsequently, the wavelengths used by the NDVIpi show greater sensitivity to changes in chlorophyll content than the RedEdge. CONCLUSION: We present a methodology for a Raspberry Pi based NDVI imaging system that utilizes low cost, off-the-shelf components, and a robust multi-reference calibration protocols that provides accurate NDVI measurements. When compared with a commercial system, comparable NDVI values were obtained, despite the fact that our system was a fraction of the cost. Our results also highlight the importance of the choice of red wavelengths in the calculation of NDVI, which resulted in differences in sensitivity between camera systems

Temporal Dynamics of Stomatal Behavior: Modeling and Implications for Photosynthesis and Water Use.

An analysis of stomatal behavior reveals the importance of modeling slow stomatal responses and the impacts on photosynthesis under dynamic light environments

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

Stomata on the abaxial and adaxial leaf surface 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 mesophyll cells associated with the adaxial leaf surface have a higher photosynthetic capacity than the abaxial, supported by a greater stomatal conductance (driven by differences in stomatal density). Blocking vertical gas flux on the abaxial leaf surface demonstrated no compensation by adaxial stomata, 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 gs that would also support greater evaporative leaf cooling to maintain optimal leaf temperatures for photosynthesis. Furthermore, abaxial gas exchange contributed approximately 50% to leaf photosynthesis, and therefore represents an important contributor to overall leaf gas photosynthesis

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 inArabidopsis thalianaand 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 theslac1Cl-channel andost2H+-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.This work was supported by Biotechnology and Biological Sciences Research Council (BBSRC) Grants BB/L019205/1 and BB/M001601/1 to M.R.B., BB/L001276/1 to M.R.B. and S.R., and BB/I001187/1 to H.G. and T.L