Role of blue and red light in stomatal dynamic behaviour

1Stomata control the flux of CO2 into the leaf and water lost through transpiration, and are crucial in maintaining plant water status, leaf temperature, and photosynthetic rates, depending on the current needs of the plant. The surface of most leaves is effectively impermeable to water and CO2; therefore, most of the CO2 fixed and water lost by plants must pass through stomatal pores, with stomata controlling the majority of gas exchange between the leaf and external environment, despite typically occupying only a small proportion (0.3–5%) of the leaf surface. The capacity of stomata to allow CO2 into or water out of the leaf is known as stomatal conductance (gs), with stomatal behaviour leading to alterations in stomatal aperture and therefore diffusional fluxes. Stomatal aperture is governed by changes in guard cell (GC) volume and turgor pressure driven by alterations in osmotic potential, and, along with stomatal density, determines gs. Adjustment in stomatal behaviour is driven by the external environmental (e.g. light) and internal signalling cues (see ), with responses to these signals varying between and within species. In general, stomatal opening is triggered by increasing light or temperatures (up to an optimum), low CO2, and low vapour pressure deficit (VPD), whilst closure is driven by the reverse; decreasing light, extreme low or high temperatures, high CO2, and high VPD. In response to changes in these environmental cues, various ion and solute channels in GCs are activated via a signalling cascade, triggering the uptake or release of ions and solutes that modify the osmotic and water potential of the cell, leading to the uptake or loss of water, changes in turgor pressure, and, therefore, changes in stomatal aperture (e.g. see;;; ). It is well established that high gs can facilitate a higher net photosynthetic rate (A); however, this is at a greater cost of water loss, making plants more vulnerable to water stress or cavitation (depending on the species), whereas low gs can limit CO2 diffusion and photosynthetic rates by up to 20% in well-watered C3 species, negatively affecting biomass accumulation and yield. Low stomatal aperture may also impact evaporative cooling and conservation of leaf temperature in an optimal range, which is important for maintaining photosynthetic rates and therefore has consequences for harvestable yield. Although it is well established that a close correlation between gs and A exists, and whilst the exact signalling pathways and mechanisms that support this relationship have not been fully established, several theories have been put forward. This correlation between A and gs is understood to exist to optimize the trade-off between carbon gain and water loss, with stomata continually adjusting aperture to balance the requirement for CO2 for photosynthesis against the need to maintain leaf hydration. However, stomatal responses tend to be an order of magnitude slower than photosynthetic responses, and this leads to non-coordinated responses in gs and A, where lags in behaviour or slow stomatal responses will often lead to a limitation in carbon gain or unnecessary increase in water loss (see Lawson and ). It has therefore been suggested that species with more rapid gs responses to changing environmental conditions will maximize both photosynthesis and water use efficiency (WUE;;;;; ) and that manipulation of stomatal kinetics could be a novel approach to enhancing CO2 uptake, maintaining optimal leaf temperature for carbon assimilation, and improving water use in important crops, particularly given the predicted changes to the climate.

2As photosynthesis and stomata do not respond with the same rapidity to changes in light intensity and spectral quality, short-term fluctuations in light lead to temporal and spatial disconnections between stomatal behaviour and photosynthesis (e.g.;;; ). Although it is possible to detect these spatial and temporal differences in gs and A (e.g. using imaging approaches; see; ), there are still major gaps in our understanding of the impact of this variation on plant carbon gain or WUE and how such patterns relate to differences in light perception, signal transduction, and stomatal behaviour. Therefore, manipulation of stomatal behaviour and/or the mechanisms that coordinate stomatal response to light quality and intensity could provide potential targets for increasing photosynthesis, WUE, and overall plant productivity in the field. However, in order to succeed, more evidence on the mechanisms and signalling pathways associated with stomatal dynamics in response to light quantity and quality is required, as well as an understanding of the influence of the mesophyll and the hierarchy of stomatal responses.

