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The local endocytosis of membrane proteins is critical for many physiological processes in plants, including the regulation of growth, development, nutrient absorption, and osmotic stress response. Much of our knowledge on the local endocytosis of plasma membrane (PM) protein only focuses on the polar growth of pollen tubes in plants and neuronal axon in animals. However, the role of local endocytosis of PM proteins in guard cells has not yet been researched. Here, we first cloned duckweed SUT2 (sucrose transporter 2) protein and then conducted subcellular and histological localization of the protein. Our results indicated that LpSUT2 (Landoltia punctata 0202 SUT2) is a PM protein highly expressed on guard cells. In vitro experiments on WT (wild type) lines treated with high sucrose concentration showed that the content of ROS (reactive oxygen species) in guard cells increased and stomatal conductance decreased. We observed the same results in the lines after overexpression of the LpSUT2 gene with newfound local endocytosis of LpSUT2. The local endocytosis mainly showed that LpSUT2 was uniformly distributed on the PM of guard cells in the early stage of development, and was only distributed in the endomembrane of guard cells in the mature stage. Therefore, we found the phenomenon of guard cell LpSUT2 local endocytosis through the changes of duckweed stomata and concluded that LpSUT2 local endocytosis might be dependent on ROS accumulation in the development of duckweed guard cells. This paper might provide future references for the genetic improvement and water-use efficiency in other crops. [ABSTRACT FROM AUTHOR]

Drought stress limits plant development and reproduction. Multiple mechanisms in plants are activated to respond to stress. The MYC2 transcription factor is a core regulator of the jasmonate (JA) pathway and plays a vital role in the crosstalk between abscisic acid (ABA) and JA. In this study, we found that SlMYC2 responded to drought stress and regulated stomatal aperture in tomato (Solanum lycopersicum). Overexpression of SlMYC2 repressed SlCHS1 expression and decreased the flavonol content, increased the reactive oxygen species (ROS) content in guard cells and promoted the accumulation of JA and ABA in leaves. Additionally, silencing the SlCHS1 gene produced a phenotype that was similar to that of the MYC2-overexpressing (MYC2-OE) strain, especially in terms of stomatal dynamics and ROS levels. Finally, we confirmed that SlMYC2 directly repressed the expression of SlCHS1. Our study revealed that SlMYC2 drove stomatal closure by modulating the accumulation of flavonol and the JA and ABA contents, helping us decipher the mechanism of stomatal movement under drought stress. [ABSTRACT FROM AUTHOR]

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a protein complex spanning the inner and outer membranes of the nuclear envelope. Outer nuclear membrane KASH proteins interact in the nuclear envelope lumen with inner nuclear membrane SUN proteins. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light–dark transitions and ABA. Previous studies have shown F-actin organization, cytoplasmic calcium (Ca2+) oscillations, and vacuolar morphology changes are involved in ABA-induced stomatal closure. Here, we show that SINE1 and SINE2 are both required for actin pattern changes during ABA-induced stomatal closure, but influence different, temporally distinguishable steps. External Ca2+ partially overrides the mutant defects. ABA-induced cytoplasmic Ca2+ oscillations are diminished in sine2-1 but not sine1-1 , and this defect can be rescued by both exogenous Ca2+ and F-actin depolymerization. We show first evidence for nuclear Ca2+ oscillations during ABA-induced stomatal closure, which are disrupted in sine2-1. Vacuolar fragmentation is impaired in both mutants and is partially rescued by F-actin depolymerization. Together, these data indicate distinct roles for SINE1 and SINE2 upstream of this network of players involved in ABA-based stomatal closure, suggesting a role for the nuclear surface in guard cell ABA signaling. [ABSTRACT FROM AUTHOR]

