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Calcium-dependent protein kinases (CDPKs), which are activated by transient changes in the Ca2+ concentration in plants, are important for various biological processes, such as growth, development, defense against biotic and abiotic stresses, and others. Mannitol is commonly used as an osmotic regulatory substance in culture medium or nutrient solutions to create water-deficit conditions. Here, we cloned the potato (Solanum tuberosum L.) StCDPK13 gene and generated stable transgenic StCDPK13-overexpression potato plants. To investigate the potential functions of StCDPK13 in response to drought stress, overexpression-transgenic (OE1, OE2, and OE7) and wild-type (WT) potato seedlings were cultured on MS solid media without or with mannitol, representing the control or drought stress, for 20 days; the elevated mannitol concentrations (150 and 200 mM) were the drought stress conditions. The StCDPK13 gene was consistently expressed in different tissues and was induced by drought stress in both OE and WT plants. The phenotypic traits and an analysis of physiological indicators revealed that the transgenic plants exhibited more tolerance to drought stress than the WT plants. The overexpression lines showed an increased plant height, number of leaves, dry shoot weight, root length, root number, root volume, number of root tips, fresh root weight, and dry root weight under drought stress. In addition, the activities of antioxidant enzymes (CAT, SOD, and POD) and the accumulation of proline and neutral sugars were significantly increased, whereas the levels of malondialdehyde (MDA) and reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and O2•−, were significantly reduced in the OE lines compared to WT plants under drought stress. Moreover, the stomatal aperture of the leaves and the water loss rate in the leaves of the OE lines were significantly reduced under drought stress compared to the WT plants. In addition, the overexpression of StCDPK13 upregulated the expression levels of stress-related genes under drought stress. Collectively, these results indicate that the StCDPK13 gene plays a positive role in drought tolerance by reducing the stomatal aperture, promoting ROS scavenging, and alleviating oxidative damage under drought stress in potatoes. [ABSTRACT FROM AUTHOR]

Eremopyrum triticeum is a typical spring ephemeral species, which in China mainly distributed in the desert regions of northern Xinjiang, and play an important role in the desert ecosystems. E. triticeum has several adaptive characteristics such as short growth rhythms, high photosynthetic efficiency, high seed production, drought and salt resistance. However, the molecular regulatory mechanism of E. triticeum in responses to abiotic stress resistance is still unknown. In this study, two NAC-like transcription factor-encoding genes, EtNAC1 and EtNAC2, were isolated from E. triticeum. The predicted EtNAC1 and EtNAC2 proteins possess a typical NAC DNA-binding domain at the N-terminal region. The qRT-PCR analysis showed that EtNAC1 and EtNAC2 were highly expressed in mature roots of E. triticeum, and were significantly up-regulated under drought, high salt and abscisic acid (ABA) stresses. Subcellular localization analysis in onion epidermal cells revealed that EtNAC1 and EtNAC2 were located in the nucleus. Expression of EtNAC1 and EtNAC2 in yeast cells improved the survival rate of yeast under low temperature, H2O2, high drought and salt stresses. Overexpression of EtNAC1 and EtNAC2 in Arabidopsis thaliana conferred enhanced tolerance to drought and salt stresses, increased ABA sensitivity, and transgenic plants showed higher proline (Pro) content, but lower malondialdehyde content, lower chlorophyll leaching, lower water loss rate and stomatal aperture (width/length) than WT plants. In conclusion, EtNAC1 and EtNAC2 play important roles in abiotic stress responses of E. triticeum, which might have significant potential in crop molecular breeding for abiotic stress tolerance. [ABSTRACT FROM AUTHOR]

Drought stress is a primary abiotic stress that causes significant losses to forestry and agricultural production. Therefore, exploring drought-responsive genes and their regulatory mechanism is crucial for plant molecular breeding for forestry and agriculture production safety. Small auxin-up RNA (SAUR) proteins are essential in plant growth and development but show functional diversity in stress response. In this study, the transcriptome sequencing data of Ammopiptanthus nanus seedlings revealed that the expression of AnSAUR50 was continuously downregulated under drought stress. Hence, the AnSAUR50 gene was cloned and functionally analyzed in drought response. The results showed that the coding sequence of AnSAUR50 was 315 bp in length and encoded 104 amino acids. The AnSAUR50 protein showed high conservation, possessed a SAUR-specific domain, and localized in the nucleus and cell membrane. The heterologous expression of the AnSAUR50 gene enhanced the drought sensitivity of the transgenic Arabidopsis with a lower survival rate, biomass, and higher malondialdehyde content and relative electrolyte leakage. Moreover, transgenic plants showed shorter root lengths and bigger stomatal apertures, resulting in facilitating water loss under drought stress. The study indicates that AnSAUR50 negatively regulates drought tolerance by inhibiting root growth and stomatal closure, which provides insights into the underlying function and regulatory mechanism of SAURs in plant stress response. [ABSTRACT FROM AUTHOR]

Main conclusion: TaMYB44-5A identified as a transcription factor negatively regulates drought tolerance in transgenic Arabidopsis. Drought can severely reduce yields throughout the wheat-growing season. Many studies have shown that R2R3-MYB transcription factors are involved in drought stress responses. In this study, the R2R3-MYB transcription factor MYB44-5A was identified in wheat (Triticum aestivum L.) and functionally analyzed. Three homologs of TaMYB44 were isolated, all of which localized to the nucleus. Overexpression of TaMYB44-5A reduced drought tolerance in Arabidopsis thaliana. Further analysis showed that TaMYB44-5A reduced the sensitivity of transgenic Arabidopsis to ABA. Genetic and transcriptional regulation analyses demonstrated that the expression levels of drought- and ABA-responsive genes were downregulated by TaMYB44-5A, and TaMYB44-5A directly bound to the MYB-binding site on the promoter to repress the transcription level of TaRD22-3A. Our results provide insights into a novel molecular pathway in which the R2R3-MYB transcription factor negatively regulates ABA signaling in response to drought stress. [ABSTRACT FROM AUTHOR]