3Plants experience light in a range of intensities and spectral properties, largely due to passing clouds, changes in canopy cover, and self-shading from overlapping leaves, and this produces unpredictable fluctuations in spectral distribution (see Fig. 1) that impact stomatal behaviour, carbon gain, and the diurnal course of WUE. The path of the sun across the sky affects both the quantity and quality of the light available to plants at any given location. At dawn and towards dusk as solar angle diminishes, sunlight negotiates an increasingly long path through the atmosphere, enhancing atmospheric light absorption and scattering, thus depleting shorter wavelengths of light. Furthermore, the contribution of direct radiation relative to diffuse radiation declines, often leading to a pronounced peak in blue light, changing the light quality and therefore plant response. Changes in the quality of light throughout the day may impact stomatal dynamic response and diurnal behaviour, and therefore affect photosynthetic efficiency and water use through gs enforced diffusional constraints on A.

4Stomata in C3 and C4 species open in response to increasing light intensity whilst closure is brought about by reductions in intensity, whereas CAM (crassulacean acid metabolism) species display the opposite response, with stomata opening in darkness for nocturnal CO2 uptake and closing in the light. The stomatal opening responses to light can be divided into two distinct pathways; termed the red or mesophyll/photosynthetic response (herein, termed the red light response) and the GC-specific blue light response. The red light response occurs at high fluence rates and saturates at similar intensities to photosynthesis, and many studies have suggested that response is the primary mechanism linking stomatal behaviour with photosynthetic rates and that it is responsible for the close correlation between A and gs. The blue light stomatal response occurs and is saturated at a low fluence rate (~5–10 µmol m–2 s–1; ), is GC specific, and is thought to be independent of mesophyll photosynthesis. Blue-light-initiated responses are not exclusive to stomata, and other responses are initiated by this signal that are important for optimal performance; including phototropism (see;; ), photomorphogenesis, flowering and circadian clock function, and the directional movement of chloroplasts in the mesophyll and GC complexes (see; ).

5Although most research on the spectral aspect of stomatal behaviour and photosynthesis focuses on red and blue light, there is also evidence that green light plays a vital role in physiological responses to the environment. It has been reported that plants may use green wavelengths as a crucial signal to determine short-term dynamic responses and long-term developmental acclimation, enabling optimization of resource use efficiency and photosynthesis to available irradiance. Furthermore, green light has been shown to inhibit blue-light-induced stomatal opening, potentially to prevent excessive leaf water loss in shade environments when photosynthetic potential is low. In this review, we focus on light-stimulated stomatal behaviour and specifically on the red-(photosynthetic) and blue-light-driven responses, what is understood about the different signalling pathways involved, and GC metabolism that facilitates these responses. We further explore how a better understanding of stomatal response to irradiance, and the influence of the mesophyll on these responses, could provide novel targets for the development of plants with improved photosynthetic carbon gain and WUE.

