Guard Cells Research Articles

Cytosolic alkalinization in guard cells: an intriguing but interesting event during stomatal closure that merits further validation of its importance

Stomatal closure is essential to conserve water and prevent microbial entry into leaves. Alkalinization of guard cells is common during closure by factors such as abscisic acid, methyl jasmonate, and even darkness. Despite reports pointing at the role of cytosolic pH, there have been doubts about whether the guard cell pH change is a cause for stomatal closure or an associated event, as changes in membrane potential or ion flux can modulate the pH. However, the importance of cytosolic alkalinization is strongly supported by the ability of externally added weak acids to restrict stomatal closure. Using genetically encoded pH sensors has confirmed the rise in pH to precede the elevation of Ca2+ levels. Yet some reports claim that the rise in pH follows the increase in ROS or Ca2+. We propose a feedback interaction among the rise in pH or ROS or Ca2+ to explain the contrasting opinions on the positioning of pH rise. Stomatal closure and guard cell pH changes are compromised in mutants deficient in vacuolar H+-ATPase (V-ATPase), indicating the importance of V-ATPase in promoting stomatal closure. Thus, cytosolic pH change in guard cells can be related to the rise in ROS and Ca2+, leading to stomatal closure. We emphasize that cytosolic pH in stomatal guard cells deserves further attention and evaluation.

Grapevine and maize: Two guard cell shaped strategies to cope with repeated drought stress

Adaptation of crops to recurrent drought stress is crucial for maintaining agricultural productivity and achieving food security under changing climate. Guard cells, pivotal regulators of plant water usage and assimilation, are central to this adaptation process. However, the metabolic dynamics of guard cells under drought stress remain poorly understood, particularly in grapevine, a prominent crop grown in arid regions, and maize, a staple crop with substantial water requirements. In this study, differences in guard cells metabolism during drought stress of grapevine and maize were investigated by performing physiological and metabolomic analyses. Metabolomic analysis highlighted differential responses in amino acids and sugars, with grapevine guard cells displaying greater stability in amino acid and sugar signatures, while maize showed marked increases in sugar levels. These findings suggest two distinct adaptive strategies, a vigorous acclimation of guard cells, as observed in maize, and an attenuated acclimation of guard cells, shown in grapevine. Understanding these metabolic adjustments is helpful for enhancing drought resilience in agricultural systems.

A putative hemicellulose gene promoter drives high levels of expression in guard cells and stem epidermis of poplar and highlights hormone regulation of cell wall formation

Forest trees generate substantial quantities of organic carbon, recognized as a renewable and economically viable resource for applications in bioenergy and industry. This study specifically focuses on xylan, a major hemicellulose component in poplar, and on the role of PtrPARVUS2—a member of the glycosyltransferase 8 family genes involved in xylan biosynthesis. PtrPARVUS2 is hypothesized to exhibit tissue-specific activity, and here we assessed its promoter via bioinformatic analysis, revealing motifs associated with responsiveness to hormones including Gibberellic Acid (GA), Methyl Jasmonate (MeJA), Auxin, and Abscisic Acid (ABA), among others. Treatment with hormones, including ABA, 1-naphthaleneacetic acid, a synthetic auxin, MeJA, and GA, demonstrated a swift inhibition of PtrPARVUS2 transcription. Analysis of expression patterns in wildtype trees indicated heightened expression in vascular tissues, particularly the bark. Transgenic lines expressing Enhanced Green Fluorescent Protein and β-glucuronidase under the control of the PtrPARVUS2 promoter confirmed tissue-specific activity, particularly in guard cells, stem epidermis, and developing xylem. These results offer understanding into the potential functions of PtrPARVUS2 in terms of phytohormone response and the formation of guard cell walls. The investigation introduces a valuable promoter for specific biotechnological applications in guard cells and stem epidermal cells. Additional research is required to elucidate the precise regulatory mechanisms of PtrPARVUS2 in the future.

Emerging roles of auxin in plant abiotic stress tolerance.