Stomata play a pivotal role in regulating gas exchange between plants and the atmosphere controlling water and carbon cycles. Accordingly, we investigated the impact of ultraviolet‐B radiation, a neglected environmental factor varying with ongoing global change, on stomatal morphology and function by a Comprehensive Meta‐Analysis. The overall UV effect at the leaf level is to decrease stomatal conductance, stomatal aperture and stomatal size, although stomatal density was increased. The significant decline in stomatal conductance is marked (6% in trees and >10% in grasses and herbs) in short‐term experiments, with more modest decreases noted in long‐term UV studies. Short‐term experiments in growth chambers are not representative of long‐term field UV effects on stomatal conductance. Important consequences of altered stomatal function are hypothesized. In the short term, UV‐mediated stomatal closure may reduce carbon uptake but also water loss through transpiration, thereby alleviating deleterious effects of drought. However, in the long term, complex changes in stomatal aperture, size, and density may reduce the carbon sequestration capacity of plants and increase vegetation and land surface temperatures, potentially exacerbating negative effects of drought and/or heatwaves. Therefore, the expected future strength of carbon sink capacity in high‐UV regions is likely overestimated. Summary statement: Ultraviolet‐B radiation reduces stomatal conductance, aperture and size while increasing density. Short‐term effects are stronger than long ones, affecting grasses more than trees. Reduced conductance may lessen drought stress, but raise leaf warming, worsening heatwave damage and reducing carbon uptake. [ABSTRACT FROM AUTHOR]

High temperatures negatively impact the yield and quality of fruit crops. Exogenous melatonin (MT) application has been shown to enhance heat tolerance, but the response of endogenous MT to heat stress, particularly in perennial fruit trees, remains unclear. The present study investigated the effects of high temperatures on transgenic apple plants overexpressing the MT biosynthesis gene N‐acetylserotonin methyltransferase 9 (MdASMT9). Endogenous MT protected transgenic plants from heat stress by increasing antioxidant enzyme activity and scavenging reactive oxygen species (ROS), and protecting the chloroplasts from damage. Application of MT and overexpression of MdASMT9 also reduced abscisic acid accumulation through promoting MdWRKY33‐mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus inducing stomatal opening for better heat dissipation. Furthermore, MT‐enhanced autophagic activity through promoting MdWRKY33‐mediated transcriptional enhancement of MdATG18a under heat stress. These findings provide new insights into the regulation of endogenous MT and its role in improving basal thermotolerance in perennial fruit trees. Summary Statement: MdASMT9‐mediated melatonin (MT) biosynthesis in apple plants enhanced autophagic activity and protected plants from heat stress. Furthermore, MT‐promoted MdWRKY33‐mediated transcriptional inhibition of MdNCED1 and MdNCED3, thus promoting stomatal opening for better heat dissipation. [ABSTRACT FROM AUTHOR]

Drought-tolerant plants have become a convenient model to study the mechanisms underlying drought tolerance in order to improve susceptible domesticated relatives. Various studies have shown that local landraces possess superior qualities that help them survive in harsh environmental conditions. One of the key mechanisms that helps with tolerance in crops is timely stomatal regulation. In this study, the physiological, morphological, and molecular stomatal responses in three drought-tolerant landraces (Clm-01–03) and hybrid (Clm-04) watermelons were evaluated under drought stress. The watermelon plants were grown under a water deficit (complete withholding of water) and non-stress conditions. The highest SPAD values were recorded for the Clm-03 and Clm-02 (50 ± 3) watermelon genotypes, and the lowest for Clm-04 (27 ± 0.37), showing this genotype's tolerance and ability to maintain its systems during drought stress. Fluorescence parameters also gave important clues to the tolerant genotypes of Clm-02 and Clm-03 under drought stress, while the domesticated genotype showed a slow response to fluorescence parameters, which could lead to damage to the photosynthesis apparatus. During the drought period, the wild watermelon was found to have a limited stomatal opening as the drought progressed, and on day 9, it had the smallest opening of 23.1 ± 1.2 µm compared to any other genotype; most importantly, upon re-watering, it showed more rapid recovery than any other genotype. This was also expressed by mRNA quantification of stomatal aperture TFs, with an eight-fold increase in Cla004380 TFs recorded for wild watermelon. All of these mechanisms have been attributed to the tolerance mechanisms of the drought-tolerant watermelon genotype. This study provides important insight into the stomatal responses of probable tolerant watermelon accessions and suggests that improving the stomatal aperture of susceptible domesticated species would also improve their tolerance. [ABSTRACT FROM AUTHOR]

Plants face multiple stresses in their natural habitats. WRKY transcription factors (TFs) play an important regulatory role in plant stress signaling, regulating the expression of multiple stress-related genes to improve plant stress resistance. In this study, we analyzed the expression profiles of 25 BnWRKY genes in three stages of ramie growth (the seedling stage, the rapid-growth stage, and the fiber maturity stage) and response to abiotic stress through qRT-PCR. The results indicated that 25 BnWRKY genes play a role in different growth stages of ramie and were induced by salt and drought stress in the root and leaf. We selected BnWRKY49 as a candidate gene for overexpression in Arabidopsis. BnWRKY49 was localized in the nucleus. Overexpression of BnWRKY49 affected root elongation under drought and salt stress at the Arabidopsis seedling stage and exhibited increased tolerance to drought stress. Further research found that BnWRKY49-overexpressing lines showed decreased stomatal size and increased cuticular wax deposition under drought compared with wild type (WT). Antioxidant enzyme activities of SOD, POD, and CAT were higher in the BnWRKY49-overexpressing lines than the WT. These findings suggested that the BnWRKY49 gene played an important role in drought stress tolerance in Arabidopsis and laid the foundation for further research on the functional analysis of the BnWRKYs in ramie. [ABSTRACT FROM AUTHOR]

Drought stress is a primary abiotic stress that causes significant losses to forestry and agricultural production. Therefore, exploring drought-responsive genes and their regulatory mechanism is crucial for plant molecular breeding for forestry and agriculture production safety. Small auxin-up RNA (SAUR) proteins are essential in plant growth and development but show functional diversity in stress response. In this study, the transcriptome sequencing data of Ammopiptanthus nanus seedlings revealed that the expression of AnSAUR50 was continuously downregulated under drought stress. Hence, the AnSAUR50 gene was cloned and functionally analyzed in drought response. The results showed that the coding sequence of AnSAUR50 was 315 bp in length and encoded 104 amino acids. The AnSAUR50 protein showed high conservation, possessed a SAUR-specific domain, and localized in the nucleus and cell membrane. The heterologous expression of the AnSAUR50 gene enhanced the drought sensitivity of the transgenic Arabidopsis with a lower survival rate, biomass, and higher malondialdehyde content and relative electrolyte leakage. Moreover, transgenic plants showed shorter root lengths and bigger stomatal apertures, resulting in facilitating water loss under drought stress. The study indicates that AnSAUR50 negatively regulates drought tolerance by inhibiting root growth and stomatal closure, which provides insights into the underlying function and regulatory mechanism of SAURs in plant stress response.