6The red-light-driven opening response of stomata resembles the carbon assimilation response to increasing light intensity and is eliminated by inhibitors of photosynthetic electron transport, including 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that it is photosynthesis dependent (e.g.;;;; ), suggesting that chlorophyll could be the receptor. This red light response is considered the primary mechanism linking stomatal behaviour with mesophyll demands for CO2, although the exact location of the red light signal has not been fully elucidated. There are suggestions that it occurs in the chloroplast, either in the GCs themselves or in the mesophyll, and that a signal is transferred from the mesophyll to the GCs. Several studies have suggested that GCs do not directly sense red light, and instead respond to the supply of CO2 in the mesophyll (see Fig. 2), coupling A and gs via Ci, although other signalling mechanisms have been suggested (discussed below). Mesophyll consumption of CO2 driven by increasing photosynthetic irradiance reduces [CO2] in the intercellular air spaces to which stomata respond by opening; low light reduces this consumption and increases the concentration, closing stomata. Support for a Ci-driven response, independent of a specific GC response, was provided by, ), who showed that a beam of blue light, but not red, induced changes in membrane potential in the GCs, altering K+ transport across the plasma membrane. Only when [CO2] around the GCs was altered under red light did these authors report that cells were hyperpolarized in CO2-free air, and switched to being depolarized when [CO2] was increased to 700 μmol mol–1, extruding K+ which resulted in stomatal closure. From these studies, it was concluded that GCs do not respond to red light directly but to the indirect changes in Ci. This is further supported by studies that have shown that stomatal opening to red light is mediated in part by a component of the low CO2 signalling network (HT1; HIGH LEAF TEMPERATURE1;; ), and that high [CO2] activates the release of Cl– ions from GCs via S-type anion channels (such as SLAC1;;; ), inducing stomatal closure (Fig. 2). Further studies showed that stomata of Vicia faba and wheat did not respond to red light when treated with the carotenoid inhibitor norflurazon (which results in albino leaves that lack functional green chloroplasts). Similarly, no response was observed in the white area of variegated Hedera helix and Chlorophytum comosum (that do contain photosynthetically active chloroplasts in the GC) when illuminated with red light. However, in both cases, stomata still responded to blue light, [CO2], and abscisic acid (ABA); therefore, it was concluded that photosynthetically active mesophyll is required for stomatal red light responses via changes in Ci. However, many other studies have suggested that stomatal aperture responses to Ci are too small to account for the changes observed in gs in response to light, and it has been reported that under red light, gs increases even when Ci is held constant, questioning Ci as the main driver of A and gs coordination. Furthermore, studies on transgenic plants in which expression levels of several enzymes associated with electron transport or the Calvin cycle were manipulated leading to reduced photosynthetic rates demonstrated that stomata opened in response to light regardless of the higher Ci values observed. The lack of coordination between A and gs in these transgenic plants raised questions concerning the mechanism(s) that links these two processes, Other studies have suggested that an as yet unidentified signal originating in the mesophyll is potentially sensed by GCs activating a stomatal response. demonstrated that stomata on the abaxial leaf surface opened more widely when the leaf was irradiated from the adaxial rather than the abaxial side with Ci kept constant, supporting the idea of a direct mesophyll signal influencing stomatal behaviour., ) were the first to propose an aqueous metabolic signal, with potential candidates such as ribulose bisphosphate (RuBP), ATP, NADPH, malate, and sugar. However, further research has suggested a gaseous vapour phase ion, sucrose metabolism, and even GC photosynthesis itself.

7The red light response (as with all stomatal responses) requires changes in osmotic potential in the GC, driven by the accumulation or loss of ions such as K+ and/or sugar accumulation (see reviews by; ) to change water flux and therefore pore width (Fig. 2). Early research demonstrated that potassium accumulation is the result of red light activation of the plasma membrane proton pump, with ATP supplied by photophosphorylation in the GC chloroplasts, although subsequent patch-clamp experiments could not replicate red light activation of the proton pump. Sugars as well as K+ have also been reported to accumulate in response to red-light-induced stomatal opening (Talbott and; ), provided either by starch breakdown, import from the mesophyll (e.g. ), or directly through GC photosynthesis (see review by ). Although early studies on red-light-induced stomatal opening in epidermal peels reported high GC sucrose concentrations, suggesting that the supply must be from GC photosynthetic carbon assimilation (Talbott and ), other studies have reported that GC photosynthesis is insufficient to produce sucrose required for osmoregulation (e.g.; ). This led to the hypothesis that apoplastic sucrose fixed in the mesophyll cells travels to the GCs via the transpiration stream, where it can be imported into the GCs via sucrose-mediated H+ symporter mechanism(s) and act to open or close stomata or replace GC carbon stores. Apoplastic sucrose accumulation at the GC has been proposed to initiate stomatal closure and provide a mechanism to coordinate A and gs. Outlaw and colleagues suggested that when mesophyll cells produce more sugar than can be loaded into the phloem, any excess will be carried to the GCs to reduce stomatal aperture (see ). This is supported by the numerous studies that have demonstrated sugar import into GCs. suggested that sucrose arriving at the GC is cleaved in the apoplasts to produce glucose and fructose that is then sensed by hexokinase, which signals stomatal closure response. Although this mechanism may explain the reported decrease in A and gs often observed over longer time scales and toward the end of the day, it cannot explain the short-term coordination of A and gs.

