Stomata: custodians of leaf gaseous exchange

Stomata are a key feature of all higher terrestrial plants and are found in different numbers and patterns on the aerial surfaces of most green tissues. The surrounding guard cells modulate the size of the stomatal pore in response to environmental conditions and internal cues in a manner that optimizes the rate of CO ₂ uptake for photosynthesis while minimizing water loss. Stomatal conductance (g _s ) is a measure of the capacity for gas exchange between the inside of the leaf and the atmosphere, and is dependent on stomatal density (SD), size (SS), and aperture. Changes in g _s regulate photosynthesis (A) and water loss, with significant implications for plant performance and crop yield. Stomata are therefore a major target for crop improvement via enhanced photosynthesis, optimal biochemical performance, and improved water use efficiency. This topic was part of the SEB centenary meeting in Edinburgh 2023, and forms the basis for this Special Issue. The meeting brought together researchers exploring a range of approaches to monitor, manipulate, and understand stomatal responses to a number of biotic and abiotic challenges with the ambition to develop plants that are more productive and more resilient in the face of climate change.

As the gatekeepers to gaseous diffusion between the internal leaf air spaces and the external atmosphere, the anatomy and behaviour of stomatal pores are fundamental to the control of CO₂ uptake for photosynthesis and water loss through transpiration and the balance between these two processes. Typically stomata only occupy 0.3–5% of the leaf surface; however, they are responsible for ~95% of all gaseous fluxes through plants in terrestrial ecosystems (Morison, 2003). As a measure for the maximum capacity for gaseous diffusion (of water), changes in g_s crucially impact on intrinsic water use efficiency (W_i), which is defined as the ratio of the photosynthetic CO₂ assimilation rate (A) relative to g_s (Blum, 2009; Lawson and Blatt, 2014; Lawson and Vialet-Chabrand, 2019). W_i is important as a constraint on plant function and has been a long-term target for crop improvement for which new opportunities are emerging (Leakey et al., 2019). The specialized guard cells surrounding stomata respond to external and environmental cues, including, for example, light intensity (and spectra), CO₂ concentration [CO₂], vapour pressure deficit (VPD), temperature, as well as mesophyll signals and hormones to adjust stomatal aperture and optimize g_s.

Stomatal responses to dynamic environments

In the field (and glasshouse environment), plants are subjected to fluctuating environmental conditions, and therefore photosynthesis and stomatal behaviour are rarely in steady state. Light is one of the most dynamic environmental inputs that impacts plant performance, with intensities changing rapidly with leaf movements, shading from overlapping leaves, and cloud cover. Research on the impact of dynamic light on the rapidity and kinetics of stomatal responses and the influence on photosynthetic induction (Long et al., 2022) and water use efficiency (Lawson and Blatt, 2014; Lawson and Vialet-Chabrand, 2019) has intensified in the last few years. It is well known that both A and g_s respond to changes in irradiance (intensity and spectra), with increasing light driving stomatal opening to support diffusional uptake of CO₂ for light-driven photosynthesis (Lawson et al., 2010). However, in general, stomatal responses to changes in irradiance can be an order of magnitude slower than A (McAusland et al., 2016), limiting CO₂ diffusion for photosynthesis, while slow closure when light intensity decreases leads to unnecessary water loss for no carbon gain (Lawson et al., 2010, 2012). This has led to a number of research efforts to try and manipulate stomata or guard cells to increase the speed of responses, with several success stories demonstrating the potential of this approach. For example, Papanatsiou et al. (2019) used optogenetics to trigger a reduction in the half-times for stomatal opening and closing with changes in blue light intensity by expressing a synthetic blue light-inducted K⁺ channel (BLINK1) in guard cells. Engineering plasma membrane GORK channels responsible for K⁺ efflux during stomatal closure resulted in plants with faster kinetics, leading to greater A and W_i (Horaruang et al., 2022).

Exploiting natural variation in stomatal traits

Anatomical and functional stomatal traits are known to vary intra- and interspecifically (McAusland et al., 2016, 2020; Faralli et al., 2019), and exploring such natural variation provides an opportunity for breeders and researcher to exploit these untapped resources to develop high-yielding crops for predicted future climates. Screening for these differences is challenging, with most studies relying on time-consuming infrared gas exchange analyses which limits throughput depending on the number of instruments. Faralli et al. (2024) used a combination of chlorophyll fluorescence and thermography in conjunction with a bespoke gas exchange chamber to not only demonstrate a rapid and effective screen for stomatal dynamics in relation to photosynthesis, but also quantify significant natural variation for these traits in bread wheat (Triticum aestivum L.) genotypes. Since stomatal conductance and rapidity of responses are important for evaporative cooling and maintenance of optimal leaf temperature, the large phenotypic variation suggests the existence of exploitable genetic variability of dynamic traits that could be used for future breeding.

