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The hormonal and environmental regulation of stomatal aperture is mediated by a complex signaling pathway found within the guard cells that surround stomata. Abscisic acid (ABA) induces stomatal closure in response to drought stress by binding to its guard cell localized receptor, initiating a signaling cascade that includes synthesis of reactive oxygen species (ROS). Genetic evidence in Arabidopsis indicates that ROS produced by plasma membrane respiratory burst oxidase homolog (RBOH) enzymes RBOHD and RBOHF modulate guard cell signaling and stomatal closure. However, ABA-induced ROS accumulates in many locations such as the cytoplasm, chloroplasts, nucleus, and endomembranes, some of which do not coincide with plasma membrane localized RBOHs. ABA-induced guard cell ROS accumulation has distinct spatial and temporal patterns that drive stomatal closure. Productive ROS signaling requires both rapid increases in ROS, as well as the ability of cells to prevent ROS from reaching damaging levels through synthesis of antioxidants, including flavonols. The relationship between locations of ROS accumulation and ABA signaling and the role of enzymatic and small molecule ROS scavengers in maintaining ROS homeostasis in guard cells are summarized in this review. Understanding the mechanisms of ROS production and homeostasis and the role of ROS in guard cell signaling can provide a better understanding of plant response to stress and could provide an avenue for the development of crop plants with increased stress tolerance.

Few evolutionary adaptations in plants were so critical as the stomatal complex. This structure allows transpiration and efficient gas exchange with the atmosphere. Plants have evolved numerous distinct stomatal architectures to facilitate gas exchange, while balancing water loss and protection from pathogens that can egress via the stomatal pore. Some plants have simple stomata composed of two kidney-shaped guard cells; however, the stomatal apparatus of many plants includes subsidiary cells. Guard cells and subsidiary cells may originate from a single cell lineage, or subsidiary cells may be recruited from cells adjacent to the guard mother cell. The number and morphology of subsidiary cells varies dramatically, and subsidiary cell function is also varied. Subsidiary cells may support guard cell function by offering a mechanical advantage that facilitates guard cell movements, and/or by acting as a reservoir for water and ions. In other cases, subsidiary cells introduce or enhance certain morphologies (such as sunken stomata) that affect gas exchange. Here we review the diversity of stomatal morphology with an emphasis on multi-cellular stomata that include subsidiary cells. We will discuss how subsidiary cells arise and the divisions that produce them; and provide examples of anatomical, mechanical and biochemical consequences of subsidiary cells on stomatal function.

Stomatal movements are crucial for the control of plant water status and protection against pathogens. Assays on epidermal peels revealed that, similar to abscisic acid (ABA), pathogen-associated molecular pattern (PAMP) flg22 requires the AtPIP2;1 aquaporin to induce stomatal closure. Flg22 also induced an increase in osmotic water permeability (P f) of guard cell protoplasts through activation of AtPIP2;1. The use of HyPer, a genetic probe for intracellular hydrogen peroxide (H₂O₂), revealed that both ABA and flg22 triggered an accumulation of H₂O₂ in wild-type but not pip2;1 guard cells. Pretreatment of guard cells with flg22 or ABA facilitated the influx of exogenous H₂O₂. Brassinosteroid insensitive 1-associated receptor kinase 1 (BAK1) and open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6) were both necessary to flg22-induced P f and both phosphorylated AtPIP2;1 on Ser121 in vitro. Accumulation of H₂O₂ and stomatal closure as induced by flg22 was restored in pip2;1 guard cells by a phosphomimetic form (Ser121Asp) but not by a phosphodeficient form (Ser121Ala) of AtPIP2;1. We propose a mechanism whereby phosphorylation of AtPIP2;1 Ser121 by BAK1 and/or OST1 is triggered in response to flg22 to activate its water and H₂O₂ transport activities. This work establishes a signaling role of plasma membrane aquaporins in guard cells and potentially in other cellular context involving H₂O₂ signaling.