Reasonable nitrogen fertilizer application is an important strategy to maintain optimal growth of grasslands, thereby enabling them to better fulfil their ecological functions while reducing environmental pollution caused by high nitrogen fertilizer production and application. Optimizing the ammonium (NH4+):nitrate (NO3-) ratio is a common approach for growth promotion in crops and vegetables, but research on this topic in grass plants has not received sufficient attention. Centipedegrass, which is widely used in landscaping and ecological protection, was used as the experimental material. Different NH4+:NO3- ratios (0: 100, 25:75, 50:50, 75:25, 100:0) were used as the experimental treatments under hydroponic conditions. By monitoring the physiological and morphological changes under each treatment, the appropriate NH4+:NO3- ratio for growth and its underlying mechanism were determined. As the proportion of ammonium increased, the growth showed a “bell-shaped” response, with the maximum biomass and total carbon and nitrogen accumulation achieved with the NH4+:NO3- ratio of 50:50 treatment. Compared with the situation where nitrate was supplied alone, increasing the ammonium proportion increased the whole plant biomass by 93.2%, 139.7%, 59.0%, and 30.5%, the whole plant nitrogen accumulation by 44.9%, 94.6%, 32.8%, and 54.8%, and the whole plant carbon accumulation by 90.4%, 139.9%, 58.7%, and 26.6% in order. As a gateway for nitrogen input, the roots treated with an NH4+:NO3- ratio of 50:50 exhibited the highest ammonium and nitrate uptake rate, which may be related to the maximum total root length, root surface area, average root diameter, root volume, and largest root xylem vessel. As a gateway for carbon input, leaves treated with an NH4+:NO3- ratio of 50:50 exhibited the highest stomatal aperture, stomatal conductance, photosynthetic rate, transpiration rate, and photosynthetic products. The NH4+:NO3- ratio of 50:50 treatment had the largest stem xylem vessel area. This structure and force caused by transpiration may synergistically facilitate root-to-shoot nutrient translocation. Notably, the change in stomatal opening occurred in the early stage (4 hours) of the NH4+:NO3- ratio treatments, indicating that stomates are structures that are involved in the response to changes in the root NH4+:NO3- ratio. In summary, we recommend 50:50 as the appropriate NH4+:NO3- ratio for the growth of centipedegrass, which not only improves the nitrogen use efficiency but also enhances the carbon sequestration capacity. [ABSTRACT FROM AUTHOR]

Deciphering drought resistance in crops is crucial for enhancing water productivity. Previous studies have highlighted the significant role of the transcription factor SlHB8 in regulating developmental processes in tomato plants but its involvement in drought resistance remains unclear. Here, gene overexpression (SlHB8-OE) and gene knockout (slhb8) tomato plants were utilized to study the role of SlHB8 in regulating drought resistance. Our findings showed that slhb8 plants exhibited a robust resistant phenotype under drought stress conditions. The stomata of slhb8 tomato leaves displayed significant closure, effectively mitigating the adverse effects of drought stress on photosynthetic efficiency. The slhb8 plants exhibited a decrease in oxidative damage and a substantial increase in antioxidant enzyme activity. Moreover, slhb8 effectively alleviated the degree of photoinhibition and chloroplast damage caused by drought stress. SlHB8 regulates the expression of numerous genes related to photosynthesis (such as SlPSAN, SlPSAL, SlPSBP, and SlTIC62) and stress signal transduction (such as SlCIPK25, SlABA4, and SlJA2) in response to drought stress. Additionally, slhb8 plants exhibited enhanced water absorption capacity and upregulated expression of several aquaporin genes including SlPIP1;3, SlPIP2;6, SlTIP3;1, SlNIP1;2, and SlXIP1;1. Collectively, our findings suggest that SlHB8 plays a negative regulatory role in the drought resistance of tomato plants. [ABSTRACT FROM AUTHOR]

During seed germination, the cotton chaperone protein HSP24.7 regulates the release, from the mitochondrial electron transport chain, of reactive oxygen species (ROS), a stimulative signal regulating germination. The function of HSP24.7 during vegetative stages remains largely unknown. Here we propose that suppression of GhHSP24.7 in cotton seedlings increases tolerance to heat and drought stress. Elevation of GhHSP24.7 was found to be positively associated with endogenous levels of ROS. We identified a new client protein of GhHSP24.7, cotton lysine deacetylase (GhHDA14), which is involved in mitochondrial protein modification. Elevated levels of GhHSP24.7 suppressed deacetylase activity in mitochondria, leading to increased acetylation of mitochondrial proteins enriched in the subunit of Ftype ATPase, V-type ATPase, and cytochrome C reductase, ultimately reducing leaf ATP content. Consequently, in combination with altered ROS content, GhHSP24.7 transgenic lines were unable to coordinate stomatal closure under stress. The regulation circuit composed of GhHSP24.7 and GhHDA14 represents a post-translation level mechanism in plant abiotic stress responses that integrates the regulation of ROS and ATP. [ABSTRACT FROM AUTHOR]

Climate change‐induced variations in temperature and precipitation negatively impact plant growth and development. To ensure future food quality and availability, a critical need exists to identify morphological and physiological responses that confer drought tolerance in agro‐economically important crop plants throughout all growth stages. In this study, two Sorghum bicolor accessions that differ in their pre‐flowering responses to drought were exposed to repeated cycles of drying and rewatering. Morphological, physiological, and histological traits were measured across both juvenile and adult developmental stages. Our results demonstrate that plant height is not predictive of metaxylem area but does influence the hydraulic path and water management in an accession‐specific manner. Further, when drought‐responsive changes to the plant architecture are unable to compensate for the hydraulic risk associated with prolonged drought exposure, tight control of stomatal aperture is crucial to further mitigate hydraulic damage and prevent xylem embolism. [ABSTRACT FROM AUTHOR]

Abstract Climate change‐induced variations in temperature and precipitation negatively impact plant growth and development. To ensure future food quality and availability, a critical need exists to identify morphological and physiological responses that confer drought tolerance in agro‐economically important crop plants throughout all growth stages. In this study, two Sorghum bicolor accessions that differ in their pre‐flowering responses to drought were exposed to repeated cycles of drying and rewatering. Morphological, physiological, and histological traits were measured across both juvenile and adult developmental stages. Our results demonstrate that plant height is not predictive of metaxylem area but does influence the hydraulic path and water management in an accession‐specific manner. Further, when drought‐responsive changes to the plant architecture are unable to compensate for the hydraulic risk associated with prolonged drought exposure, tight control of stomatal aperture is crucial to further mitigate hydraulic damage and prevent xylem embolism.