Another approach for high-throughput assessment of W_i includes leaf carbon isotope analysis (δ¹³Cleaf) (Farquhar and Sharkey, 1982; Farquhar, 1983; Farquhar and Richards, 1984; Rebetzke et al., 2002; Condon et al., 2004), which reflects photosynthetic discrimination of ¹³C against ¹²C relative to CO₂ input through stomata. When g_s is lower, the supply of ¹²C for photosynthesis is reduced relative to demands, resulting in the fixation of the less-preferred C¹³ alternative, which decreases the isotope fractionation (Barbour et al., 2011). The majority of studies exploiting this approach have focused on C₃ plants, due to the complexity of the carbon-concentrating mechanism on this signature in C₄ crops. Crawford et al. (2024) provided further evidence that carbon isotope discrimination is a useful proxy for W_i in C₄ plants and that differences in stomatal kinetics were responsible for genotypic differences in W_i, supporting this as a key target for crop breeding (Condon et al., 2004; Leakey et al., 2019). While advances in phenotyping functional stomatal characteristics have depended mostly on the development of techniques or construction of new instrumentation, phenotyping anatomical features, such as size and density, has traditionally relied on the tedious process of visual quantification using microscopy. The innovation of deep learning (DL) models has revolutionized the rapidity, accuracy, and throughput of image analysis to phenotype stomatal traits, which opens up considerable opportunities for more rapid application of modern genetic methods for plant science and crop improvement. This work is reviewed as a case study for the enabling capabilities of DL, while providing an introductory primer on DL methods for biologists, by Tan et al. (2024). The various pipelines, opportunities, and uses have been extensively reviewed by Gibbs and Burgess (2024) while providing an overview of future applications.

Natural variation in g_s has been reported for several crops, with some genomic regions identified (Faralli et al., 2019), although these have often focused on SD or SS, while functional responses such as the rapidity or magnitudes of change have received less attention. Such responses may be dependent on a number of factors including guard cell biochemistry, sensitivity, or membrane transporters, with little known regarding the natural variation in these traits (Faralli et al., 2019). Yoshiyama et al. (2024) explored differences in stomatal kinetics and anatomy in wild and cultivated tomato, reporting faster induction rates in the wild species which resulted in greater daily photosynthetic carbon gain. Differences in water use efficiency in response to water stresses were also observed in a panel of 89 sorghum genotypes; however, this was attributed to maintenance of photosynthesis, independent of hydraulics (Al-Salman et al., 2024). Under natural dynamic light conditions, faster stomatal responses facilitated a greater A; however, the associated higher g_s, while supporting gaseous diffusion, did so at the expense W_i. It is often the case that manipulating g_s to be higher or faster increases A but also leads to greater water loss, reducing W_i, while lowering g_s can save water but limit A and growth (Tanaka et al., 2010). In the ideal situation, the dynamic responses of g_s and A would be fast and tightly correlated to ensure that photosynthetic demands are matched with supply, and that when demands drop unnecessary water loss is avoided (Lawson and Blatt, 2014; McAusland et al., 2016). Sorghum, a C₄ crop, demonstrates these desirable traits, with rapid g_s kinetics observed in a population of 48 natural variants. This rapid g_s response resulted in an exceptionally tight coupling of A and g_s under both dynamic and steady-state conditions, ensuring that W_i remained near constant (Battle et al., 2024). These findings not only highlight rapid g_s and its tight coupling with A as desirable traits, but may also offer valuable insights into the underlying mechanisms that link these two processes. It is generally widely accepted that stomatal responses to internal CO₂ concentration inside the leaf (C_i) link g_s with A. Any increase in A lowers C_i, to which stomata respond and open, while lowering A increases C_i, initiating stomatal closure. However, several studies have shown that the sensitivity of stomata to C_i is insufficient to be the only mechanism for this coordination (Sharkey and Raschke, 1981) and that there must be another C_i-independent mechanism, with several metabolites, metabolite pools, and substrates proposed over the last couple of decades (see Lawson et al., 2018), although to date there is still no consensus. In a study using a range of Arabidopsis mutants, Taylor et al. (2024) quantified the C_i-dependent and independent contribution to stomatal responses to red light (also known as photosynthetic light; Matthews et al., 2020) and showed that both mechanisms contributed equally. However, at high C_i concentrations, the C_i-independent component was suppressed, which supports earlier studies that suggested that the redox state of the plastoquinone pool was a key component that linked A and g_s (Busch, 2014; Kromdijk et al., 2019).