Sucrose-non-fermenting-1-related protein kinase-2s (SnRK2s) are critical for plant abiotic stress responses, including abscisic acid (ABA) signaling. Here, we develop a genetically encoded reporter for SnRK2 kinase activity. This sensor, named SNACS, shows an increase in the ratio of yellow to cyan fluorescence emission by OST1/SnRK2.6-mediated phosphorylation of a defined serine residue in SNACS. ABA rapidly increases FRET efficiency in N. benthamiana leaf cells and Arabidopsis guard cells. Interestingly, protein kinase inhibition decreases FRET efficiency in guard cells, providing direct experimental evidence that basal SnRK2 activity prevails in guard cells. Moreover, in contrast to ABA, the stomatal closing stimuli, elevated CO2 and MeJA, did not increase SNACS FRET ratios. These findings and gas exchange analyses of quintuple/sextuple ABA receptor mutants show that stomatal CO2 signaling requires basal ABA and SnRK2 signaling, but not SnRK2 activation. A recent model that CO2 signaling is mediated by PYL4/PYL5 ABA-receptors could not be supported here in two independent labs. We report a potent approach for real-time live-cell investigations of stress signaling.

N-myristoylation and S-acylation promote protein membrane association, allowing regulation of membrane proteins. However, how widespread this targeting mechanism is in plant signaling processes remains unknown. Through bioinformatics analyses, we determined that among plant protein kinase families, the occurrence of motifs indicative for dual lipidation by N-myristoylation and S-acylation is restricted to only five kinase families, including the Ca2+-regulated CDPK-SnRK and CBL protein families. We demonstrated N-myristoylation of CDPK-SnRKs and CBLs by incorporation of radiolabeled myristic acid. We focused on CPK6 and CBL5 as model cases and examined the impact of dual lipidation on their function by fluorescence microscopy, electrophysiology and functional complementation of Arabidopsis mutants. We found that both lipid modifications were required for proper targeting of CBL5 and CPK6 to the plasma membrane. Moreover, we identified CBL5–CIPK11 complexes as phosphorylating and activating the guard cell anion channel SLAC1. SLAC1 activation by CPK6 or CBL5–CIPK11 was strictly dependent on dual lipid modification, and loss of CPK6 lipid modification prevented functional complementation of cpk3 cpk6 guard cell mutant phenotypes. Our findings establish the general importance of dual lipid modification for Ca2+ signaling processes, and demonstrate their requirement for guard cell anion channel regulation.

Stomata are produced by a controlled series of epidermal cell divisions. The molecular underpinnings of this process are becoming well understood, but mechanisms that determine plasticity of stomatal patterning to many exogenous and environmental cues remain less clear. Light quantity and quality, vapour pressure deficit, soil water content, and CO2 concentration are detected by the plant, and new leaves adapt their stomatal densities accordingly. Mature leaves detect these environmental signals and relay messages to immature leaves to tell them how to adapt and grow. Stomata on mature leaves may act as stress signal-sensing and transduction centres, locally by aperture adjustment, and at long distance by optimizing stomatal density to maximize future carbon gain while minimizing water loss. Although mechanisms of stomatal aperture responses are well characterized, the pathways by which mature stomata integrate environmental signals to control immature epidermal cell fate, and ultimately stomatal density, are not. Here we evaluate current understanding of the latter through the influence of the former. We argue that mature stomata, as key portals by which plants coordinate their carbon and water relations, are controlled by abscisic acid (ABA), both metabolically and hydraulically, and that ABA is also a core regulator of environmentally determined stomatal development.

Open Stomata 1 (OST1) (SnRK2.6 or SRK2E), a serine/threonine protein kinase, is a positive regulator in abscisic acid (ABA)-mediated stomatal response, but OST1-regulation of K+ and Ca2+ currents has not been studied directly in guard cells and it is unknown whether OST1 activity is limiting in ABA-mediated stomatal responses. We employed loss-of-function and gain-of-function approaches to study native ABA responses of Arabidopsis guard cells. We performed stomatal aperture bioassays, patch clamp analyses and reactive oxygen species (ROS) measurements. ABA inhibition of inward K+ channels and light-induced stomatal opening are reduced in ost1 mutants while transgenic plants overexpressing OST1 show ABA hypersensitivity in these responses. ost1 mutants are insensitive to ABA-induced stomatal closure, regulation of slow anion currents, Ca2+-permeable channel activation and ROS production while OST1 overexpressing lines are hypersensitive for these responses, resulting in accelerated stomatal closure in response to ABA. Overexpression of OST1 in planta in the absence of ABA application does not affect basal apertures or ion currents. Moreover, we demonstrate the physical interaction of OST1 with the inward K+ channel KAT1, the anion channel SLAC1, and the NADPH oxidases AtrbohD and AtrbohF. Our findings support OST1 as a critical limiting component in ABA regulation of stomatal apertures, ion channels and NADPH oxidases in Arabidopsis guard cells.