8It has recently been demonstrated that Arabidopsis mutants with reduced GC chlorophyll content have reduced gs, implying that GC photosynthesis is crucial for energetics and stomatal movements (Fig. 2; ). Support for the involvement of GC electron transport in light-induced stomatal opening also comes from work on the ‘crumpled leaf’ mutants, which lack GC chloroplasts. These mutants exhibited reduced levels of GC ATP and stomatal aperture in response to white light. also demonstrated that lower ATP levels were observed in epidermal peels incubated (for 2 h in light) in isolation from the mesophyll, compared with peels that had been collected from intact leaf material after the incubation period. These findings strongly suggest that both GCs and mesophyll cells provide ATP for stomatal opening. Therfore, as there is currently no evidence for ATP import into GCs, mesophyll cells are likely to indirectly supply ATP by providing sugars that are utilized by the mitochondria. The ATP from GC electron transport provides additional energy to that produced by glycolysis and mitochondrial respiration, which can be directly used for proton pumping or other metabolic processes involved in osmoregulation for stomatal opening in response to red (and blue) light (see,;; ). Interestingly, a recent report has demonstrated that red-light-induced plasma membrane H+-ATPase phosphorylation correlated with stomatal opening, a process previously thought to be blue light dependent and under the control of the photoreceptor protein kinases, phototropins (see below). Using knockout mutants of one of the major isoforms of plasma membrane H+-ATPase in GCs, aha1-9, revealed that red-light-dependent stomatal opening was delayed in whole leaves. An immunohistochemical technique to detect phosphorylation demonstrated that DCMU inhibited plasma membrane H+-ATPase phosphorylation and red-light-induced stomatal opening. However, the lack of this response in isolated epidermal peels further suggests that mesophyll photosynthesis is required for the red light response. Furthermore, the authors did not rule out that GC chloroplasts might have the potential to induce partial phosphorylation of the plasma membrane H+-ATPase, and that this could be the underlying cause for interspecific differences in red light sensitivity in GCs. Moreover, as electron transport in the GCs and mesophyll chloroplasts is essentially the same, this could be involved in the regulation and coordination of A and gs responses. Interestingly, the redox state of the chloroplastic plastoquinone pool (QA) has been put forward as a signal that coordinates red light stomatal responses with the mesophyll at a range of light intensities. This was experimentally tested using tobacco mutants with reduced expression levels of PSII subunit S (PsbS), which directly effects the redox state of QA and results in a strong correlation with gs when measured under a range of light intensities. As tobacco generally lacks the GC-specific blue light response (see below), this enabled a direct relationship, driven by the red or photosynthetic light response, between QA and gs to be evaluated.

9Blue-light-induced stomatal opening has been demonstrated in isolated epidermal peels and GC protoplasts; therefore, all the components required for this response are located in the GCs themselves and, unlike the red light response, does not require the involvement of a mesophyll signal. Blue-light-induced stomatal opening is saturated at a low (~5–10 µmol m–2 s–1) fluence rate, too low to drive photosynthetic carbon gain, and is 20 times more effective at opening stomata than red light. It has been proposed that the stomatal blue light response is important for morning pore opening to facilitate photosynthetic carbon gain early in the diel period, when the irradiance spectrum is enriched in blue wavelengths (Fig. 1; ). Additionally, this response could be important in rapid stomatal responses to sun flecks to maximize opportunistic periods of photosynthesis, particularly in understorey environments.

10Although the stomatal blue light response has been reported as not requiring photosynthesis, it has been demonstrated that two kinases found in the GC blue light signalling pathway—CBC1 and CBC2 (CONVERGENCE OF BLUE LIGHT AND CO2)—are actually involved in linking blue light responses (via phototropins) to low CO2 concentrations in the GC (; see Fig. 2). This therefore suggests that photosynthesis is indirectly involved, and in fact it has been shown previously that the sensitivity and magnitude of the stomatal response to blue light depend on the intensity of background red light. In their review on light regulation of stomatal movement, reported an increased gs rate when blue light was applied to a background of red light compared with red alone, and virtually no response to weak blue light was observed when red light was absent, although this response could be species specific. Figure 3 highlights the impact of blue light on the magnitude of gs compared with red light alone, demonstrating the independent and synergistic behaviour of the two distinct signals.