Manipulating stomatal anatomical features to improve water use efficiency

Due to climate change, drought episodes are likely to become more frequent and severe in some geographical locations, severely impacting yield (Webber et al., 2018). Plant resilience to climate-induced stresses, such as drought, are intricately linked to g_s responses, and understanding the regulation of relevant stomatal characteristics could provide invaluable targets to improve plant water use efficiency and maintain productivity in future climates, for example if g_s could be lowered with no reduction of A.

Strong proof-of-concept exists for reducing SD via the gene network responsible for stomatal development, such as EPIDERMAL PATTERNING FACTOR 1 and 2 (EPF1/2), in C₃ plants (Harrison et al., 2020). However, lower SD may coincide with an increase in SS in some species (Zhu et al., 2015; Wang et al., 2016). The impact on g_s is therefore unclear, especially as plants may also adjust stomatal aperture to compensate for anatomical differences (Büssis et al., 2006). The possibility of engineering C₄ plants with lower SD and g_s is particularly appealing as, under current atmospheric CO₂ concentrations, C₄ crops are CO₂ saturated (Leakey et al., 2019). Thus, any reductions in g_s that do not lower C_i below the inflection point of the A/C_i curve would avoid limitations on A, that are often seen in C₃ plants (Wang et al., 2016; Hughes et al., 2017; Caine et al., 2019; Dunn et al., 2019), while lowering water loss via transpiration and improving W_i. The potential of this approach has been demonstrated by Ferguson et al. (2024) who showed that reductions in SD in sorghum by 45% via EPF manipulation resulted in reduced g_s, with no change in A, and therefore greater W_i. However, these outcomes might not occur consistently because compensatory behavioural adjustments of stomatal aperture that result in g_s being maintained even when SD is reduced have occasionally been observed (Büssis et al., 2006). Lunn et al. (2024) have proposed sugarcane plants expressing EPF2, with lowered SD, as a system for further analysis of stomatal behavioural changes, as these plants had reductions in SD without any differences in g_s, indicating a compensatory increase in stomatal aperture and no impact on transpiration, A, or W_i that otherwise can trigger whole-plant feedback effects on carbon and water relations. Meta-analysis of 10 plant species across functional types (C₃ and C₄) indicated that this compensatory mechanism was widely conserved across phylogenetic space and that a reduction of SD of >25% was generally required to result in a lower g_s (Lunn et al., 2024). Improvement of W_i in crops via reductions in SD clearly has enormous potential; however, it should be considered alongside other approaches manipulating the control of stomatal aperture by guard cells, as well as changing the size of the stomatal complex (Des Marais et al., 2014; Lawson and Blatt, 2014; Leakey et al., 2019; Nunes et al., 2023).

Understanding plant resilience: importance of regulation of stomatal responses via internal signals

