Guard Cells Integrate Light and Temperature Signals to Control Stomatal Aperture

Barl L., Debastiani Benato B., Genze N., Grimm D.G., Gigl M., Dawid C., Schön C., Avramova V.

Abstract Stomata play a crucial role in balancing carbon dioxide uptake and water vapor loss, thereby regulating plant water use efficiency (WUE). Enhancing WUE is important for sustainable agriculture and food security, particularly for crops such as maize (Zea mays L.), as climate change and growing global food demand exacerbate limitations on water availability. Genetic factors controlling stomatal density and levels of the plant hormone abscisic acid (ABA) in leaves, which affect stomatal aperture, are key determinants of stomatal conductance (gs) and intrinsic WUE (iWUE). In this study, we demonstrate that stomatal density and stomatal aperture have a combined effect on gs and iWUE in maize. Using near-isogenic lines (NILs) and CRISPR/Cas9 mutants, we show that combining reduced stomatal density and reduced stomatal aperture can improve iWUE without compromising photosynthesis. This effect is pronounced at both, optimal and high temperatures. These findings highlight the potential of targeting multiple stomatal traits through genetic stacking to enhance WUE, offering a promising strategy for crop adaptation to water-limited environments.

Zhou J., Hao D., Gu Z.

Increasing the stomatal aperture is a crucial strategy for enhancing the rate of CO2 absorption, which ultimately contributes to increased plant yield through improved photosynthetic activity. The successful implementation of this strategy depends on the rapid identification of positive regulatory environmental stimuli that promote stomatal opening. However, current research on stomatal opening regulation has predominantly focused on Arabidopsis and other crops, with comparatively less attention given to leafy vegetables. In this study, Chinese cabbage was selected as the experimental material. A suitable method for isolating stomata from Chinese cabbage was developed by comparing the advantages and disadvantages of several commonly used stomatal isolation techniques. Subsequently, an effective method for observing stomatal aperture was established through an investigation of the time and concentration dependence on potassium-containing solutions. Utilizing this observation method, the stomatal aperture response to twelve environmental stimuli was examined to facilitate the rapid screening of a formula to enhance stomatal opening. The stomatal aperture observation protocol involved incubating the abaxial epidermis, obtained via the epidermal peeling method, in an opening solution containing 0.5% KCl (pH 6.0) under light for 5 h. The results indicated that stomatal opening is concentration dependent on external environmental stimuli. The exogenous application of 100 µM Ca2+ (33.5%), 50 µM brassinosteroid (43.5%), and 10 µM cytokinin (43.4%) resulted in an increase in stomatal aperture of over 30%. This research provides a foundation for manipulating the stomatal opening of Chinese cabbage to enhance production.

Liu X., Wu T., Jiang Q., Ao X., Zhu L., Qiao R.

Can H., Dogan I., Uras M.E., Tabanli F., Hocaoglu-Ozyigit A., Ozyigit I.I.

Li L., Li Q., Pan A., Chen F., He Z., Li Y., Bai Z., Zhang R.

Yu H., Xiao H., Abou‐Elwafa S.F., Qiao Y., Chen L., Alshehri M.A., Wu Y., Jiang W., Tan W.

AbstractReactive oxygen species (ROS) serve as crucial signaling molecules in plants, enabling rapid responses to environmental stresses such as abiotic factors. ROS production primarily stems from the activation of enzymes such as nicotinamide adenine dinucleotide phosphate (NADPH) oxidases and peroxidases, as well as disruptions in the respiratory and photosynthetic electron transport chains. This oxidative stress triggers signaling pathways that involve in calcium ion (Ca2+) influx across cell membranes, altering ionic conductance. ROS encompass hydroxyl radicals (OH•) and hydrogen peroxide (H2O2), which activate hyperpolarization‐activated Ca2+ channels and influence ion transport dynamics. Our review focuses on the mechanisms driving ROS generation and ion transport during plant responses to abiotic stress. We explore the regulation, characteristics, and potential structures of ROS‐activated ion channels in plants. Specifically, we examine the molecular responses and evolutionary adaptations of Shaker‐type K+ channels (AKT/KAT/GORK/SKOR) under stress conditions. Comparative genetic analyses highlight the conservation of these channels and other ROS‐regulated proteins (e.g., MDHAR, POX, and RBOH), suggesting their essential roles in plant to adapt to diverse stresses. This study underscores the significance of ROS‐regulated proteins in plant stress responses, advocating for further research to elucidate their fundamental roles.

Zhao Q., Lv Q., Fu X., Wu Q., Liu Y., Xue L., Zhao X.