The linker of nucleoskeleton and cytoskeleton (LINC) complex is a protein complex spanning the inner and outer membranes of the nuclear envelope. Outer nuclear membrane KASH proteins interact in the nuclear envelope lumen with inner nuclear membrane SUN proteins. The paralogous Arabidopsis KASH proteins SINE1 and SINE2 function during stomatal dynamics induced by light–dark transitions and ABA. Previous studies have shown F-actin organization, cytoplasmic calcium (Ca2+) oscillations, and vacuolar morphology changes are involved in ABA-induced stomatal closure. Here, we show that SINE1 and SINE2 are both required for actin pattern changes during ABA-induced stomatal closure, but influence different, temporally distinguishable steps. External Ca2+ partially overrides the mutant defects. ABA-induced cytoplasmic Ca2+ oscillations are diminished in sine2-1 but not sine1-1, and this defect can be rescued by both exogenous Ca2+ and F-actin depolymerization. We show first evidence for nuclear Ca2+ oscillations during ABA-induced stomatal closure, which are disrupted in sine2-1. Vacuolar fragmentation is impaired in both mutants and is partially rescued by F-actin depolymerization. Together, these data indicate distinct roles for SINE1 and SINE2 upstream of this network of players involved in ABA-based stomatal closure, suggesting a role for the nuclear surface in guard cell ABA signaling.

On sensing the microbial elicitor molecules, the guard cells trigger a rise in ROS, NO, Ca 2+, and H 2 S. These secondary messengers cause ion-efflux from guard cells and stomatal closure. Something smells bad to plant pathogens: production of hydrogen sulfide in plants and its role in plant defence responses. Keywords: guard cells; innate immunity; peroxisomes; photorespiration; secondary messengers; transpiration EN guard cells innate immunity peroxisomes photorespiration secondary messengers transpiration 1 6 6 09/29/21 20210927 NES 210927 Introduction Stomata are the main gateways for the entry of microbial pathogens into leaves (Melotto et al., [37]). [Extracted from the article]

There are more than 100 GDSL lipases in Arabidopsis , but only a few members have been functionally investigated. Moreover, no reports have ever given a comprehensive analysis of GDSLs in stomatal biology. Here, we systematically investigated the expression patterns of 19 putative G uard-cell-enriched G DSL L ipases (GGLs) at various developmental stages and in response to hormone and abiotic stress treatments. Gene expression analyses showed that these GGLs had diverse expression patterns. Fifteen GGLs were highly expressed in guard cells, with seven preferentially in guard cells. Most GGLs were localized in endoplasmic reticulum, and some were also localized in lipid droplets and nucleus. Some closely homologous GGLs exhibited similar expression patterns at various tissues and in response to hormone and abiotic stresses, or similar subcellular localization, suggesting the correlation of expression pattern and biological function, and the functional redundancy of GGLs in plant development and environmental adaptations. Further phenotypic identification of ggl mutants revealed that GGL7 , GGL14 , GGL22 , and GGL26 played unique and redundant roles in stomatal dynamics, stomatal density and morphology, and plant water relation. The present study provides unique resources for functional insights into these GGLs to control stomatal dynamics and development, plant growth, and adaptation to the environment. [ABSTRACT FROM AUTHOR]

There are more than 100 GDSL lipases in Arabidopsis, but only a few members have been functionally investigated. Moreover, no reports have ever given a comprehensive analysis of GDSLs in stomatal biology. Here, we systematically investigated the expression patterns of 19 putative Guard-cell-enriched GDSL Lipases (GGLs) at various developmental stages and in response to hormone and abiotic stress treatments. Gene expression analyses showed that these GGLs had diverse expression patterns. Fifteen GGLs were highly expressed in guard cells, with seven preferentially in guard cells. Most GGLs were localized in endoplasmic reticulum, and some were also localized in lipid droplets and nucleus. Some closely homologous GGLs exhibited similar expression patterns at various tissues and in response to hormone and abiotic stresses, or similar subcellular localization, suggesting the correlation of expression pattern and biological function, and the functional redundancy of GGLs in plant development and environmental adaptations. Further phenotypic identification of ggl mutants revealed that GGL7, GGL14, GGL22, and GGL26 played unique and redundant roles in stomatal dynamics, stomatal density and morphology, and plant water relation. The present study provides unique resources for functional insights into these GGLs to control stomatal dynamics and development, plant growth, and adaptation to the environment.