Plants are continuously attacked by several biotic and abiotic factors. Among abiotic factors, heat, cold, drought, and salinity are common stresses. Plants produce several hormones as their main weapon in fightback against these stresses. Among these hormones, the role of auxin is well established in regulating plant growth and development at various scales. However, in recent literature, the important role of auxin in abiotic stress tolerance has emerged. Several auxin signalling and transport mutants exhibit heat, drought, and salinity-related phenotypes. Among them, auxin-mediated hypocotyl elongation and root growth in response to increased heat are of importance due to the continuous rise in global temperature. Auxin is also involved in regulating and recruiting specialized metabolites like aliphatic glucosinolate to defend themselves from drought stress. Aliphatic glucosinolate (A-GLS) regulates guard cell closure using auxin, which is independent of the major abiotic stress hormone abscisic acid. This regulatory mechanism serves as an additional layer of guard cell movement to protect plants from drought. Transferring the aliphatic glucosinolate pathway into non-brassica plants such as rice and soybean holds the promise to improve drought tolerance. In addition to these, post-translational modification of auxin signalling components and redistribution of auxin efflux transporters are also playing important roles in drought and salt tolerance and, hence, may be exploited to breed drought-tolerant crops. Also, reactive oxygen species, along with peptide hormone and auxin signalling, are important in root growth under stress. In conclusion, we summarize recent discoveries that suggest auxin is involved in various abiotic stresses.

Image-Based Quantitative Analysis of Epidermal Morphology in Wild Potato Leaves.

The epidermal leaf patterns of plants exhibit remarkable diversity in cell shapes, sizes, and arrangements, driven by environmental interactions that lead to significant adaptive changes even among closely related species. The Solanaceae family, known for its high diversity of adaptive epidermal structures, has traditionally been studied using qualitative phenotypic descriptions. To advance this, we developed a workflow combining multi-scale computer vision, image processing, and data analysis to extract digital descriptors for leaf epidermal cell morphology. Applied to nine wild potato species, this workflow quantified key morphological parameters, identifying descriptors for trichomes, stomata, and pavement cells, and revealing interdependencies among these traits. Principal component analysis (PCA) highlighted two main axes, accounting for 45% and 21% of variance, corresponding to features such as guard cell shape, trichome length, stomatal density, and trichome density. These axes aligned well with the historical and geographical origins of the species, separating southern from Central American species, and forming distinct clusters for monophyletic groups. This workflow thus establishes a quantitative foundation for investigating leaf epidermal cell morphology within phylogenetic and geographic contexts.

Linking stomatal size and density to water use efficiency and leaf carbon isotope ratio in juvenile and mature trees.

Water-use efficiency (WUE) is affected by multiple leaf traits, including stomatal morphology. However, the impact of stomatal morphology on WUE across different ontogenetic stages of tree species is not well-documented. Here, we investigated the relationship between stomatal morphology, intrinsic water-use efficiency (iWUE) and leaf carbon isotope ratio (δ13C). We sampled 190 individuals, including juvenile and mature trees belonging to 18 temperate broadleaved tree species and 9 genera. We measured guard cell length (GCL), stomatal density (SD), specific leaf area (SLA), iWUE and bulk leaf δ13C as a proxy for long-term WUE. Leaf δ13C correlated positively with iWUE across species in both juvenile and mature trees, while GCL showed a negative and SD a positive effect on iWUE and leaf δ13C. Within species, however, only GCL was significantly associated with iWUE and leaf δ13C. SLA had a minor negative influence on iWUE and leaf δ13C, but this effect was inconsistent between juvenile and mature trees. We conclude that GCL and SD can be considered functional morphological traits related to the iWUE and leaf δ13C of trees, highlighting their potential for rapid phenotyping approaches in ecological studies.

Guard Cell Activity of PIF4 Represses Disease Resistance in Arabidopsis.