Plants face multiple stresses in their natural habitats. WRKY transcription factors (TFs) play an important regulatory role in plant stress signaling, regulating the expression of multiple stress-related genes to improve plant stress resistance. In this study, we analyzed the expression profiles of 25 BnWRKY genes in three stages of ramie growth (the seedling stage, the rapid-growth stage, and the fiber maturity stage) and response to abiotic stress through qRT-PCR. The results indicated that 25 BnWRKY genes play a role in different growth stages of ramie and were induced by salt and drought stress in the root and leaf. We selected BnWRKY49 as a candidate gene for overexpression in Arabidopsis. BnWRKY49 was localized in the nucleus. Overexpression of BnWRKY49 affected root elongation under drought and salt stress at the Arabidopsis seedling stage and exhibited increased tolerance to drought stress. Further research found that BnWRKY49-overexpressing lines showed decreased stomatal size and increased cuticular wax deposition under drought compared with wild type (WT). Antioxidant enzyme activities of SOD, POD, and CAT were higher in the BnWRKY49-overexpressing lines than the WT. These findings suggested that the BnWRKY49 gene played an important role in drought stress tolerance in Arabidopsis and laid the foundation for further research on the functional analysis of the BnWRKYs in ramie.

Drought-tolerant plants have become a convenient model to study the mechanisms underlying drought tolerance in order to improve susceptible domesticated relatives. Various studies have shown that local landraces possess superior qualities that help them survive in harsh environmental conditions. One of the key mechanisms that helps with tolerance in crops is timely stomatal regulation. In this study, the physiological, morphological, and molecular stomatal responses in three drought-tolerant landraces (Clm-01–03) and hybrid (Clm-04) watermelons were evaluated under drought stress. The watermelon plants were grown under a water deficit (complete withholding of water) and non-stress conditions. The highest SPAD values were recorded for the Clm-03 and Clm-02 (50 ± 3) watermelon genotypes, and the lowest for Clm-04 (27 ± 0.37), showing this genotype’s tolerance and ability to maintain its systems during drought stress. Fluorescence parameters also gave important clues to the tolerant genotypes of Clm-02 and Clm-03 under drought stress, while the domesticated genotype showed a slow response to fluorescence parameters, which could lead to damage to the photosynthesis apparatus. During the drought period, the wild watermelon was found to have a limited stomatal opening as the drought progressed, and on day 9, it had the smallest opening of 23.1 ± 1.2 µm compared to any other genotype; most importantly, upon re-watering, it showed more rapid recovery than any other genotype. This was also expressed by mRNA quantification of stomatal aperture TFs, with an eight-fold increase in Cla004380 TFs recorded for wild watermelon. All of these mechanisms have been attributed to the tolerance mechanisms of the drought-tolerant watermelon genotype. This study provides important insight into the stomatal responses of probable tolerant watermelon accessions and suggests that improving the stomatal aperture of susceptible domesticated species would also improve their tolerance.

Plants are frequently subjected to a broad spectrum of abiotic stresses including drought, salinity and extreme temperatures and have evolved both common and stress-specific responses to promote fitness and survival. Understanding the components and mechanisms that underlie both common and stress-specific responses can enable development of crop plants tolerant to different stresses. Here, we report a rice heat stress-tolerant 1 (hst1) mutant with increased heat tolerance. HST1 encodes the DST transcription factor, which also regulates drought and salinity tolerance. Increased heat tolerance of hst1 was associated with suppressed expression of reactive oxygen species (ROS)-scavenging peroxidases and increased ROS levels, which reduced water loss by decreasing stomatal aperture under heat stress. In addition, increased ROS levels enhanced expression of genes encoding heat shock protein (HSPs) including HSP80, HSP74, HSP58 and small HSPs. HSPs promote stabilization of proteins and protein refolding under heat stress and accordingly mutation of HST1 also improved reproductive traits including pollen viability and seed setting under high temperature. These results broaden the negative roles of DST in abiotic stress tolerance and provide important new insights into DST-regulated tolerance to diverse abiotic stresses through both shared and stress-specific mechanisms. [ABSTRACT FROM AUTHOR]

Stomata are microscopic pores on epidermal cells of leaves and stems that regulate water loss and gas exchange between the plant and its environment. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase that is involved in plant growth and development and multiple abiotic stress responses by regulating the stability of various target proteins. However, little is known about how COP1 controls stomatal aperture and leaf temperature under various environmental conditions. Here, we show that COP1 participates in leaf temperature and stomatal closure regulation under normal and stress conditions in Arabidopsis. Leaf temperature of cop1 mutants was relatively lower than that of wild type (WT) under drought, salt, and heat stress and after abscisic acid (ABA), CaCl2, and H2O2 treatments. However, leaf temperature was generally higher in both WT and cop1 mutants after abiotic stress and chemical treatment than that of untreated WT and cop1 mutants. Stomatal aperture was wider in cop1 mutants than that in WT under all conditions tested, although the extent of stomatal closure varied between WT and cop1 mutants. Under dark conditions, leaf temperature was also lower in cop1 mutants than that in WT. Expression of the genes encoding ABA receptors, ABA biosynthesis proteins, positive regulators of stomatal closure and heat tolerance, and ABA‐responsive proteins was lower in cop1 mutants that that in WT. In addition, expression of respiration‐related genes was lower in cop1 mutants that that in WT. Taken together, the data provide evidence that mutations in COP1 lead to wider stomatal aperture and higher leaf temperature under normal and stress conditions, indicating that leaf temperature is highly correlated with stomatal aperture. [ABSTRACT FROM AUTHOR]