Stomata arose about 400 million years ago when plants left their aquatic environment. The last step of stomatal development is shared by all plant groups, and it implies a symmetrical cell division from the guard mother cell (GMC) to produce two guard cells (GCs) flanking a pore. In Arabidopsis, the basic helix-loop-helix transcription factor MUTE controls this step, upregulating cell-cycle regulators of the GMC division, and immediately afterward, repressors of theses regulators like FAMA and FOUR LIPS. Recently, three grass MUTE orthologs (BdMUTE from Brachypodium distachyon, OsMUTE from rice, and ZmMUTE from maize) have been identified and characterized. Mutations in these genes disrupt GMC fate, with bdmute also blocking GC morphogenesis. However, because these genes also regulate subsidiary cell recruitment, which takes place before GMC division, their functions regulating GMC division and GC morphogenesis could be an indirect consequence of that inducing the recruitment of subsidiary cells. Comprehensive data evaluation indicates that BdMUTE, and probably grass MUTE orthologs, directly controls GMC fate. Although grass MUTE proteins, whose genes are expressed in the GMC, move between cells, they regulate GMC fate from the cells where they are transcribed. Grass MUTE genes also regulate GC morphogenesis. Specifically, OsMUTE controls GC shape by inducing OsFAMA expression. In addition, while SCs are not required for GMC fate progression, they are for GC maturation.

Stomata arose about 400 million years ago when plants left their aquatic environment. The last step of stomatal development is shared by all plant groups, and it implies a symmetrical cell division from the guard mother cell (GMC) to produce two guard cells (GCs) flanking a pore. In Arabidopsis, the basic helix-loop-helix transcription factor MUTE controls this step, upregulating cell-cycle regulators of the GMC division, and immediately afterward, repressors of theses regulators like FAMA and FOUR LIPS. Recently, three grass MUTE orthologs (BdMUTE from Brachypodium distachyon , OsMUTE from rice, and ZmMUTE from maize) have been identified and characterized. Mutations in these genes disrupt GMC fate, with bdmute also blocking GC morphogenesis. However, because these genes also regulate subsidiary cell recruitment, which takes place before GMC division, their functions regulating GMC division and GC morphogenesis could be an indirect consequence of that inducing the recruitment of subsidiary cells. Comprehensive data evaluation indicates that BdMUTE , and probably grass MUTE orthologs, directly controls GMC fate. Although grass MUTE proteins, whose genes are expressed in the GMC, move between cells, they regulate GMC fate from the cells where they are transcribed. Grass MUTE genes also regulate GC morphogenesis. Specifically, OsMUTE controls GC shape by inducing OsFAMA expression. In addition, while SCs are not required for GMC fate progression, they are for GC maturation. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is a stress hormone that accumulates under different abiotic and biotic stresses. A typical effect of ABA on leaves is to reduce transpirational water loss by closing stomata and parallelly defend against microbes by restricting their entry through stomatal pores. ABA can also promote the accumulation of polyamines, sphingolipids, and even proline. Stomatal closure by compounds other than ABA also helps plant defense against both abiotic and biotic stress factors. Further, ABA can interact with other hormones, such as methyl jasmonate (MJ) and salicylic acid (SA). Such cross-talk can be an additional factor in plant adaptations against environmental stresses and microbial pathogens. The present review highlights the recent progress in understanding ABA’s multifaceted role under stress conditions, particularly stomatal closure. We point out the importance of reactive oxygen species (ROS), reactive carbonyl species (RCS), nitric oxide (NO), and Ca2+ in guard cells as key signaling components during the ABA-mediated short-term plant defense reactions. The rise in ROS, RCS, NO, and intracellular Ca2+ triggered by ABA can promote additional events involved in long-term adaptive measures, including gene expression, accumulation of compatible solutes to protect the cell, hypersensitive response (HR), and programmed cell death (PCD). Several pathogens can counteract and try to reopen stomata. Similarly, pathogens attempt to trigger PCD of host tissue to their benefit. Yet, ABA-induced effects independent of stomatal closure can delay the pathogen spread and infection within leaves. Stomatal closure and other ABA influences can be among the early steps of defense and a crucial component of plants’ innate immunity response. Stomatal guard cells are quite sensitive to environmental stress and are considered good model systems for signal transduction studies. Further research on the ABA-induced stomatal closure mechanism can help us design strategies for plant/crop adaptations to stress.

