Phytohormones play important roles in regulating various aspects of plant growth and development as well as in biotic and abiotic stress responses. Stomata are openings on the surface of land plants that control gas exchange with the environment. Accumulating evidence shows that various phytohormones, including abscisic acid, jasmonic acid, brassinosteroids, auxin, cytokinin, ethylene, and gibberellic acid, play many roles in the regulation of stomatal development and patterning, and that the cotyledons/leaves and hypocotyls/stems of Arabidopsis exhibit differential responsiveness to phytohormones. In this review, we first discuss the shared regulatory mechanisms controlling stomatal development and patterning in Arabidopsis cotyledons and hypocotyls and those that are distinct. We then summarize current knowledge of how distinct hormonal signaling circuits are integrated into the core stomatal development pathways and how different phytohormones crosstalk to tailor stomatal density and spacing patterns. Knowledge obtained from Arabidopsis may pave the way for future research to elucidate the effects of phytohormones in regulating stomatal development and patterning in cereal grasses for the purpose of increasing crop adaptive responses.© Crown copyright 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Stomata are crucial for the productivity and survival of land plants. Until recently, little was known about the events and molecular pathways required for stomatal development. Emerging data indicate that cell-cell signaling conveys spatial information about cell identity and location. Such information might pattern stomata by orienting the plane of asymmetric division and might control stomatal number by regulating division frequency. This pathway also provides an accessible model system for studying post-apical meristem stem cells that generate specific tissues.

Coordinated growth of organs requires communication among cells within and between tissues. In plants, leaf growth is largely dictated by the epidermis; here, asymmetric and self-renewing divisions of the stomatal lineage create two essential cell types-pavement cells and guard cells-in proportions reflecting inputs from local, systemic, and environmental cues. The transcription factor SPEECHLESS (SPCH) is the prime regulator of divisions, but whether and how it is influenced by external cues to provide flexible development is enigmatic. Here, we show that the phytohormone cytokinin (CK) can act as an endogenous signal to affect the extent and types of stomata! lineage divisions and forms a regulatory circuit with SPCH. Local domains of low CK signaling are created by SPCH-dependent cell-type-specific activity of two repressive type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR16 and ARR17, and two secreted peptides, CLE9 and CLE10, which, together with SPCH, can customize epidermal cell-type composition.

Stomatal formation is regulated by multiple developmental and environmental signals, but how these signals are integrated to control this process is not fully understood. In Arabidopsis thaliana, the basic helix-loop-helix transcription factor SPEECHLESS (SPCH) regulates the entry, amplifying and spacing divisions that occur during stomatal lineage development. SPCH activity is negatively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation. Here, we show that in addition to MAPKs, SPCH activity is also modulated by brassinosteroid (BR) signalling. The GSK3/SHAGGY-like kinase BIN2 (BR INSENSITIVE2) phosphorylates residues overlapping those targeted by the MAPKs, as well as four residues in the amino-terminal region of the protein outside the MAPK target domain. These phosphorylation events antagonize SPCH activity and limit epidermal cell proliferation. Conversely, inhibition of BIN2 activity in vivo stabilizes SPCH and triggers excessive stomatal and non-stomatal cell formation. We demonstrate that through phosphorylation inputs from both MAPKs and BIN2, SPCH serves as an integration node for stomata and BR signalling pathways to control stomatal development in Arabidopsis.

Stomatal development is tightly regulated through internal and external factors that are integrated by a complex signalling network. Light represents an external factor that strongly promotes stomata formation. Here, we show that auxin-resistant aux/iaa mutants, e.g. axr3-1, exhibit a de-repression of stomata differentiation in dark-grown seedlings. The higher stomatal index in dark-grown axr3-1 mutants when compared with the wild type is due to increased cell division in the stomatal lineage. Excessive stomata in dark-grown seedlings were also observed in mutants defective in auxin biosynthesis or auxin perception and in seedlings treated with the polar auxin transport inhibitor NPA. Consistent with these findings, exogenous auxin repressed stomata formation in light-grown seedlings. Taken together, these results indicate that auxin is a negative regulator of stomatal development in dark-grown seedlings. Epistasis analysis revealed that axr3-1 acts genetically upstream of the bHLH transcription factors SPCH, MUTE and FAMA, as well as the YDA MAP kinase cascade, but in parallel with the repressor of photomorphogenesis COP1 and the receptor-like protein TMM. The effect of exogenous auxin required the ER family of leucine-rich repeat receptor-like kinases, suggesting that auxin acts at least in part through the ER family. Expression of axr3-1 in the stomatal lineage was insufficient to alter the stomatal index, implying that cell-cell communication is necessary to mediate the effect of auxin. In summary, our results show that auxin signalling contributes to the suppression of stomatal differentiation observed in dark-grown seedlings. © 2014. Published by The Company of Biologists Ltd.