11The stomatal blue light response is mediated by blue light photoreceptor protein kinases, known as phototropins (phot1 and phot2;;; ). Under blue light, phototropins within the GCs are activated via autophosphorylation and initiate a signalling cascade that eventually results in stomatal opening (see Fig. 2;;;; ). The protein kinase BLUE LIGHT SIGNALLING 1 (BLUS1) is directly phosphorylated by the activated phototropins, and has been shown to indirectly transmit a signal to a type 1 protein phosphatase (PP1) and regulatory subunit PRSL1. This blue-light-driven BLUS1 signal activates plasma membrane H+-ATPase in GCs via phosphorylation of a penultimate C-terminal residue (Thr) and through subsequent binding of a 14-3-3 protein. Further recent research on the stomatal blue light pathway has identified that a Raf-like (receptor kinase involved in cell cycle regulation) protein kinase, BLUE LIGHT-DEPENDENT H+-ATPase PHOSPHORYLATION (BHP), binds to BLUS1 and forms a signalling complex with phototropins to mediate phosphorylation of plasma membrane H+-ATPase. However, these authors suggest that BHP does not directly phosphorylate the penultimate Thr of membrane H+-ATPase, and another as yet unidentified signalling kinase may exist that directly controls phosphorylation of H+-ATPase, and stomatal opening in the blue light signalling cascade. Blue-light-activated plasma membrane H+-ATPase initiates hyperpolarization of the membrane and drives H+ transport out of the GC. This hyperpolarization further activates inward-rectifying K+ channels, resulting in the influx and accumulation of K+ in the cytosol. Transport and accumulation of K+ and the counter-ions Cl– and malate2– into the vacuole occurs via K+/H+ exchangers (NHX1 and NHX2), chloride channel c (CLCc), and chloride channel malate transporters (ALMT9), which decrease GC water potential, increasing water uptake and turgor pressure, and ultimately leads to stomatal opening (Fig. 2;;;; ). It has been suggested that among the phototropin-mediated responses, BLUS1 defines signalling specificity of stomatal opening and, as BLUS1 expression is only found in the GC and not in mesophyll cells, is not involved in other important phototropin-mediated responses (including phototropism and chloroplast movement), and therefore could be an unexploited target for manipulating stomatal behaviour.

12In addition, the phototropin-dependent blue light signalling cascade and activation of plasma membrane H+-ATPase has been reported to be involved in carbon metabolism in GCs. A study by revealed a novel pathway of starch degradation involving synergistic activities of β-amylase 1 (BAM1) and α-amylase 3 (AMY3) in GCs. This is in contrast to the mesophyll starch metabolism in which BAM3 is the major isoform and BAM1 has limited involvement (see ). Using the phot1/phot2 double mutant and the BLUS1 mutant, these authors showed that the blue light signalling pathway was required for starch breakdown, in order to produce maltose which is subsequently turned into malate (which acts as a counter-ion for K+ uptake) through glycolysis and the activity of phosphoenolpyruvate carboxylase (PEPc; ). This is is agreement with other studies that have suggested a role for malate and PEPc in GC osmoregulation. This novel starch degradation pathway proposed by highlighted that BAM1 and AMY3 are redox regulated (unlike the starch degradation enzymes associated with mesophyll activity), and suggested that GC electron transport could provide the reduced environment that would support BAM1 and AMY3 activation as well as providing a further connection between increasing light and stomatal opening, and a possible link between red and blue light responses (see below).

13The fact that the blue light response has been observed in isolated tissues and occurs at low fluence rates has led to the suggestion that the ATP required for the proton pumps is most probably provided by GC mitochondria. This is supported by the lower number of chloroplasts (although this is species specific; ) and high concentration of mitochondria reported for GCs along with high rates of respiration. Furthermore, when respiration was repressed with the inhibitors oligomycin and KCN or low [O2], ATP levels were greatly reduced in GCs, blue-light-dependent proton pumping was reduced, and stomatal opening was inhibited (Schwartz and ). These findings demonstrate that the signalling pathways and at least some of the energetics for osmoregulation lie within the GCs themselves. In addition, photosynthetic electron transport within GC chloroplasts has been proposed to directly provide ATP for blue-light-induced H+ transport via plasma membrane H+-ATPase. In their experiment, showed that red light enhanced blue-light-dependent H+ pumping in protoplasts, and that this was eliminated by DCMU, and in intact leaves DCMU inhibited both red and blue light stomatal opening. From this work, they concluded that ATP and/or reducing equivalents from GC electron transport is involved in fuelling blue-light-dependent stomatal opening.