Abscisic acid (ABA) is a stress hormone that accumulates under different abiotic and biotic stresses. A typical effect of ABA on leaves is to reduce transpirational water loss by closing stomata and parallelly defend against microbes by restricting their entry through stomatal pores. ABA can also promote the accumulation of polyamines, sphingolipids, and even proline. Stomatal closure by compounds other than ABA also helps plant defense against both abiotic and biotic stress factors. Further, ABA can interact with other hormones, such as methyl jasmonate (MJ) and salicylic acid (SA). Such cross-talk can be an additional factor in plant adaptations against environmental stresses and microbial pathogens. The present review highlights the recent progress in understanding ABA's multifaceted role under stress conditions, particularly stomatal closure. We point out the importance of reactive oxygen species (ROS), reactive carbonyl species (RCS), nitric oxide (NO), and Ca2+ in guard cells as key signaling components during the ABA-mediated short-term plant defense reactions. The rise in ROS, RCS, NO, and intracellular Ca2+ triggered by ABA can promote additional events involved in long-term adaptive measures, including gene expression, accumulation of compatible solutes to protect the cell, hypersensitive response (HR), and programmed cell death (PCD). Several pathogens can counteract and try to reopen stomata. Similarly, pathogens attempt to trigger PCD of host tissue to their benefit. Yet, ABA-induced effects independent of stomatal closure can delay the pathogen spread and infection within leaves. Stomatal closure and other ABA influences can be among the early steps of defense and a crucial component of plants' innate immunity response. Stomatal guard cells are quite sensitive to environmental stress and are considered good model systems for signal transduction studies. Further research on the ABA-induced stomatal closure mechanism can help us design strategies for plant/crop adaptations to stress. [ABSTRACT FROM AUTHOR]

Plant stomata which consist of a pair of guard cells, are not only finely controlled to balance water loss as transpiration and CO2 absorption for photosynthesis, but also serve as the major sites to defend against pathogen attack, thus allowing plants to respond appropriately to abiotic and biotic stress conditions. The regulatory signaling network for stomatal movement is complex in nature, and plant peptides have been shown to be involved in signaling processes. Arabidopsis secreted peptide PIP1 was previously identified as an endogenous elicitor, which induced immune response through its receptor, RLK7. PIP1-RLK7 can activate stomatal immunity against the bacterial strain Pst DC3118. However, the molecular mechanism of PIP1 in stomatal regulation is still unclear and additional new factors need to be discovered. In this study, we further clarified that PIP1 could function as an important regulator in the induction of stomatal closure. The results showed that PIP1 could promote stomata to close in a certain range of concentrations and response time. In addition, we uncovered that PIP1-RLK7 signaling regulated stomatal response by activating S-type anion channel SLAC1. PIP1-induced stomatal closure was impaired in bak1, mpk3, and mpk6 mutants, indicating that BAK1 and MPK3/MPK6 were required for PIP1-regulated stomatal movement. Our research further deciphered that OST1 which acts as an essential ABA-signaling component, also played a role in PIP1-induced stomatal closure. In addition, ROS participated in PIP1-induced stomatal closure and PIP1 could activate Ca2+ permeable channels. In conclusion, we reveal the role of peptide PIP1 in triggering stomatal closure and the possible mechanism of PIP1 in the regulation of stomatal apertures. Our findings improve the understanding of the role of PIP1 in stomatal regulation and immune response.

Because stomata in bryophytes are uniquely located on sporangia, the physiological and evolutionary constraints placed on bryophyte stomata are fundamentally different from those on leaves of tracheophytes. Although losses of stomata have been documented in mosses, the extent to which this evolutionary process occurred remains relatively unexplored. We initiated this study by plotting the known occurrences of stomata loss and numbers per capsule on the most recent moss phylogeny. From this, we identified 40 families and 74 genera that lack stomata, of which at least 63 are independent losses. No trends in stomata losses or numbers are evident in any direction across moss diversity. Extant taxa in early divergent moss lineages either lack stomata or produce pseudostomata that do not form pores. The earliest land plant macrofossils from 400 ma exhibit similar sporangial morphologies and stomatal distribution to extant mosses, suggesting that the earliest mosses may have possessed and lost stomata as is common in the group. To understand why stomata are expendable in mosses, we conducted comparative anatomical studies on a range of mosses with and without stomata. We compared the anatomy of stomate and astomate taxa and the development of intercellular spaces, including substomatal cavities, across mosses. Two types of intercellular spaces that develop differently are seen in peristomate mosses, those associated with stomata and those that surround the spore sac. Capsule architecture in astomate mosses ranges from solid in the taxa in early divergent lineages to containing an internal space that is directly connected to the conducing tissue and is involved in capsule expansion and the nourishment, hydration and development of spores. This anatomy reveals there are different architectural arrangements of tissues within moss capsules that are equally effective in accomplishing the essential processes of sporogenesis and spore dispersal. Stomata are not foundational to these processes.