Phytochrome Interacting Factor 4 (PIF4) plays a central role in coordinating plant growth regulation by integrating multiple environmental cues. However, studies on whether and how PIF4 regulates plant immunity have inconsistent findings. In this study, we investigated the role of PIF4 in disease resistance against Pst DC3000 by characterizing its loss-of-function mutants using different inoculation strategies. Our findings reveal that pif4 mutants exhibit enhanced disease resistance with spray inoculation but not with infiltration inoculation compared to wild-type plants, and that mutants displayed more closed stomata apertures, indicating that PIF4 promotes stomatal opening. Importantly, expression of PIF4 by a guard-cell-specific promoter was sufficient to restore disease resistance to the wild-type level in the pif4 mutant. Additionally, PIF4 overexpression enhances disease symptom development independent of disease resistance and chlorophyll degradation, while the loss of PIF4 function leads to higher chlorophyll accumulation. Thus, our findings highlight a crucial function of PIF4 in regulating stomata-mediated disease resistance and chlorophyll accumulation, providing new insights into the connection of growth and defense in plants.

Robust Truncated Statistics Constant False Alarm Rate Detection of UAVs Based on Neural Networks

With the rapid popularity of unmanned aerial vehicles (UAVs), airspace safety is facing tougher challenges, especially for the identification of non-cooperative target UAVs. As a vital approach for non-cooperative target identification, radar signal processing has attracted continuous and extensive attention and research. The constant false alarm rate (CFAR) detector is widely used in most current radar systems. However, the detection performance will sharply deteriorate in complex and dynamical environments. In this paper, a novel truncated statistics- and neural network-based CFAR (TSNN-CFAR) algorithm is developed. Specifically, we adopt a right truncated Rayleigh distribution model combined with the characteristics of pattern recognition using a neural network. In the simulation environments of four different backgrounds, the proposed algorithm does not need guard cells and outperforms the traditional mean level (ML) and ordered statistics (OS) CFAR algorithms. Especially in high-density target and clutter edge environments, since utilizing 19 statistics obtained from the numerical calculation of two reference windows as the input characteristics, the TSNN-CFAR algorithm has the best adaptive decision ability, accurate background clutter modeling, stable false alarm regulation property and superior detection performance.

Stomatal development in the changing climate.

Stomata, microscopic pores flanked by symmetrical guard cells, are vital regulators of gas exchange that link plant processes with environmental dynamics. The formation of stomata involves the multi-step progression of a specialized cell lineage. Remarkably, this process is heavily influenced by environmental factors, allowing plants to adjust stomatal production to local conditions. With global warming set to alter our climate at an unprecedented pace, understanding how environmental factors impact stomatal development and plant fitness is becoming increasingly important. In this Review, we focus on the effects of carbon dioxide, high temperature and drought - three environmental factors tightly linked to global warming - on stomatal development. We summarize the stomatal response of a variety of plant species and highlight the existence of species-specific adaptations. Using the model plant Arabidopsis, we also provide an update on the molecular mechanisms involved in mediating the plasticity of stomatal development. Finally, we explore how knowledge on stomatal development is being applied to generate crop varieties with optimized stomatal traits that enhance their resilience against climate change and maintain agricultural productivity.

Transcriptomic dynamics of ABA response in Brassica napus guard cells

Drought has a significant, negative impact on crop production; and these effects are poised to increase with climate change. Plants acclimate to drought and water stress through diverse physiological responses, primarily mediated by the hormone abscisic acid (ABA). Because plants lose the majority of their water through stomatal pores on aerial surfaces of plants, stomatal closure is one of the rapid responses mediated by ABA to reduce transpirational water loss. The dynamic changes in the transcriptome of stomatal guard cells in response to ABA have been investigated in the model plant Arabidopsis thaliana. However, guard cell transcriptomes have not been analyzed in agronomically valuable crops such as a major oilseed crop, rapeseed. In this study, we investigated the dynamics of ABA-regulated transcriptomes in stomatal guard cells of Brassica napus and conducted comparison analysis with the transcriptomes of A. thaliana. We discovered changes in gene expression indicating alterations in a host of physiological processes, including stomatal movement, metabolic reprogramming, and light responses. Our results suggest the existence of both immediate and delayed responses to ABA in Brassica guard cells. Furthermore, the transcription factors and regulatory networks mediating these responses are compared to those identified in Arabidopsis. Our results imply the continuing evolution of ABA responses in Brassica since its divergence from a common ancestor, involving both protein-coding and non-coding nucleotide sequences. Together, our results will provide a basis for developing strategies for molecular manipulation of drought tolerance in crop plants.