Responses of plant leaf stomatal conductance and photosynthesis to water deficit have been extensively reported; however, little is known concerning the relationships of stomatal density with regard to water status and gas exchange. The responses of stomatal density to leaf water status were determined, and correlation with specific leaf area (SLA) in a photosynthetic study of a perennial grass, Leymus chinensis, subjected to different soil moisture contents. Moderate water deficits had positive effects on stomatal number, but more severe deficits led to a reduction, described in a quadratic parabolic curve. The stomatal size obviously decreased with water deficit, and stomatal density was positively correlated with stomatal conductance (gs), net CO₂ assimilation rate (An), and water use efficiency (WUE). A significantly negative correlation of SLA with stomatal density was also observed, suggesting that the balance between leaf area and its matter may be associated with the guard cell number. The present results indicate that high flexibilities in stomatal density and guard cell size will change in response to water status, and this process may be closely associated with photosynthesis and water use efficiency.

Summary Stomatal closure is an important process to prevent water loss in plants response to drought stress, which is finely modulated by ion channels together with their regulators in guard cells, especially the S‐type anion channel AtSLAC1 in Arabidopsis. However, the functional characterization and regulation analyses of anion channels in gramineous crops, such as in maize guard cells are still limited. In this study, we identified an S‐type anion channel ZmSLAC1 that was preferentially expressed in maize guard cells and involved in stomatal closure under drought stress. We found that two Ca2+‐dependent protein kinases ZmCPK35 and ZmCPK37 were expressed in maize guard cells and localized on the plasma membrane. Lesion of ZmCPK37 resulted in drought‐sensitive phenotypes. Mutation of ZmSLAC1 and ZmCPK37 impaired ABA‐activated S‐type anion currents in maize guard cells, while the S‐type anion currents were increased in the guard cells of ZmCPK35‐ and ZmCPK37‐overexpression lines. Electrophysiological characterization in maize guard cells and Xenopus oocytes indicated that ZmCPK35 and ZmCPK37 could activate ZmSLAC1‐mediated Cl‐ and NO3‐ currents. The maize inbred and hybrid lines overexpressing ZmCPK35 and ZmCPK37 exhibited enhanced tolerance and increased yield under drought conditions. In conclusion, our results demonstrate that ZmSLAC1 plays crucial roles in stomatal closure in maize, whose activity is regulated by ZmCPK35 and ZmCPK37. Elevation of ZmCPK35 and ZmCPK37 expression levels is a feasible way to improve maize drought tolerance as well as reduce yield loss under drought stress.

• Soil salinity is an increasingly global problem which hampers plant growth and crop yield. Plant productivity depends on optimal water-use efficiency and photosynthetic capacity balanced by stomatal conductance. Whether and how stomatal behavior contributes to salt sensitivity or tolerance is currently unknown. This work identifies guard cell-specific signaling networks exerted by a salt-sensitive and salt-tolerant plant under ionic and osmotic stress conditions accompanied by increasing NaCl loads. • We challenged soil-grown Arabidopsis thaliana and Thellungiella salsuginea plants with short- and long-term salinity stress and monitored genome-wide gene expression and signals of guard cells that determine their function. • Arabidopsis plants suffered from both salt regimes and showed reduced stomatal conductance while Thellungiella displayed no obvious stress symptoms. The salt-dependent gene expression changes of guard cells supported the ability of the halophyte to maintain high potassium to sodium ratios and to attenuate the abscisic acid (ABA) signaling pathway which the glycophyte kept activated despite fading ABA concentrations. • Our study shows that salinity stress and even the different tolerances are manifested on a single cell level. Halophytic guard cells are less sensitive than glycophytic guard cells, providing opportunities to manipulate stomatal behavior and improve plant productivity.