14Although the red and blue regions of the spectrum are considered the main drivers of photosynthesis and stomatal behaviour in higher plants, it is important to consider the influence of other spectral qualities of light, including green light. Green light (~500–560 nm) has been reported to inhibit blue-light-induced stomatal opening across a number of plant species, but seems to depend almost entirely on the light environment experienced by the plant during growth. Stomatal responses to green light were observed in plants grown under conditions reproducing an understorey environment, and the magnitude of green-light-driven stomatal responses decreased over the course of the day. Although the receptors and exact mechanism of stomatal response to green light have not been identified, green light is known to deactivate the blue light cryptochrome photoreceptors via removal of the signal that suppresses ABA production in GCs, promoting a decrease in stomatal aperture. Green light has also been shown to contribute to photosynthesis, often at a more efficient rate than red or blue light due to non-photosynthetic absorption of blue light by carotenoids, and particularly in strong white light, and therefore can impact photosynthetic-dependent stomatal opening. Moreover, observed a green-light-driven stomatal response in sunflower leaves, which, similarly to the stomatal response to red light, was photosynthesis dependent as it was partly eliminated when DCMU was applied. This implicates the potential existence of a green light receptor, with cryptochromes suggested as being involved in this DCMU-independent fraction, and that any further signal transduction is photosynthetically dependent, although further work would be required to elucidate a green light photoreceptor. It has been suggested that a possible role of the green light reversal effect on blue-light-driven stomatal opening could be the prevention of excessive leaf water loss through stomata under (green light-rich) vegetational shade, where, within a crop or other terrestrial plant canopy, photosynthetic potential is greatly reduced.

15Interestingly stomatal responses to blue light are not universal, with fern species of Polypodiopsida and Adiantum capillus-veneris, along with several species from the family Solanaceae, exhibiting a lack of stomatal response to blue light. The facultative CAM plant Mesembryanthemum crystallinum loses its stomatal blue light response when the plant shifts from C3 metabolism to CAM. In some species, including the gymnosperm Cycas revoluta and the ferns Equiestum hyemale and Psilotum nudum, blue light is essential for stomata to open, suggesting that these differences may be due to various evolutionary pressures, whilst the loss of a stomatal blue light response in Polypodiopsida may be the result of adaption to understorey canopy environments. Moreover, it should be kept in mind that growth conditions, such as drought and high temperatures that may alter the water status of the plant, may impact stomatal sensitivity to blue and red light. This is because plants will balance the need to maintain leaf turgor and/or maximize carbon gain and evaporative cooling, and finding the balance between these factors is ultimately dependent on species specificity to the growth environment (Lawson and ). Although several species have been reported to respond to blue light (e.g. Arabidopsis thaliana and Vicia faba; Table 1), those that do not respond or exhibit a diminished or slow response have generally not been reported (e.g. Nicotiana tabacum; ). This is further complicated by the different protocols used to assess red and blue light responses, making comparison almost impossible, with different red and blue light intensities, ratios, and even durations being applied. Furthermore, the photosynthesis dependence of stomatal response to blue light is species specific (see;; ), with some species known to respond to blue light even in the dark (with no red light background), whilst in others stomatal blue light is only apparent on a background of red light. This has significance, as it is presumed that the response of stomata to blue light does not necessarily require photosynthesis, and that different species may use different sources of energy for blue-light-induced stomatal opening. Given the evidence that indicates that species specificity of stomatal response to blue light exists, there is a need for standardization of the protocols to be able to accurately compare the biological importance of the stomatal blue light response between and within species.

16Although some species do not exhibit a stomatal blue light response, little is known about diversity in the magnitude and/or speed of these responses. This is especially important when considering the impact stomatal behaviour to blue light might have on carbon uptake and water use in major crop species (see;; ). The fact that stomata in some species open to blue light even when photosynthesis is already saturated with red light means that gs may be higher than required to achieve maximum CO2 diffusion for photosynthesis, and therefore WUE is greatly reduced. Conversely, the impact of stomatal opening response to blue light on carbon uptake depends on the degree of diffusional limitation of gs for photosynthesis. Figure 4 illustrates the influence of blue light on gs over the diurnal period, and how this greatly affects WUE even when photosynthesis is saturated. From this, we can infer that reducing stomatal sensitivity to blue light may potentially be beneficial for optimizing crop resource use, whereby photosynthetic rates are maintained whilst using water more efficiently. However, this may only be beneficial under certain environmental conditions, as reduced gs could lead to increased leaf temperature, which, depending on the species and environment, could be detrimental to photosynthetic rates and overall plant productivity. Here we show, via thermal imaging, the impact of blue-light-dependent gs response on leaf cooling, and how the addition of ~10% of blue light facilities greater leaf evaporative cooling even when light intensity is held constant (Fig. 5). Decreasing water loss during early stages of growth in crops such as wheat would facilitate greater water availability later in the season, that in turn would enable sustained photosynthetic rates through the grain-filling period when water is a major limiting factor, potentially increasing overall grain yield.