Eremopyrum triticeum is a typical spring ephemeral species, which in China mainly distributed in the desert regions of northern Xinjiang, and play an important role in the desert ecosystems. E. triticeum has several adaptive characteristics such as short growth rhythms, high photosynthetic efficiency, high seed production, drought and salt resistance. However, the molecular regulatory mechanism of E. triticeum in responses to abiotic stress resistance is still unknown. In this study, two NAC-like transcription factor-encoding genes, EtNAC1 and EtNAC2, were isolated from E. triticeum. The predicted EtNAC1 and EtNAC2 proteins possess a typical NAC DNA-binding domain at the N-terminal region. The qRT-PCR analysis showed that EtNAC1 and EtNAC2 were highly expressed in mature roots of E. triticeum, and were significantly up-regulated under drought, high salt and abscisic acid (ABA) stresses. Subcellular localization analysis in onion epidermal cells revealed that EtNAC1 and EtNAC2 were located in the nucleus. Expression of EtNAC1 and EtNAC2 in yeast cells improved the survival rate of yeast under low temperature, H 2 O 2 , high drought and salt stresses. Overexpression of EtNAC1 and EtNAC2 in Arabidopsis thaliana conferred enhanced tolerance to drought and salt stresses, increased ABA sensitivity, and transgenic plants showed higher proline (Pro) content, but lower malondialdehyde content, lower chlorophyll leaching, lower water loss rate and stomatal aperture (width/length) than WT plants. In conclusion, EtNAC1 and EtNAC2 play important roles in abiotic stress responses of E. triticeum, which might have significant potential in crop molecular breeding for abiotic stress tolerance., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests. Ethical approval: No Ethical statement was reported., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Stomata play critical roles in gas exchange and immunity to pathogens. While many genes regulating early stomatal development up to the production of young guard cells (GCs) have been identified in Arabidopsis, much less is known about how young GCs develop into mature functional stomata. Here we perform a maturomics study on stomata, with "maturomics" defined as omics analysis of the maturation process of a tissue or organ. We develop an integrative scheme to analyze three public stomata-related single-cell RNA-seq datasets and identify a list of 586 genes that are specifically up-regulated in all three datasets during stomatal maturation and function formation. The list, termed sc_586, is enriched with known regulators of stomatal maturation and functions. To validate the reliability of the dataset, we selected two candidate G2-like transcription factor genes, MYS1 and MYS2, to investigate their roles in stomata. These two genes redundantly regulate the size and hoop rigidity of mature GCs, and the mys1 mys2 double mutants cause mature GCs with severe defects in regulating their stomatal apertures. Taken together, our results provide a valuable list of genes for studying GC maturation and function formation., Competing Interests: Conflict of interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Abstract Stomata are microscopic pores on epidermal cells of leaves and stems that regulate water loss and gas exchange between the plant and its environment. Constitutive photomorphogenic 1 (COP1) is an E3 ubiquitin ligase that is involved in plant growth and development and multiple abiotic stress responses by regulating the stability of various target proteins. However, little is known about how COP1 controls stomatal aperture and leaf temperature under various environmental conditions. Here, we show that COP1 participates in leaf temperature and stomatal closure regulation under normal and stress conditions in Arabidopsis. Leaf temperature of cop1 mutants was relatively lower than that of wild type (WT) under drought, salt, and heat stress and after abscisic acid (ABA), CaCl2, and H2O2 treatments. However, leaf temperature was generally higher in both WT and cop1 mutants after abiotic stress and chemical treatment than that of untreated WT and cop1 mutants. Stomatal aperture was wider in cop1 mutants than that in WT under all conditions tested, although the extent of stomatal closure varied between WT and cop1 mutants. Under dark conditions, leaf temperature was also lower in cop1 mutants than that in WT. Expression of the genes encoding ABA receptors, ABA biosynthesis proteins, positive regulators of stomatal closure and heat tolerance, and ABA‐responsive proteins was lower in cop1 mutants that that in WT. In addition, expression of respiration‐related genes was lower in cop1 mutants that that in WT. Taken together, the data provide evidence that mutations in COP1 lead to wider stomatal aperture and higher leaf temperature under normal and stress conditions, indicating that leaf temperature is highly correlated with stomatal aperture.

Cold stress limits plant growth and development; however, the precise mechanisms underpinning plant acclimation to cold stress remain largely unknown. In this study, the Ser/Thr protein kinase SOS2-LIKE PROTEIN KINASE5 (PKS5) was shown to play a positive role in plant responses to cold stress. A PKS5 loss-of-function mutant (pks5-1) exhibited elevated sensitivity to cold stress, as well as a lower survival rate and increased ion leakage. Conversely, PKS5 gain-of-function mutants (pks5-3, pks5-4) were more tolerant to cold stress and exhibited higher survival rates and decreased ion leakage. Stomatal aperture analysis revealed that stomatal closure was slower during the first 25 min after cold exposure in pks5-1 compared to wild-type, whereas pks5-3 and pks5-4 displayed accelerated stomatal closure over the same time period. Further stomatal aperture analysis under an abscisic acid (ABA) treatment showed slower closure in pks5-1 and more rapid closure in pks5-3 and pks5-4. Finally, expression levels of cold-responsive genes were regulated by PKS5 under cold stress conditions, while cold stress and ABA treatment can regulate PKS5 expression. Taken together, these results suggest that PKS5 plays a positive role in short-term plant acclimation to cold stress by regulating stomatal aperture, possibly via ABA pathways, and in long-term acclimation by regulating cold-responsive genes. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the nitrogen (N) and phosphorus (P) fertilization of continuous addition effects plant biomass, the physiological properties of leaves and the antioxi-dant enzyme activities of alfalfa (Medicago sativa L) in the northern Xinjiang region; including the no fertilization (CK), nitrogen fertilization (N, 120 kg·ha−1), phosphorus fertilization (with low amount of N) (P, 100 kg·ha−1 P and 23.5 kg·ha−1 N) and combined nitrogen and phosphorus fertilization (NP, 120 kg·ha−1 N and 100 kg·ha−1 P) on the K well supplied soil. After three consecutive years of the supply of N and P fertilization, samples were taken at the first flowering of alfalfa (four clippings in the total year) to determine its pigment concentration, stomatal aperture, antioxidant enzyme activity and hay yield. The results showed that NP fertilization promoted growth with a higher number of branches and hay yield of alfalfa, while N or P fertilization alone had a positive effect on the growth of alfalfa. However, P fertilization significantly increased the carotenoid (Car) content at the early flowering stage of alfalfa leaves (during four clippings) (p < 0.05), In addition, NP ferti-lization enhanced stomatal aperture, increased the antioxidant enzyme activity and decreased the oxidized substance at the early flowering stage of alfalfa leaves. The results showed that a N and P balance rather than an absolute amount of either enhanced the growth of alfalfa, and N or P fertili-zation affects physiological traits differently. We propose that NP fertilization increases the nutri-tional characteristics and physiological characteristics, enhancing the adaptive capacity of alfalfa and making it better adapted to external environmental changes. [ABSTRACT FROM AUTHOR]

The Arabidopsis thaliana aluminum-activated malate transporter 9 (AtALMT9) functions as a vacuolar chloride channel that regulates the stomatal aperture. Here, we present the cryoelectron microscopy (cryo-EM) structures of AtALMT9 in three distinct states. AtALMT9 forms a dimer, and the pore is lined with four positively charged rings. The apo-AtALMT9 state shows a putative endogenous citrate obstructing the pore, where two W120 constriction residues enclose a gate with a pore radius of approximately 1.8 Å, representing an open state. Interestingly, channel closure is solely controlled by W120. Compared to wild-type plants, the W120A mutant exhibits more sensitivity to drought stress and is unable to restore the visual phenotype on leaves upon water recovery, reflecting persistent stomatal opening. Furthermore, notable variations are noted in channel gating and substrate recognition of Glycine max ALMT12, AtALMT9, and AtALMT1. In summary, our investigation enhances comprehension of the interplay between structure and function within the ALMT family. [Display omitted] • Structures of AtALMT9 in the putative citrate-blocked open state and closed state • W120 and R143 are indispensable for the binding of citrate and malate, respectively • Channel gating is regulated by conformational changes occurring at W120 Qian et al. present the cryo-EM structures of the Arabidopsis vacuolar chloride channel ALMT9, offering insights into the molecular mechanisms underlying channel gating and citrate-mediated inhibition of AtALMT9. This study enhances our understanding of the intricate relationship between structure and function within the ALMT family. [ABSTRACT FROM AUTHOR]