In vitro cytotoxic evaluation of Hypericum perforatum and molecular docking and dynamic analysis of PINK-1 inhibitors on model organism Tribolium castaneum and Homo sapiens

Hypericum species are especially known for their pharmacological characteristic. One of the major component of the plant is hypericin that can be used in tumor inhibition with its potent cytotoxic effects. In this study, the anticancer effect of H. perforatum essential oil on the MCF-7 breast cancer cell line was examined, and the binding potential of the compounds of Hypericum perforatum L., hypericin, hyperoside and hyperforin, to the PINK1 protein of both human and model organism, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) was investigated in silico. Recently, many insect species have been proposed as model organisms, including T. castaneum, that the first species with whole genome sequenced. Worlwide distribution of insects, their environmental importance and realtively inexpensive cultivation have increased the interest in them Therefore, in in silico studies, T. castaneum was used as a model to compare binding similarities with humans and model organisms. In the study, the IC50 concentration of H. perforatum L. on MCF-7 cells was determined to be 98.765 μg/ml. Based on in silico findings, the most favorable binding affinity of -12.5 kcal/mol was observed between the Hypericin molecule and the insect PINK1 protein. The fact that these plant components bind with high energy to the PINK1 protein, which is believed to guard cells from mitochondrial dysfunction triggered by stress, is promising for the development of plant-based medical drugs and biopesticides.

Fungal toxin fusicoccin enhances plant growth by upregulating 14-3-3 interaction with plasma membrane H+-ATPase.

Fusicoccin-A (FC-A) is a diterpene glucoside produced by a pathogenic fungus. Since its discovery, FC-A has been widely recognized as a phytotoxin that induces stomatal opening and leaf wilting, eventually leading to plant death. In this study, we present the first evidence that FC-A enhances plant growth by stabilizing the protein-protein interaction between plasma membrane (PM) H+-ATPase and 14-3-3 in guard cells. Long-term treatment of Arabidopsis plants with FC-A resulted in ~ 30% growth enhancement. Structurally similar fusicoccin-J (FC-J) showed a similar degree of growth-promotion activity as FC-A, whereas the more hydrophilic fusicoccin-H (FC-H) exhibited no effect on plant growth, indicating that the enhancement of plant growth observed with FC-A and FC-J involves upregulation of the protein-protein interaction between PM H+-ATPase and 14-3-3 in guard cells, which promotes stomatal opening and photosynthesis.

Gaining or cutting SLAC: the evolution of plant guard cell signalling pathways.

The evolution of adjustable stomatal pores, enabling CO2 acquisition, was one of the most significant events in the development of life on land. Here, we investigate how the guard cell signalling pathways that regulate stomatal movements evolved. We compare fern and angiosperm guard cell transcriptomes and physiological responses, and examine the functionality of ion channels from diverse plant species. We find that, despite conserved expression in guard cells, fern anion channels from the SLAC/SLAH family are not activated by the same abscisic acid (ABA) pathways that provoke stomatal closure in angiosperms. Accordingly, we find an insensitivity of fern stomata to ABA. Moreover, our analysis points to a complex evolutionary history, featuring multiple gains and/or losses of SLAC activation mechanisms, as these channels were recruited to a role in stomatal closure. Our results show that the guard cells of flowering and nonflowering plants share similar core features, with lineage-specific and ecological niche-related adaptations, likely underlying differences in behaviour.

Metabolic modelling reveals distinct roles of sugars and carboxylic acids in stomatal opening and uncovers unexpected carbon fluxes.

Guard cell metabolism is crucial for stomatal dynamics, but a full understanding of its role is hampered by experimental limitations and the flexible nature of the metabolic network. To tackle this challenge, we constructed a time-resolved stoichiometric model of guard cell metabolism that accounts for energy and osmolyte requirements and which is integrated with the mesophyll. The model resolved distinct roles for starch, sugars, and malate in guard cell metabolism and revealed several unexpected flux patterns in central metabolism. During blue light-mediated stomatal opening, starch breakdown was the most efficient way to generate osmolytes with downregulation of glycolysis allowing starch-derived glucose to accumulate as a cytosolic osmolyte. Maltose couldalso accumulate as a cytosolic osmoticum, although this made the metabolic system marginally less efficient. The metabolic energy for stomatal opening was predicted to be derived independently of starch, using nocturnally accumulated citrate which was metabolised in the tricarboxylic acid cycle to malate to provide mitochondrial reducing power for ATP synthesis. In white light-mediated stomatal opening, malate transferred reducing equivalents from guard cell photosynthesis to mitochondria for ATP production. Depending on the capacity for guard cell photosynthesis, glycolysis showed little flux during the day but was crucial for energy metabolism at night. In summary, our analyses have corroborated recent findings in Arabidopsis guard cell research, resolved conflicting observations by highlighting the flexibility of guard cell metabolism, and proposed new metabolic flux modes for further experimental testing.