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release.

Plant stomata which consist of a pair of guard cells, are not only finely controlled to balance water loss as transpiration and CO2 absorption for photosynthesis, but also serve as the major sites to defend against pathogen attack, thus allowing plants to respond appropriately to abiotic and biotic stress conditions. The regulatory signaling network for stomatal movement is complex in nature, and plant peptides have been shown to be involved in signaling processes. Arabidopsis secreted peptide PIP1 was previously identified as an endogenous elicitor, which induced immune response through its receptor, RLK7. PIP1-RLK7 can activate stomatal immunity against the bacterial strain Pst DC3118. However, the molecular mechanism of PIP1 in stomatal regulation is still unclear and additional new factors need to be discovered. In this study, we further clarified that PIP1 could function as an important regulator in the induction of stomatal closure. The results showed that PIP1 could promote stomata to close in a certain range of concentrations and response time. In addition, we uncovered that PIP1-RLK7 signaling regulated stomatal response by activating S-type anion channel SLAC1. PIP1-induced stomatal closure was impaired in bak1 , mpk3 , and mpk6 mutants, indicating that BAK1 and MPK3/MPK6 were required for PIP1-regulated stomatal movement. Our research further deciphered that OST1 which acts as an essential ABA-signaling component, also played a role in PIP1-induced stomatal closure. In addition, ROS participated in PIP1-induced stomatal closure and PIP1 could activate Ca2+ permeable channels. In conclusion, we reveal the role of peptide PIP1 in triggering stomatal closure and the possible mechanism of PIP1 in the regulation of stomatal apertures. Our findings improve the understanding of the role of PIP1 in stomatal regulation and immune response. [ABSTRACT FROM AUTHOR]

Because stomata in bryophytes are uniquely located on sporangia, the physiological and evolutionary constraints placed on bryophyte stomata are fundamentally different from those on leaves of tracheophytes. Although losses of stomata have been documented in mosses, the extent to which this evolutionary process occurred remains relatively unexplored. We initiated this study by plotting the known occurrences of stomata loss and numbers per capsule on the most recent moss phylogeny. From this, we identified 40 families and 74 genera that lack stomata, of which at least 63 are independent losses. No trends in stomata losses or numbers are evident in any direction across moss diversity. Extant taxa in early divergent moss lineages either lack stomata or produce pseudostomata that do not form pores. The earliest land plant macrofossils from 400 ma exhibit similar sporangial morphologies and stomatal distribution to extant mosses, suggesting that the earliest mosses may have possessed and lost stomata as is common in the group. To understand why stomata are expendable in mosses, we conducted comparative anatomical studies on a range of mosses with and without stomata. We compared the anatomy of stomate and astomate taxa and the development of intercellular spaces, including substomatal cavities, across mosses. Two types of intercellular spaces that develop differently are seen in peristomate mosses, those associated with stomata and those that surround the spore sac. Capsule architecture in astomate mosses ranges from solid in the taxa in early divergent lineages to containing an internal space that is directly connected to the conducing tissue and is involved in capsule expansion and the nourishment, hydration and development of spores. This anatomy reveals there are different architectural arrangements of tissues within moss capsules that are equally effective in accomplishing the essential processes of sporogenesis and spore dispersal. Stomata are not foundational to these processes. [ABSTRACT FROM AUTHOR]

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana , moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release. [ABSTRACT FROM AUTHOR]

Water is a limiting resource for many land plants. Most of the water taken up by plants is lost to the atmosphere through the stomata, which are adjustable pores on the leaf surface that allow for gas exchange between the plant and the atmosphere. Modulating stomatal activity might be an effective way to reduce plants’ water consumption and enhance their productivity under normal, as well as water-limiting conditions. Our recent discovery of stomatal regulation by sugars that is mediated by guard-cell hexokinase (HXK), a sugar-sensing enzyme, has raised the possibility that HXK might be used to increase plant water-use efficiency (WUE; i.e., carbon gain per unit of water). We show here that transgenic tomato and Arabidopsis plants with increased expression of HXK in their guard cells (GCHXK plants) exhibit reduced transpiration and higher WUE without any negative effects on growth under normal conditions, as well as drought avoidance and improved photosynthesis and growth under limited-water conditions. Our results demonstrate that exclusive expression of HXK in guard cells is an effective tool for improving WUE, and plant performance under drought.