The spectral composition of some light-emitting diodes (LEDs) reportedly results in higher crop yield, prevents wilting, and reduces thermal damage to plants. The use of LEDs for postharvest storage and shelf-life extension has been limited, but the potential of this technology will allow for greater applications in horticulture and the food industry. In this experiment, 'Winterbor' kale (Brassica oleracea) and 'Melody' spinach (Spinacia oleracea) plants were measured for the light compensation point and stomatal response under 14 different wavelengths of light ranging from 405 to 661 nm. Data collected from these measurements were used to select two different wavelengths of LEDs and determine the proper irradiance levels for an LED irradiance storage test on spinach and kale. Treatments comprising blue, red, and amber lights were effective at increasing the stomatal opening, while the green light resulted in reduced stomatal opening. For spinach, the light response curve showed that light compensation points at 500 nm and 560 nm were 65.3 and 64.7 μmol m−2 s−1, respectively. For kale, the light compensation points at 500 nm and 560 nm were 50.8 and 44.1 μmol m−2 s−1, respectively. For the storage test experiment at room temperature, kale and spinach were stored under four different treatments: dark treatment (control), standard white fluorescent light, 500 nm, and 560 nm LED wavelengths. For spinach, the moisture content was 70.1% at 560 nm and 53.7% for dark, moisture losses of 41.5% under the 560-nm treatment and 52.0% for the dark treatment. The fresh basis moisture content was 74.6% at 560 nm and 59.3% in the dark. Moisture loss under the 560 nm treatment was 39.6% while the dark treatment had a 54.0% moisture loss. A visual assessment scale was monitored, 560 nm resulted in the top visual quality for kale compared to the other treatments with the lowest visual quality under the dark treatment at day 4. For spinach, the visual quality for 560 nm treatment was statistically the standard white fluorescent light and 500 nm, with poor-quality product occurring by day 4 and the lowest-quality product occurring at day 5. The LED treatments improved the shelf life of spinach and kale, likely as a result of stomatal aperture closure, photosynthetic rate near the light compensation point and stability of the atmospheric moisture content. This study provides valuable information on the extension of the shelf life of leafy greens during storage. Reducing fresh produce waste in grocery stores will increase revenue, thereby benefiting the Canadian economy while providing social and environmental benefits that entail increased food security and reduced food waste. [Display omitted] • Light compensation points were higher for green LEDs (500-nm to 560-nm). • Blue, red, and amber LEDs increased stomatal opening. • Green (500-nm to 560-nm) LEDs resulted in reduced stomatal opening. • Visual assessment of kale showed that 560-nm resulted in top quality. • Reduced quality of spinach was observed after day 5 for all light treatments. [ABSTRACT FROM AUTHOR]

Grasslands, the largest carbon pool in China, possess enormous potential for carbon sequestration. Increasing the stomatal aperture to increase the CO 2 absorption capacity is a potential method to improve plant photosynthetic efficiency and ultimately enhance the carbon sequestration capacity of grass plants. Research on stomatal aperture regulation has focused mostly on Arabidopsis or crops, while research on grass plants in these areas is scarce, which seriously restricts the implementation of this grassland carbon sequestration strategy. Here, a widely used ecological grass, centipedegrass, was used as the experimental material. First, a convenient method for observing the stomatal aperture was developed. The leaves were floated in a potassium ion-containing open solution (67 mM KCl, pH 6.0) with the adaxial surface rather than the abaxial surface in contact with the solution and were cultivated under light for 1.5 h. Then, nail polish was applied on the adaxial surface, and a large number of open stomata were imprinted. Second, with the help of this improved method, the concentration‒response characteristics of the stomatal aperture to eleven environmental stimuli were tested. The stomatal aperture is dependent on these environmental stimuli in a concentration-dependent manner. The addition of 100 μM brassinolide led to the maximal stomatal aperture. This study provided a technical basis for manipulating stomatal opening to increase the carbon sequestration capacity of centipedegrass. • Established a convenient method for detecting the stomatal aperture of centipedegrass. • Optimal open solution for the stomatal aperture investigation is 67 mM KCl at pH 6.0 • The addition of 100 μM brassinolide led to the maximal stomatal aperture. [ABSTRACT FROM AUTHOR]

GTP is an important signaling molecule involved in the growth, development, and stress adaptability of plants. The functions are mediated via binding to GTPases which are in turn regulated by GTPase-activating proteins (GAPs). Satellite reports have suggested the positive roles of GAPs in regulating ABA signaling and pathogen resistance in plants. However, the molecular mechanisms that bring forth the pathogen resistance have remained unclear. In this study, we demonstrated that the expression of AtGAP1 was inducible by Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The overexpression of AtGAP1 in Arabidopsis promoted the expression of PR1 and the resistance to Pst DC3000. Proteomic analyses revealed the enhanced accumulation of cell-wall-modifying proteins as a result of AtGAP1 overexpression. By microscopic analyses, we showed that the overexpression of AtGAP1 resulted in increased thickness of the mesophyll cell wall and reduced stomatal aperture, which are effective strategies for restricting the entry of foliar pathogens. Altogether, we demonstrated that AtGAP1 increases the resistance to Pst DC3000 in Arabidopsis by promoting cellular strategies that restrict the entry of pathogens into the cells. These results point to a future direction for studying the modes of action of GAPs in regulating plant cell structures and disease resistance. [ABSTRACT FROM AUTHOR]

Salicylic acid (SA) is an endogenous growth regulator. It is vital for the growth and development, photosynthesis, disease resistance, and abiotic stress tolerance of plants. This study aimed to investigate the effects of exogenous SA on photosynthetic characteristics and antioxidant system in the four-leaf-stage waxy corn inbred seedlings SY-MY13 (waterlogging-resistant) and SY-XT1 (waterlogging-sensitive) under waterlogging stress. Waterlogging stress negatively influenced the morphology, plant height, biomass, photosynthetic characteristics, and chlorophyll content of waxy corn seedlings, obstructing their growth and development. SY-MY13 exhibited better growth and photosynthesis than SY-XT1 under waterlogging, this was due to SY-MY13 possessed excellent scavenging ability to reactive oxygen species (ROS). However, the application of exogenous SA could not only reduce the accumulation of ROS and regulate the activities of antioxidant enzymes but also improve photosynthesis in leaves and promote the growth of seedlings. Hence, the results revealed that the potential of SA as a promising exogenous growth regulator mediated physiological and photosynthetic adaptation of waxy corn seedlings under waterlogging stress, which might prove helpful for other plants suffering from waterlogging stress.