Gamma-Aminobutyric Acid (GABA) as a Defense Booster for Wheat against Leaf Rust Pathogen (Puccinia triticina).

Wheat leaf rust, caused by Puccinia triticina, poses a growing threat to global wheat production, necessitating alternative strategies for effective disease management. This study investigated the potential of gamma-aminobutyric acid (GABA) to enhance resistance to leaf rust in two wheat cultivars: the susceptible Morocco and moderately resistant Sakha 94 cultivar. Our findings revealed that GABA significantly improved resistance in both cultivars to P. triticina, particularly in Morocco, by mitigating disease severity and reducing pustule density and size while extending both incubation and latent periods. This study assessed the effectiveness of two GABA application methods: plants received 1 mM GABA treatment, as a foliar spray, twenty-four hours prior to infection (pre-GABA), and plants received 1 mM GABA treatment both 24 h before and after infection (pre-/post-GABA), with the latter yielding significantly better results in reducing infection severity and improving plant resilience. Additionally, GABA application influenced stomatal behavior, promoting closure that may enhance resilience against leaf rust. GABA application on plants also modulated the production of reactive oxygen species (ROS). This led to a stronger oxidative burst in both susceptible and moderately resistant cultivars. GABA increased O2●- levels in guard cells and surrounding stomata, enhancing stomatal closure and the hypersensitive response. GABA enhanced the accumulation of soluble phenols and increased the activity of key antioxidant enzymes, catalase (CAT) and peroxidase (POX), which are vital for managing oxidative stress. To the best of our knowledge, this investigation represents the first report into the impact of GABA on wheat leaf rust disease.

Guard cells count the number of unitary cytosolic Ca2+ signals to regulate stomatal dynamics.

Transient stimulus-specific increases in the cytosolic Ca2+ concentration ("calcium signatures") of guard cells have been proposed to regulate the opening and closure of stomatal pores on plant leaves. However, the mechanism by which these Ca2+ signatures are generated and translated into stomatal movement is still largely unresolved. We used a light-gated, Ca2+-permeable variant of ChannelRhodopsin 2 (ChR2-XXM2.0) that was stimulated by tailored light pulses to investigate this phenomenon. We found that activation of the ChR2-XXM2.0 channel provoked characteristic increases in the cytosolic concentration of Ca2+. We also demonstrated that the endoplasmic reticulum (ER) was involved in the generation of these calcium signatures. Using ChR2-XXM2.0 technology, we showed that transient increases in Ca2+ activated S-type anion channels and determined the extent and speed of stomatal closure with their number and frequency. Our data reveal that guard cells are capable of counting Ca2+ transients in order to optimize stomatal aperture in the prevailing environmental conditions.

Warming triggers stomatal opening by enhancement ofphotosynthesis and ensuing guard cell CO2 sensing,whereashigher temperatures induce a photosynthesis-uncoupled response.