Water is a limiting resource for many land plants. Most of the water taken up by plants is lost to the atmosphere through the stomata, which are adjustable pores on the leaf surface that allow for gas exchange between the plant and the atmosphere. Modulating stomatal activity might be an effective way to reduce plants' water consumption and enhance their productivity under normal, as well as water-limiting conditions. Our recent discovery of stomatal regulation by sugars that is mediated by guard-cell hexokinase (HXK), a sugar-sensing enzyme, has raised the possibility that HXK might be used to increase plant water-use efficiency (WUE; i.e., carbon gain per unit of water). We show here that transgenic tomato and Arabidopsis plants with increased expression of HXK in their guard cells (GCHXK plants) exhibit reduced transpiration and higher WUE without any negative effects on growth under normal conditions, as well as drought avoidance and improved photosynthesis and growth under limited-water conditions. Our results demonstrate that exclusive expression of HXK in guard cells is an effective tool for improving WUE, and plant performance under drought. [ABSTRACT FROM AUTHOR]

Guard cells are pairs of epidermal cells that control gas diffusion by regulating the opening and closure of stomatal pores. Guard cells, like other types of plant cells, are surrounded by a three-dimensional, extracellular network of polysaccharide-based wall polymers. In contrast to the walls of diffusely growing cells, guard cell walls have been hypothesized to be uniquely strong and elastic to meet the functional requirements of withstanding high turgor and allowing for reversible stomatal movements. Although the walls of guard cells were long underexplored as compared to extensive studies of stomatal development and guard cell signaling, recent research has provided new genetic, cytological, and physiological data demonstrating that guard cell walls function centrally in stomatal development and dynamics. In this review, we highlight and discuss the latest evidence for how wall polysaccharides are synthesized, deposited, reorganized, modified, and degraded in guard cells, and how these processes influence stomatal form and function. We also raise open questions and provide a perspective on experimental approaches that could be used in the future to shed light on the composition and architecture of guard cell walls.

Guard cells are pairs of epidermal cells that control gas diffusion by regulating the opening and closure of stomatal pores. Guard cells, like other types of plant cells, are surrounded by a three-dimensional, extracellular network of polysaccharide-based wall polymers. In contrast to the walls of diffusely growing cells, guard cell walls have been hypothesized to be uniquely strong and elastic to meet the functional requirements of withstanding high turgor and allowing for reversible stomatal movements. Although the walls of guard cells were long underexplored as compared to extensive studies of stomatal development and guard cell signaling, recent research has provided new genetic, cytological, and physiological data demonstrating that guard cell walls function centrally in stomatal development and dynamics. In this review, we highlight and discuss the latest evidence for how wall polysaccharides are synthesized, deposited, reorganized, modified, and degraded in guard cells, and how these processes influence stomatal form and function. We also raise open questions and provide a perspective on experimental approaches that could be used in the future to shed light on the composition and architecture of guard cell walls. [ABSTRACT FROM AUTHOR]

Plants use stomatal closure mediated by elicitors as the first step of the innate immune response to restrict the microbial entry. We present a comprehensive study of the effect of cryptogein and harpin, two elicitors from microbial pathogens of tobacco, on stomatal closure and guard cell signaling components in Arabidopsis thaliana, a model plant. Cryptogein as well as harpin induced stomatal closure, while elevating the levels of reactive oxygen species (ROS) and nitric oxide (NO) in the guard cells of A. thaliana. Kinetic studies with fluorescent dyes revealed that the rise in ROS levels preceded that of NO in guard cells, when treated with these two elicitors. The restriction of NO levels in guard cells, even by ROS modulators indicates the essentiality of ROS for NO production during elicitor-triggered stomatal closure. The signaling events during elicitor-induced stomatal closure appear to converge at NADPH oxidase and ROS production. Our results provide the first report on stomatal closure associated with rise in ROS/NO of guard cells by cryptogein and harpin in A. thaliana. Our results establish that A. thaliana can be used to study stomatal responses to the typical elicitors from microbial pathogens of other plants. The suitability of Arabidopsis opens up an excellent scope for further studies on signaling events leading to stomatal closure by microbial elicitors.