Plant stems grow towards the incident light in response to unilateral blue light to optimize photosynthesis. However, our findings reveal that unilateral high-intensity blue light (HBL) triggers backlit lodging in etiolated cotton (Gossypium hirsutum) hypocotyls when they are pulled approximately 1.5 cm from the soil. Phenotypic analysis indicated that stomata on the lit side were open, while those on the shaded side were closed under unilateral HBL. To investigate the relationship between stomatal movement and backlit lodging, we applied abscisic acid (ABA), hydrogen peroxide (H2O2), and lanthanum chloride (LaCl3) to the lit side, and cytokinins (6-BA) and ascorbic acid (ASA) to the shaded side. Results showed that all these treatments inhibited the backlit lodging phenomenon, specifically, ABA, H2O2, and LaCl3 reduced stomatal opening on the lit side, while 6-BA and ASA enhanced stomatal opening on the shade side. These results demonstrate that HBL-induced asymmetrical stomatal opening on the lit and shade side of hypocotyl supports the backlit lodging phenomenon. Notably, maize (Zea mays), which lack stomata in the hypocotyl did not exhibit HBL-induced backlit lodging, whereas soybean (Glycine max), which has stomata in its etiolated hypocotyl, displayed a similar phenotype to that of cotton. Additionally, while both red light and low-intensity blue light (LBL) can induce stomatal opening, they do not trigger the backlit lodging phenomenon. These findings suggest that backlit lodging is a unique HBL-dependent response, but the mechanism need further investigation.

Razzaq K., Du J.

Plants have developed adaptive mechanisms to maximize their productivity and fitness under varying light conditions. They react to diverse environmental cues and make adjustments to regulate their growth and developmental patterns. Light is one of the crucial environmental factors that directly affects plant development. Light signals are critical for seed germination, photomorphogenesis, and flowering; they assist in light collection through architectural modifications during phototropism and shade avoidance. Photoreceptors detect light signals, activating downstream signaling pathways. Phytohormone-mediated signaling integrates physiological responses to light perception while regulating photoreceptors and their primary interactions. Hormone levels and signaling fluctuations are often employed to trigger growth and development to adjust appropriately in response to changes in light. Phytohormones are critical for a variety of developmental processes, from organ formation to senescence, because they are growth and developmental regulators. Phytohormones such as auxin, cytokinin, abscisic acid, brassinosteroid, ethylene, gibberellin, jasmonic acid, strigolactone, and karrikin regulate and coordinate developmental processes in response to fluctuations in light conditions. Furthermore, the circadian clock regulates plant development by combining light signals with several hormone pathways. This review aims to provide a comprehensive overview of light-regulated processes in plants throughout their life cycle, including the way light signals influence phytohormonal regulation. We provide insight into the mechanisms that allow plants to flourish in environments of variable light by looking into well-established connections between perception of light and hormone fluctuations.

Xu X., Liu H., Praat M., Pizzio G.A., Jiang Z., Driever S.M., Wang R., Van De Cotte B., Villers S.L., Gevaert K., Leonhardt N., Nelissen H., Kinoshita T., Vanneste S., Rodriguez P.L., et. al.

Plants continuously respond to changing environmental conditions to prevent damage and maintain optimal performance. To regulate gas exchange with the environment and to control abiotic stress relief, plants have pores in their leaf epidermis, called stomata. Multiple environmental signals affect the opening and closing of these stomata. High temperatures promote stomatal opening (to cool down), and drought induces stomatal closing (to prevent water loss). Coinciding stress conditions may evoke conflicting stomatal responses, but the cellular mechanisms to resolve these conflicts are unknown. Here we demonstrate that the high-temperature-associated kinase TARGET OF TEMPERATURE 3 directly controls the activity of plasma membrane H+-ATPases to induce stomatal opening. OPEN STOMATA 1, which regulates stomatal closure to prevent water loss during drought stress, directly inactivates TARGET OF TEMPERATURE 3 through phosphorylation. Taken together, this signalling axis harmonizes stomatal opening and closing under high temperatures and/or drought. In the context of global climate change, understanding how different stress signals converge on stomatal regulation allows the development of climate-change-ready crops. Stomata regulate gas exchange and help plants cope with abiotic stress. The authors identify a signalling pathway that coordinates the balance between stomatal opening and closing under high-temperature and/or drought conditions.

Naawe E.K., Yavuz C., Demirel U., Çaliskan M.E.