We compared how stomatal morphology and physiology control intrinsic leaf water use efficiency (iWUE) in two C3 and six C4 grasses grown at ambient (400 µmol mol–1) or glacial CO2 (180 µmol mol–1) and high (1000 µmol m–2 s–1) or low light intensity (200 µmol m–2 s–1). C4 grasses tended to have higher iWUE and CO2 assimilation rates, and lower stomatal conductance (g s), operational stomatal aperture (a op), and guard cell K+ influx rate relative to C3 grasses, while stomatal size (SS) and stomatal density (SD) did not vary according to the photosynthetic type. Overall, iWUE and g s depended most on a op and density of open stomata. In turn, a op correlated with K+ influx, stomatal opening speed on transition to high light, and SS. Species with higher SD had smaller and faster-opening stomata. Although C4 grasses operated with lower g s and a op at ambient CO2, they showed a greater potential to open stomata relative to maximal stomatal conductance (g max), indicating heightened stomatal sensitivity and control. We uncovered promising links between a op, g s, iWUE, and K+ influx among C4 grasses, and differential K+ influx responses of C4 guard cells to low light, revealing molecular targets for improving iWUE in C4 crops. [ABSTRACT FROM AUTHOR]

Gram-negative bacteria inject type III secreted effectors (T3SEs) into host cells to manipulate the immune response. The YopJ family effector HopZ1a produced by the plant pathogen Pseudomonas syringae possesses acetyltransferase activity and acetylates plant proteins to facilitate infection. Using mass spectrometry, we identified a threonine residue, T346, as the main autoacetylation site of HopZ1a. Two neighboring serine residues, S349 and S351, are required for the acetyltransferase activity of HopZ1a in vitro and are indispensable for the virulence function of HopZ1a in Arabidopsis thaliana. Using proton nuclear magnetic resonance (NMR), we observed a conformational change of HopZ1a in the presence of inositol hexakisphosphate (IP6), which acts as a eukaryotic co-factor and significantly enhances the acetyltransferase activity of several YopJ family effectors. S349 and S351 are required for IP6-binding-mediated conformational change of HopZ1a. S349 and S351 are located in a conserved region in the C-terminal domain of YopJ family effectors. Mutations of the corresponding serine(s) in two other effectors, HopZ3 of P. syringae and PopP2 of Ralstonia solanacerum, also abolished their acetyltransferase activity. These results suggest that, in addition to the highly conserved catalytic residues, YopJ family effectors also require conserved serine(s) in the C-terminal domain for their enzymatic activity.

Light plays a pivotal role in plant growth, development, and stress responses. Green light has been reported to enhance plant drought tolerance via stomatal regulation. However, the mechanisms of green light-induced drought tolerance in plants remain elusive. To uncover those mechanisms, we investigated the molecular responses of tomato plants under monochromatic red, blue, and green light spectrum with drought and well-water conditions using a comparative transcriptomic approach. The results showed that compared with monochromatic red and blue light treated plants, green light alleviated the drought-induced inhibition of plant growth and photosynthetic capacity, and induced lower stomatal aperture and higher ABA accumulation in tomato leaves after 9 days of drought stress. A total of 3,850 differentially expressed genes (DEGs) was identified in tomato leaves through pairwise comparisons. Functional annotations revealed that those DEGs responses to green light under drought stress were enriched in plant hormone signal transduction, phototransduction, and calcium signaling pathway. The DEGs involved in ABA synthesis and ABA signal transduction both participated in the green light-induced drought tolerance of tomato plants. Compared with ABA signal transduction, more DEGs related to ABA synthesis were detected under different light spectral treatments. The bZIP transcription factor- HY5 was found to play a vital role in green light-induced drought responses. Furthermore, other transcription factors, including WRKY46 and WRKY81 might participate in the regulation of stomatal aperture and ABA accumulation under green light. Taken together, the results of this study might expand our understanding of green light-modulated tomato drought tolerance via regulating ABA accumulation and stomatal aperture.

Light plays a pivotal role in plant growth, development, and stress responses. Green light has been reported to enhance plant drought tolerance via stomatal regulation. However, the mechanisms of green light-induced drought tolerance in plants remain elusive. To uncover those mechanisms, we investigated the molecular responses of tomato plants under monochromatic red, blue, and green light spectrum with drought and well-water conditions using a comparative transcriptomic approach. The results showed that compared with monochromatic red and blue light treated plants, green light alleviated the drought-induced inhibition of plant growth and photosynthetic capacity, and induced lower stomatal aperture and higher ABA accumulation in tomato leaves after 9 days of drought stress. A total of 3,850 differentially expressed genes (DEGs) was identified in tomato leaves through pairwise comparisons. Functional annotations revealed that those DEGs responses to green light under drought stress were enriched in plant hormone signal transduction, phototransduction, and calcium signaling pathway. The DEGs involved in ABA synthesis and ABA signal transduction both participated in the green light-induced drought tolerance of tomato plants. Compared with ABA signal transduction, more DEGs related to ABA synthesis were detected under different light spectral treatments. The bZIP transcription factor- HY5 was found to play a vital role in green light-induced drought responses. Furthermore, other transcription factors, including WRKY46 and WRKY81 might participate in the regulation of stomatal aperture and ABA accumulation under green light. Taken together, the results of this study might expand our understanding of green light-modulated tomato drought tolerance via regulating ABA accumulation and stomatal aperture. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the nitrogen (N) and phosphorus (P) fertilization of continuous addition effects plant biomass, the physiological properties of leaves and the antioxi-dant enzyme activities of alfalfa (Medicago sativa L) in the northern Xinjiang region; including the no fertilization (CK), nitrogen fertilization (N, 120 kg·ha−1), phosphorus fertilization (with low amount of N) (P, 100 kg·ha−1 P and 23.5 kg·ha−1 N) and combined nitrogen and phosphorus fertilization (NP, 120 kg·ha−1 N and 100 kg·ha−1 P) on the K well supplied soil. After three consecutive years of the supply of N and P fertilization, samples were taken at the first flowering of alfalfa (four clippings in the total year) to determine its pigment concentration, stomatal aperture, antioxidant enzyme activity and hay yield. The results showed that NP fertilization promoted growth with a higher number of branches and hay yield of alfalfa, while N or P fertilization alone had a positive effect on the growth of alfalfa. However, P fertilization significantly increased the carotenoid (Car) content at the early flowering stage of alfalfa leaves (during four clippings) (p < 0.05), In addition, NP ferti-lization enhanced stomatal aperture, increased the antioxidant enzyme activity and decreased the oxidized substance at the early flowering stage of alfalfa leaves. The results showed that a N and P balance rather than an absolute amount of either enhanced the growth of alfalfa, and N or P fertili-zation affects physiological traits differently. We propose that NP fertilization increases the nutri-tional characteristics and physiological characteristics, enhancing the adaptive capacity of alfalfa and making it better adapted to external environmental changes.