Plants integrate environmental stimuli to optimize photosynthesis vs water loss by controlling stomatal apertures. However, stomatal responses to temperature elevation and the underlying molecular genetic mechanisms remain less studied. We developed an approach for clamping leaf-to-air vapor pressure difference (VPDleaf) to fixed values, and recorded robust reversible warming-induced stomatal opening in intact plants. We analyzed stomatal temperature responses of mutants impaired in guard cell signaling pathways for blue light, abscisic acid (ABA), CO2, and the temperature-sensitive proteins, Phytochrome B (phyB) and EARLY-FLOWERING-3 (ELF3). We confirmed that phot1-5/phot2-1 leaves lacking blue-light photoreceptors showed partially reduced warming-induced stomatal opening. Furthermore, ABA-biosynthesis, phyB, and ELF3 were not essential for the stomatal warming response. Strikingly, Arabidopsis (dicot) and Brachypodium distachyon (monocot) mutants lacking guard cell CO2 sensors and signaling mechanisms, including ht1, mpk12/mpk4-gc, and cbc1/cbc2 abolished the stomatal warming response, suggesting a conserved mechanism across diverse plant lineages. Moreover, warming rapidly stimulated photosynthesis, resulting in a reduction in intercellular (CO2). Interestingly, further enhancing heat stress caused stomatal opening uncoupled from photosynthesis. We provide genetic and physiological evidence that the stomatal warming response is triggered by increased CO2 assimilation and stomatal CO2 sensing. Additionally, increasing heat stress functions via a distinct photosynthesis-uncoupled stomatal openingpathway.

Molecular characterization of a phytomelatonin receptor and its overexpression as a ‘one-stop’ solution to nullify the toxic effects of hazardous inorganic agro-pollutants

Inorganic toxicants like arsenic, copper, lead, nickel and fluoride are notorious agro-pollutants, impeding plant-productivity due to high bioaccumulation. Consumption of such contaminated plant-parts causes irreversible health hazards. We identified a G-protein coupled receptor, serving as melatonin receptor (MelR) in Nicotiana tabacum (NtMelR), that relayed downstream-signaling after binding melatonin, a potent growth regulator and antioxidant. Using inhibitors against G-protein-α and NADPH oxidase (NOX), and by supplementing epidermal strips with exogenous melatonin and H2O2, we established that NtMelR acted upstream of reactive oxygen species (ROS) production in guard cells. Transgenic lines of N. benthamiana overexpressing NtMelR maintained constitutive melatonin-signaling via MelR, leading to efficient stomatal closure for preventing desiccation during oxidative stress. Melatonin biosynthesis was stimulated in the transgenic lines, exposed to different agro-pollutant stress, providing a steady-abundance of ligand for NtMelR binding and activating the defence machinery, comprising of enzymatic-antioxidants like superoxide dismutase, catalase, peroxidases and glyoxalases. Due to increased antioxidant capacity, the transgenics exhibited less molecular injuries (electrolyte leakage, methylglyoxal accumulation and NOX activity), generated less ROS and bioaccumulated significantly lower levels of toxicants. Unlike the wild-type counterparts, the transgenics maintained high relative water content, photosynthetic efficiency, could flower abundantly and even produce seeds. Overall, we established that overexpression of NtMelR is a single-window strategy to generate multiple-stress tolerant genotypes.

Dual role of BdMUTE during stomatal development in the model grass Brachypodium distachyon.

Grasses form morphologically derived, four-celled stomata, where two dumbbell-shaped guard cells (GCs) are flanked by two lateral subsidiary cells (SCs). This innovative form enables rapid opening and closing kinetics and efficient plant-atmosphere gas exchange. The mobile bHLH transcription factor MUTE is required for SC formation in grasses. Yet whether and how MUTE also regulates GC development and whether MUTE mobility is required for SC recruitment is unclear. Here, we transgenically impaired BdMUTE mobility from GC to SC precursors in the emerging model grass Brachypodium distachyon. Our data indicate that reduced BdMUTE mobility severely affected the spatiotemporal coordination of GC and SC development. Furthermore, although BdMUTE has a cell-autonomous role in GC division orientation, complete dumbbell morphogenesis of GCs required SC recruitment. Finally, leaf-level gas exchange measurements showed that dosage-dependent complementation of the four-celled grass morphology was mirrored in a gradual physiological complementation of stomatal kinetics. Together, our work revealed a dual role of grass MUTE in regulating GC division orientation and SC recruitment, which in turn is required for GC morphogenesis and the rapid kinetics of grass stomata.

Guard cell K+ channels of Kalanchoë follow the diel cycle of crassulacean acid metabolism.

The activity of outward-rectifying but not inward-rectifying K + channels of Kalanchoë stomata follows the diel cycle of crassulacean acid metabolism.