Stomata play a pivotal role in maintaining plant water status during drought through closure to limit transpiration when water is scarce and opening following such stress events to recover photosynthetic carbon assimilation. Abscisic acid (ABA) is a well-established plant hormone important in stomatal regulation during dehydration (Brodribb and McAdam, 2011; Gong et al., 2021; Hasan et al., 2021). However, a lack of consensus still exists surrounding the role of ethylene, which has been shown to act both synergistically (Desikan et al., 2006) and antagonistically (Tanaka et al., 2005; Sharipova et al., 2012; Chen et al., 2013; Watkins et al., 2014) with ABA to induce stomatal closure, therefore potentially aiding and hindering drought-induced responses during dehydration. Ethylene inhibition of ABA-induced stomatal closure during drought appears to require high concentrations of both ABA and ethylene (Beguerisse-Díaz et al., 2012). A link with reactive oxygen species (ROS) production has been suggested, whereby ethylene may cause ROS accumulation and stomatal closure when ROS are low, but inhibit ROS synthesis when ABA-induced ROS are high, hence preventing stomatal closure (Hasan et al., 2024). It is clear that the complex interactions between drought-induced responses described here currently prevent a clear consensus of the role of ethylene in stomatal closure during dehydration events. Similarly, both ABA and ethylene have been implicated in slowing the recovery of stomatal conductance during rehydration (Yao et al., 2021; Bi et al., 2023). In addition, stomata can also exhibit a passive non-hormonal response to drought, driven by a decline in water status, which varies across species (McAdam and Brodribb, 2012; Gong et al., 2021). In seed-free plants, stomata rapidly re-open following instantaneous rehydration of leaves after drought, even when ABA levels are high, indicating a limited role for the hormone in stomatal closure in these plants in response to drought (Cardoso and McAdam, 2019), which contrasts with findings in seed plant species (McAdam and Brodribb, 2012, 2014), although another studies have suggested conserved stomatal behaviour in early land plants (Chater et al., 2011). However, McAdam et al. (2024) recently demonstrated that, when ABA levels are lowest during extreme drought, stomata of angiosperms also re-opened rapidly on instantaneous rehydration, suggesting that passive closure occurred based on leaf water status alone. Further study of ethylene, ABA, as well as interactions with other hormones and external factors and stressors, is therefore essential given the urgent need to find targets to improve plant resilience to climate-induced stresses and ensure crop yield gains in future environments.

As mentioned earlier, stomata also respond to CO₂ concentrations [CO₂], with high [CO₂] initiating stomatal closure by indirectly activating specific anion channels (Meyer et al., 2010), reducing water loss via transpiration (Lawson et al., 2014), whereas low [CO₂] signals stomatal opening (Roelfsema and Hedrich, 2005; Hiyama et al., 2017). Significant interactions between environmental and internal signals, as well as different internal signals, exist. For example, Xu et al. (2021) discovered that the guard cell signal γ-aminobutyric acid (GABA) affected stomatal responses to ABA as well as interacting with multiple abiotic and biotic signalling pathways, while Piechatzek et al. (2024) investigated the link between GABA and the CO₂ signalling pathway in guard cells, revealing a weakened CO₂ response in GABA-deficient mutant plants that was not preserved at low [CO₂]. The authors concluded that GABA plays a role in modulating stomatal opening and closing but not in direct response to [CO₂]. As part of plant responses to biotic and abiotic stress, plants release green leaf volatiles (GLVs) that can be perceived as a signal by neighbouring plants to initiate defence mechanisms. Although the primary entry of GLVs is via stomata, Maleki et al. (2024) have demonstrated that stress-induced stomatal closure impedes the potential effectiveness of GLVs. All of these studies illustrate the importance of elucidating the interaction between internal and external signals in order to fully understand the complexities and hierarchy of stomatal responses.

Conclusions

We have highlighted current knowledge and understanding of the impact of stomatal anatomy and behaviour on g_s and the implication for A and W_i. The importance of natural variation and technological advances in screening is also discussed by papers within this Special Issue as well as the links with climate change. Stomata play a key role in meeting the demands for future food production as custodians of leaf gaseous exchange, controlling not only CO₂ uptake for photosynthesis, but also water loss which is pivotal in maintaining plant water status, leaf temperature, nutrient uptake, and translocation. Although challenging and complex, advances in manipulating stomatal form and function have emerged as key targets to re-engineer crops for future environments.

Conflict of interest

The authors declare no conflicts of interest.

Funding

This research was supported by the: UKRI BBSRC funding BB/T0042741/1; BB/S005080/1; BB/Y000722/1; DOE Office of Science, Office of Biological and Environmental Research (BER), grant nos DE-SC0023160 and DE-SC0018277; DOE Center for Advanced Bioenergy and Bioproducts Innovation (U.S. Department of Energy, Office of Science, Biological and Environmental Research Program under award number DE-SC0018420); Artificial Intelligence for Future Agricultural Resilience, Management and Sustainability Institute (Agriculture and Food Research Initiative (AFRI) grant no. 2020-67021-32799/project accession no. 1024178 from the USDA National Institute of Food and Agriculture); and NSF Division of Integrative Organismal Systems (2034777). TL was supposed by a Royal Society Leverhulme Trusts Senior Research Fellowship (SF/R1/231041).