17Generally, the mechanisms behind the speed of stomatal response refer to short-term responses (seconds to minutes), and are not necessarily sufficient to explain diurnal behaviour of A and gs. Even in constant light conditions, decreases in gs are often seen towards the end of the day, and it has been suggested that mechanisms such as increases in the amount of sucrose from photosynthesis mediate this response, and that sucrose content and metabolism play a major role in the longer term coordination of A and gs. This sugar accumulation at high photosynthetic rates associated with high photosynthetically active radiation (PAR) conveys long-term photosynthetic feedback on gs over the course of the day, which may theoretically alter stomatal dynamic behaviour over the diurnal period. It should also be noted that toward the end of the day, some species exhibit a slower gs response to changes in light intensity, with a slow closing response resulting in continued high gs, leading to substantial water loss and reduced WUE over the diurnal period. It has already been reported that in wheat the magnitude or sensitivity of stomata to blue light is enhanced under a ‘strong’ red light background, with this behaviour potentially being dose dependent where the intensity and even the duration of the background red light determines the extent to which stomata respond to a blue light signal. Furthermore, as the stomatal response to blue light is GC specific, it may be suggested that the speed of the gs response is increased under blue light. It can therefore be proposed that altering the sensitivity and diurnal behaviour of the gs blue light response, via either breeding techniques or genetic modification, could lead to a reduction in the limitation of A by gs, and the slow decrease in A and gs through the day may be prevented. This paves the way for potential improvements in photosynthetic carbon assimilation over the diurnal period, whilst positively influencing WUE and plant productivity. As most studies have been carried out under ‘ideal’ well-watered conditions, there is little information describing the influence of drought, water status, or temperature on the temporal response of gs. As a consequence, it is currently unknown how manipulation of stomatal sensitivity to blue and red light may impact plant fitness and productivity. However, as the frequency and intensity of periods of drought are set to increase globally in the near future, water availability and its transport from roots to stomata will be a major limiting factor for crop and terrestrial ecosystems moving forward. As such, improving our understanding of the dynamic response of stomata to different spectra of light and how manipulation of the stomatal sensitivity will impact spatial and temporal stomatal responses (;; Lawson and; ), remains an unexploited avenue in which to improve plant performance and crop productivity.

18Stomatal research over the past few decades has revealed a complicated network of osmoregulatory and signalling pathways in GCs (e.g.;; ) that are species specific and influenced by the growth environment. Although significant progress has been made over the past few decades, understanding of stomatal responses to various environmental signals (including irradiance) and substantial advances in GC metabolism and osmoregulatory pathways, many gaps remain regarding the integration and hierarchy of these diverse processes and the extent to which each contributes to stomatal function. As we have illustrated throughout this review, there is extensive evidence for both mitochondrial and photosynthetic electron transport ATP supply for the H+-ATPase, ion channel activation, and stomatal opening in response to both blue and red illumination. However, the extent to which each energetic supply contributes to stomatal movements has yet to be fully elucidated. It is also apparent that manipulating one pathway may result in the up-regulation of an alternative pathway to compensate, increasing the difficulty and complexity for determining the involvement and extent of each. The role and participation of GC electron transport and photosynthetic processes remain continuing subjects of debate, despite the fact that chloroplasts are a key feature of most GCs. Furthermore, the extent to which mesophyll signals play a role and the origins and nature of these signals need clarification. Therefore, understanding the mechanisms and signal transduction pathways that operate in GCs and the influence of mesophyll photosynthesis on these processes (and subsequent stomatal responses) is essential if we are to fully exploit the relationship between A and gs in order to improve gaseous fluxes, maximize CO2 uptake, and optimize water use in fluctuating environments given the predicted changes in climate.