Heat stress represents a significant consequence of global climate dynamics, emerging as a primary focus in cultivar breeding objectives and sustainability efforts in many crops, including potato. This requires the need to evaluate the response of potato genotypes to heat stress under field conditions. This study evaluated potato genotypes’ physiological and biochemical responses to elevated temperatures. The experiments were conducted under field conditions in the 2022 and 2023 growing seasons in a split-plot design. Two temperature treatments were applied: a control treatment in which the plants received the field temperature of the experimental station and a heat treatment in which the plants received a temperature of + 6.0–10.0 °C depending on the time of the day. The results showed that elevated temperature significantly (P < 0.05) increased the leaf canopy temperature (CT), leaf area index (LAI), leaf chlorophyll content (SPAD), malondialdehyde (MDA), and proline while inducing a significant reduction in hydrogen peroxide (H2O2). The photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate differed significantly (P < 0.001) between treatments in both seasons. Pn and Gs increased significantly with temperatures up to 37.0–39.0 °C, while further temperature increases resulted in heat shock and burning of potato leaves. Heat stress enhanced the activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbic peroxidase (APX) while decreasing the peroxidase (POD). The results suggest that heat-tolerant potato genotypes may possess an efficient defence mechanism against reactive oxygen species (ROS) by enhancing the antioxidant enzyme activity under heat stress and may be used as physiological and biochemical markers in screening heat-tolerance potato varieties.

Wang Z., Perez V., Hua J.

ABSTRACTPhytochrome 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.

Pankasem N., Hsu P., Lopez B.N., Franks P.J., Schroeder J.I.

Summary 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 opening pathway.

Gustavsson M., Hill L., Franklin K.A., Pridgeon A.J.

AbstractObtaining sufficient light for photosynthesis and avoiding desiccation are two key challenges faced by seedlings during early establishment. Perception of light quality via specialised photoreceptors signals the availability of sunlight for photosynthesis. Canopy shade is depleted in red (R) and enriched in far-red (FR) light, lowering R:FR ratio, while direct sunlight and sunflecks contain UV-B. The balance of these wavelengths can determine the developmental strategy adopted by seedlings to either avoid shade, via stem elongation, or promote the expansion of photosynthetic organs. How seedlings regulate stomatal movements in different light environments is poorly understood. Using FR and UV-B supplementation to mimic aspects of canopy shade and sunlight respectively, we monitored stomatal apertures inArabidopsis thalianacotyledons. We show that low R:FR inhibits stomatal opening via a mechanism involving PHYTOCHROME INTERACTING FACTOR 4 (PIF4) and increased abscisic acid (ABA). In contrast, UV-B perceived by the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor antagonises this response to promote stomatal opening in a response requiring phototropin photoreceptors. The convergence of phytochrome and UVR8 signalling to control ABA abundance enables plants to coordinate stem elongation and water use during seedling establishment in dynamic light environments.

Jansen M.J., Jansma S.Y., Kuipers K.M., Vriezen W.H., Millenaar F.F., Montoro T., de Kovel C.G., van Eeuwijk F.A., Visser E.J., Rieu I.

AbstractEarly pollen development is a bottleneck for plant fertility in heatwave conditions, thus affecting yield stability. Mechanisms that protect this process and explain variation in tolerance level between genotypes are poorly understood. Here we show that sepal transpiration in young, still closed, flower buds reduces the impact of heat on developing tomato pollen and that this mechanism is enhanced by the major tomato pollen thermotolerance QTL, qPV11. By direct measurement of the flower bud core temperature and transpiration we show this process, which we term ‘flower bud cooling’, depends on heat-induced opening of sepal stomata and that the transpiration enhancing effect of qPV11 requires functional stomatal regulation and is specific to the sepals. Large-scale evaluation of populations in both a production field and greenhouse showed that qPV11 improves pollen viability and fruit set in heatwave-affected complex cultivation environments. These findings highlight enhanced flower bud cooling as a naturally evolved protection mechanism against heatwaves and qPV11 as genetic component in the differential regulation of transpiration between reproductive and vegetative tissues and candidate variant for the breeding of climate-resilient tomato cultivars.

Guo C., Tao R., Zhu M., Zhou M., Zhao C.

AbstractHigh sensitivity and rapid closure of wheat stomata to environmental stimuli make it difficult and inconvenient when investigating stomatal physiology and morphology using epidermal peels. This is due to inevitable mechanical stress to stomatal guard cells when separating epidermis from mesophyll cells, which induced a vast majority of stomatal closure in wheat. Stomata are more open and active in detached leaves than in epidermal peels. Based on these observations, we proposed a simple method, which promotes stomatal opening using detached leaves rather than epidermis for physiological observations. Stomatal response to stimuli was significantly increased when using intact leaf segment. The method was used to investigate stomatal behaviours of two wheat genotypes with contrasting salt tolerance to salinity stress. The effects of salt stress and exogenous abscisic acid (ABA) treatment on stomatal behaviours were also assessed. The salt-tolerant genotype, H-135, demonstrated a greater stomatal closure rate than the salt-sensitive genotype, H-093, in response to exogenous ABA under salt stress, highlighting the potential of stomatal responsiveness as an indicator for breeding salt-resistant crops. This method not only facilitates the effective initiation of stomatal opening but also ensures the continued responsiveness of stomata to subsequent treatments in wheat.