High relative air humidity (RH) promotes stomatal opening in tomato leaves. This study examined the role of the plant hormones abscisic acid (ABA) and ethylene in high RH induced stomatal opening. Plants were grown in high (90%) and moderate (60%) RH or transferred from moderate to high RH. ABA levels were only slightly, but significantly decreased during darkness by increasing RH. However, a significantly higher ethylene evolution was found in high RH compared with moderate RH. Ethephon increased conductance and stomatal aperture in moderate RH. Treatment with amino-ethoxyvinylglycine (AVG) suppressed stomatal opening when plants were transferred from moderate to high RH. Similarly, blocking the ethylene receptor or using an ethylene-insensitive mutant (NR) reduced the response to high RH. These results demonstrate that both ethylene production and sensitivity play a role in high RH-induced stomatal opening in tomato leaves. The increased conductance found when plants were transferred to high RH could be counteracted by exogenous ABA spray. The ABA deficient mutant 'Flacca' produced high levels of ethylene irrespective of the RH and the difference in water loss and conductance between high and moderate grown 'Flacca' plants was attenuated compared with WT. The results indicate that both ABA and ethylene play a role in air humidity control of stomatal movement in tomato.

Under drought conditions the growth and survival of a plant depend on its adaptive characteristics and acclimation ability. Adaptation refers to inherent morpho-physiological characters providing protection against water losses. Acclimation, however, is a special case of phenotypic plasticity: environment-dependent phenotypic expression resulting to a 'new' phenotype through drought-induced modulations in leaf morphology, anatomy and physiology. Given that phenotypic plasticity influences environmental tolerance, a multi-trait plasticity index could be of great importance. Therefore, we examined the acclimation processes of three different barley genotypes using a multi-trait plasticity assessment with emphasis on the leaf water economy-related traits. Our results showed that (i) the structure-function co-ordination during long-term drought acclimation follows the trade-off between carbon gain and water saving as well as the competition between investments in photosynthesis vs synthesis of protective compounds; (ii) the genotypes with smaller leaf area, narrower and denser veins, as well as smaller and denser stomata i.e. traits providing tolerance, exhibited less drastic adjustments under stress conditions, suggesting a trade-off between acclimation and tolerance-adaptation; and (iii) the slope values of a multi-trait 'reaction norm' based on regression analysis of PCA scores were indicative of the degree of plasticity for each genotype, providing an accurate representation of a complex set of data with single numeric results easily comparable.

Stomata are central players in the hydrological and carbon cycles, regulating the uptake of carbon dioxide (CO) for photosynthesis and transpirative loss of water (HO) between plants and the atmosphere. The necessity to balance water-loss and CO-uptake has played a key role in the evolution of plants, and is increasingly important in a hotter and drier world. The conductance of CO and water vapour across the leaf surface is determined by epidermal and stomatal morphology (the number, size, and spacing of stomatal pores) and stomatal physiology (the regulation of stomatal pore aperture in response to environmental conditions). The proportion of the epidermis allocated to stomata and the evolution of amphistomaty are linked to the physiological function of stomata. Moreover, the relationship between stomatal density and [CO] is mediated by physiological stomatal behaviour; species with less responsive stomata to light and [CO] are most likely to adjust stomatal initiation. These differences in the sensitivity of the stomatal density-[CO] relationship between species influence the efficacy of the 'stomatal method' that is widely used to infer the palaeo-atmospheric [CO] in which fossil leaves developed. Many studies have investigated stomatal physiology or morphology in isolation, which may result in the loss of the 'overall picture' as these traits operate in a coordinated manner to produce distinct mechanisms for stomatal control. Consideration of the interaction between stomatal morphology and physiology is critical to our understanding of plant evolutionary history, plant responses to on-going climate change and the production of more efficient and climate-resilient food and bio-fuel crops.© 2021. The Author(s).