19We are all fully aware that global demand for food is growing, and, due to a growing world population, it has been estimated that a >50% increase in major crop yield is required by 2050. This situation is further exacerbated by predicted increases in atmospheric temperature and heat wave frequency experienced by crop and terrestrial ecosystems. These variable changes in climate conditions are often linked to changes in precipitation patterns, water availability, and drought, and are set to aggravate crop losses and increase the agricultural water supply requirements by an estimated 17%. When water is limiting, plants close stomata to avoid excessive water loss, even during periods of high light when mesophyll demands for CO2 are high, and long-term damage to photosynthetic machinery may be induced. In crop and forest ecosystems this reduces transpiration but at a cost of reduced evaporative cooling, which greatly impacts biochemical and metabolic mechanisms of photosynthesis; including but not limited to the activity of the temperature-sensitive enzyme Rubisco activase and ATP synthesis. Finding the ‘ideal’ balance between carbon gain, evaporative cooling, and maintenance of hydraulic status is crucial for maximizing crop performance and productivity, whether it be for field- or greenhouse-grown crops.

20As mentioned above, blue light induces a GC-specific stomatal response that enables stomatal opening, increasing the magnitude of gs. Given the importance of the blue light response of gs for evaporative cooling and carbon gain in major crop species (see above), manipulation of this blue-light-triggered response represents a novel and generally unexploited target as a strategy to increase carbon uptake and/or maintenance of optimal leaf temperature, and conversely for crop water use. An analogous approach has recently been used by who expressed the synthetic light-gated K+ channel BLINK1 specifically in the GCs to enhance solute fluxes, and produced plants with stomata that opened and closed more rapidly, resulting in greater WUE and biomass.

21Understanding the mechanisms behind the blue- and red-light-driven responses of stomata would potentially enable greater control of the synchronicity between A and gs under dynamic light conditions, and therefore optimize the relationship between water use and carbon gain. Enabling a blue light gs response in species in which it is absent increases the potential for cultivating crop ideotypes for specific climate conditions. In fact, it has already been demonstrated that blue light, even at low intensities, reduced stomatal oscillations often seen under drought conditions. These oscillations, the cyclic opening and closing of stomata, are presumed to initiate from hydraulic mismatch between water supply and transpiration rate, and therefore it is suggested that a blue-light-driven gs response helps recover this synchronicity and improve plant performance under drought. On the other hand, reducing stomatal sensitivity to blue light could provide a route to producing plants with reduced levels of gs, potentially enhancing water saving at crucial stages during plant development (e.g. grain filling). However, this could be to the detriment of CO2 diffusion and leaf cooling, but could provide ideotypes for specific growth environments.

22Given the increase in alternative growth spaces (e.g. vertical farming) for ‘indoor’ crops, a new generation of smart LED lighting allowing for more precise control of light quality and quantity has recently become available. Several recent studies have demonstrated the importance of blue light for vegetable crop growers, as a way of improving WUE and even overall yield. For example, a recent study demonstrated that different ratios of red and blue light optimized growth, yield, and WUE in basil. This is interesting, as the optimal ratios for these targets were shown to be different from the ratios of light spectra observed in nature, whether it is direct or diffuse light. This highlights the potential to engineer the tapestry of plant pigments to utilize more of the sunlight’s spectrum, to maximize light absorption, and to overcome light saturation of the downstream photosynthetic processes, as it is already known that more than half of the energy in the solar spectrum is not utilized by the plant.

23In this review, we have focused on stomatal response to different light qualities. Emphasis is placed on the response of gs to blue and red spectra of light, and how plants use these independent light responses to enforce GC movement to maximize plant performance. With recent research highlighting the importance of the rapidity of gs responses to light for plant water status and photosynthetic carbon gain, we emphasize the need to re-assess the role of stomatal behaviour under blue and red light between and within species, as a means to understand the importance of stomata in crop performance. Additionally, with increases in global temperature and water demand for agricultural practices predicted to intensify crop losses in the future, we outline the potential of manipulating stomatal response to light quality to maximize drought tolerance, water-saving strategies, and yield.