Plants use stomatal closure mediated by elicitors as the first step of the innate immune response to restrict the microbial entry. We present a comprehensive study of the effect of cryptogein and harpin, two elicitors from microbial pathogens of tobacco, on stomatal closure and guard cell signaling components in Arabidopsis thaliana, a model plant. Cryptogein as well as harpin induced stomatal closure, while elevating the levels of reactive oxygen species (ROS) and nitric oxide (NO) in the guard cells of A. thaliana. Kinetic studies with fluorescent dyes revealed that the rise in ROS levels preceded that of NO in guard cells, when treated with these two elicitors. The restriction of NO levels in guard cells, even by ROS modulators indicates the essentiality of ROS for NO production during elicitor-triggered stomatal closure. The signaling events during elicitor-induced stomatal closure appear to converge at NADPH oxidase and ROS production. Our results provide the first report on stomatal closure associated with rise in ROS/NO of guard cells by cryptogein and harpin in A. thaliana. Our results establish that A. thaliana can be used to study stomatal responses to the typical elicitors from microbial pathogens of other plants. The suitability of Arabidopsis opens up an excellent scope for further studies on signaling events leading to stomatal closure by microbial elicitors. [ABSTRACT FROM AUTHOR]

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

Our current understanding of guard cell signalling pathways is derived from studies in a small number of model species. The ability to study stomatal responses in isolated epidermis has been an important factor in elucidating the mechanisms by which the stomata of these species respond to environmental stresses. However, such approaches have rarely been applied to study guard cell signalling in the stomata of graminaceous species (including many of the world’s major crops), in which the guard cells have a markedly different morphology to those in other plants. Our understanding of guard cell signalling in these important species is therefore much more limited. Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon. We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures. We use the epidermal peel assay technique to investigate in more detail interactions between different environmental factors in barley guard cells, and demonstrate that stomatal closure in response to external CO2 is inhibited at higher temperatures, whilst sensitivity to ABA is enhanced at 30°C compared to 20°C and 40°C.

Dynamic regulation of stomatal aperture is essential for plants to optimize water use and CO2 uptake. Stomatal opening or closure is accompanied by the modulation of guard cell turgor. Among the events leading to stomatal closure by plant hormones or microbial elicitors, three signaling components stand out as the major converging points. These are reactive oxygen species (ROS), cytosolic free Ca2+, and ion channels. Once formed, the ROS and free Ca2+ of guard cells regulate both downstream and upstream events. A major influence of ROS is to increase the levels of NO and cytosolic free Ca2+ in guard cells. Although the rise in NO is an important event during stomatal closure, the available evidences do not support the description of NO as the point of convergence. The rise in ROS and NO would cause an increase of free Ca2+ and modulate ion channels, through a network of events, in such a way that the guard cells lose K+/Cl-/anions. The efflux of these ions decreases the turgor of guard cells and leads to stomatal closure. Thus, ROS, NO, and cytosolic free Ca2+ act as points of divergence. The other guard cell components, which are modulated during stomatal closure are G-proteins, cytosolic pH, phospholipids, and sphingolipids. However, the current information on the role of these components is not convincing so as to assign them as the points of convergence or divergence. The interrelationships and interactions of ROS, NO, cytosolic pH, and free Ca2+ are quite complex and need further detailed examination. Our review is an attempt to critically assess the current status of information on guard cells, while emphasizing the convergence and divergence of signaling components during stomatal closure. The existing gaps in our knowledge are identified to stimulate further research. [ABSTRACT FROM AUTHOR]

The article focuses on the dual role of Nitric Oxide in the Guard Cells by promoting and attenuating the ABA and Phospholipid-Derived signals leading to the regulation of stomatal movement.

The local endocytosis of membrane proteins is critical for many physiological processes in plants, including the regulation of growth, development, nutrient absorption, and osmotic stress response. Much of our knowledge on the local endocytosis of plasma membrane (PM) protein only focuses on the polar growth of pollen tubes in plants and neuronal axon in animals. However, the role of local endocytosis of PM proteins in guard cells has not yet been researched. Here, we first cloned duckweed SUT2 (sucrose transporter 2) protein and then conducted subcellular and histological localization of the protein. Our results indicated that LpSUT2 ( Landoltia punctata 0202 SUT2) is a PM protein highly expressed on guard cells. In vitro experiments on WT (wild type) lines treated with high sucrose concentration showed that the content of ROS (reactive oxygen species) in guard cells increased and stomatal conductance decreased. We observed the same results in the lines after overexpression of the LpSUT2 gene with newfound local endocytosis of LpSUT2. The local endocytosis mainly showed that LpSUT2 was uniformly distributed on the PM of guard cells in the early stage of development, and was only distributed in the endomembrane of guard cells in the mature stage. Therefore, we found the phenomenon of guard cell LpSUT2 local endocytosis through the changes of duckweed stomata and concluded that LpSUT2 local endocytosis might be dependent on ROS accumulation in the development of duckweed guard cells. This paper might provide future references for the genetic improvement and water-use efficiency in other crops., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Liu, Fang, Tan, Hu, Jin, Yi, He, Wei, Chen and Zhao.)

Two highly specialized cells, the guard cells that surround the stomatal pore, are able to integrate environmental and endogenous signals in order to control the stomatal aperture and thereby the gas exchange. The uptake of CO2 is associated with a loss of water by leaves. Control of the size of the stomatal aperture optimizes the efficiency of water use through dynamic changes in the turgor of the guard cells. The opening and closing of stomata is regulated by the integration of environmental signals and endogenous hormonal stimuli. The various different factors to which the guard cells respond translates into the complexity of the network of signaling pathways that control stomatal movements. The perception of an abiotic stress triggers the activation of signal transduction cascades that interact with or are activated by phytohormones. Among these, abscisic acid (ABA), is the best-known stress hormone that closes the stomata, although other phytohormones, such as jasmonic acid, brassinosteroids, cytokinins, or ethylene are also involved in the stomatal response to stresses. As a part of the drought response, ABA may interact with jasmonic acid and nitric oxide in order to stimulate stomatal closure. In addition, the regulation of gene expression in response to ABA involves genes that are related to ethylene, cytokinins, and auxin signaling. In this paper, recent findings on phytohormone crosstalk, changes in signaling pathways including the expression of specific genes and their impact on modulating stress response through the closing or opening of stomata, together with the highlights of gaps that need to be elucidated in the signaling network of stomatal regulation, are reviewed. [ABSTRACT FROM AUTHOR]

Drought stress limits plant development and reproduction. Multiple mechanisms in plants are activated to respond to stress. The MYC2 transcription factor is a core regulator of the jasmonate (JA) pathway and plays a vital role in the crosstalk between abscisic acid (ABA) and JA. In this study, we found that SlMYC2 responded to drought stress and regulated stomatal aperture in tomato ( Solanum lycopersicum ). Overexpression of SlMYC2 repressed SlCHS1 expression and decreased the flavonol content, increased the reactive oxygen species (ROS) content in guard cells and promoted the accumulation of JA and ABA in leaves. Additionally, silencing the SlCHS1 gene produced a phenotype that was similar to that of the MYC2- overexpressing ( MYC2 -OE) strain, especially in terms of stomatal dynamics and ROS levels. Finally, we confirmed that SlMYC2 directly repressed the expression of SlCHS1 . Our study revealed that SlMYC2 drove stomatal closure by modulating the accumulation of flavonol and the JA and ABA contents, helping us decipher the mechanism of stomatal movement under drought stress., Competing Interests: X-BQ is employed by Bei Jing Bei Nong Enterprise Management Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Xu, Wang, Peng, Huang, Sun, Yang, Suo, Wang and Zhao.)

Our current understanding of guard cell signaling pathways is derived from studies in a small number of model species. The ability to study stomatal responses in isolated epidermis has been an important factor in elucidating the mechanisms by which the stomata of these species respond to environmental stresses. However, such approaches have rarely been applied to study guard cell signaling in the stomata of graminaceous species (including many of the world's major crops), in which the guard cells have a markedly different morphology to those in other plants. Our understanding of guard cell signaling in these important species is therefore much more limited. Here, we describe a procedure for the isolation of abaxial epidermal peels from barley, wheat and Brachypodium distachyon. We show that isolated epidermis from these species contains viable guard cells that exhibit typical responses to abscisic acid (ABA) and CO2, as determined by measurements of stomatal apertures. We use the epidermal peel assay technique to investigate in more detail interactions between different environmental factors in barley guard cells, and demonstrate that stomatal closure in response to external CO2 is inhibited at higher temperatures, whilst sensitivity to ABA is enhanced at 30°C compared to 20 and 40°C.