Yellowhorn (Xanthoceras sorbifolium) is an important edible woody oil tree species that is endemic to China. Drought and heat stresses are factors severely limiting the high-quality development of the yellowhorn industry. Transcription factors (TFs) play critical roles in regulating the response of woody plant species to water deficit or high temperature. However, the MYB TFs that respond to combined drought and heat stress in yellowhorn remain unclear. Here, we first investigated the physiological changes in 5 yellowhorn varieties in response to combined stress treatments. We observed significant changes in antioxidant enzyme activities and photosynthesis. The Maigaiti variety yielded the best results and was selected for subsequent experiments. An R2R3-type MYB TF, designated XsMYB44, was isolated from the leaves of yellowhorn. XsMYB44 expression was strongly induced by combined stress. Suppression of XsMYB44 expression via virus-induced gene silencing weakened yellowhorn tolerance to both individual and combined drought and heat stress, and the increased susceptibility was coupled with decreased plant height, fresh weight and relative water content and inhibited stomatal closure. Moreover, compared with the individual stresses, the combined stress caused increased reactive oxygen species levels and decreased antioxidant enzyme activities and proline content in XsMYB44 -silenced plants. Furthermore, the expression levels of several defense-related genes were reduced in the XsMYB44 -silenced plants. Overall, we studied the physiological characteristics of 5 yellowhorn varieties, and the results demonstrated that XsMYB44 acts as a positive regulator in the yellowhorn response to combined stress by triggering stomatal closure to maintain water levels and by modulating ROS homeostasis. ● XsMYB44 was induced by combined drought and heat stress, salt, ABA, SA and JA ● XsMYB44 acts as a positive regulator in yellowhorn response to combined drought and heat stress ● XsMYB44 regulates stress tolerance by control stomatal closure and ROS homeostasis [ABSTRACT FROM AUTHOR]

Stomatal closure is a common adaptation response of plants to the onset of drought condition and its regulation is controlled by transcription factors. MYB60, a transcription factor involved in the regulation of light-induced stomatal opening, has been characterized in arabidopsis and grapevine. In this work, we studied the role of MYB60 homolog SIMYB60 in tomato plants. We identified, isolated, and sequenced the SIMYB60 coding sequence, and found domains and motifs characteristic of other MYB60 proteins. We determined that SlMYB60 is mainly expressed in leaves, and its expression is repressed by abscisic acid. Next, we isolated a putative promoter region containing regulatory elements responsible for guard cell expression and other putative regulatory elements related to ABA repression and vascular tissue expression. Protein localization assays demonstrated that SlMYB60 localizes to the nucleus. Finally, SlMYB60 is able to complement the mutant phenotype of atmyb60-1 in Arabidopsis. Together, these results indicate that SlMYB60 is the homologous gene in tomato and potentially offer a molecular target to improve crops. [ABSTRACT FROM AUTHOR]

Stomata are pores surrounded by a pair of specialized cells, called guard cells, that play a central role in plant physiology through the regulation of gas exchange between plants and the environment. Guard cells have features like cell-autonomous responses and easily measurable readouts that have turned them into a model system to study signal transduction mechanisms in plants. Here, we provide a detailed protocol to analyze different physiological responses specifically in guard cells. We describe, in detail, the steps and conditions to isolate epidermal peels with tweezers and to analyze i) stomatal aperture in response to different stimuli, ii) cytosolic parameters such as hydrogen peroxide (H 2 O 2 ), glutathione redox potential ( E GSH ), and MgATP -2 in vivo dynamics using fluorescent biosensors, and iii) gene expression in guard cell-enriched samples. The importance of this protocol lies in the fact that most living cells on epidermal peels are guard cells, enabling the preparation of guard cell-enriched samples. Key features • Isolation of epidermal peels as a monolayer enriched in guard cells • Measurement of cytosolic guard cell signaling component dynamics in isolated epidermal peels through fluorescent biosensor analysis • Gene expression analysis of guard cell-enriched isolated tissue., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Glycerol-3-phosphate dehydrogenase (GPDH) is a key enzyme in plant glycerol synthesis and metabolism, and plays an important role in plant resistance to abiotic stress. Here, we identified 6, 7, 14 and 14 GPDH genes derived from Gossypium arboreum, Gossypium raimondii, Gossypium barbadense and Gossypium hirsutum, respectively. Phylogenetic analysis assigned these genes into three classes, and most of the genes within the family were expanded by whole-genome duplication (WGD) and segmental duplications. Moreover, determination of the nonsynonymous substitution rate/synonymous substitution rate (Ka/Ks) ratio showed that the GPDH had an evolutionary preference for purifying selection. Transcriptome data revealed that GPDH genes were more active in the early stages of fiber development. Additionally, numerous stress-related cis-elements were identified in the potential promoter region. Then, a protein–protein-interaction (PPI) network of GPDH5 in G. hirsutum was constructed. In addition, we predicted 30 underlying miRNAs in G. hirsutum. Functional validation results indicated that silencing GhGPDH5 diminished drought tolerance in the upland cotton TM-1 line. In summary, this study provides a fundamental understanding of the GPDH gene family in cotton, GhGPDH5 exerts a positive effect during drought stress and is potentially involved in stomatal closure movements.

Salicylic acid (SA) is an endogenous growth regulator. It is vital for the growth and development, photosynthesis, disease resistance, and abiotic stress tolerance of plants. This study aimed to investigate the effects of exogenous SA on photosynthetic characteristics and antioxidant system in the four-leaf-stage waxy corn inbred seedlings SY-MY13 (waterlogging-resistant) and SY-XT1 (waterlogging-sensitive) under waterlogging stress. Waterlogging stress negatively influenced the morphology, plant height, biomass, photosynthetic characteristics, and chlorophyll content of waxy corn seedlings, obstructing their growth and development. SY-MY13 exhibited better growth and photosynthesis than SY-XT1 under waterlogging, this was due to SY-MY13 possessed excellent scavenging ability to reactive oxygen species (ROS). However, the application of exogenous SA could not only reduce the accumulation of ROS and regulate the activities of antioxidant enzymes but also improve photosynthesis in leaves and promote the growth of seedlings. Hence, the results revealed that the potential of SA as a promising exogenous growth regulator mediated physiological and photosynthetic adaptation of waxy corn seedlings under waterlogging stress, which might prove helpful for other plants suffering from waterlogging stress. [ABSTRACT FROM AUTHOR]

Transpiration occurs through stomata. The alteration of stomatal apertures in response to drought stress is an important process associated with water use efficiency (WUE). Trehalose-6-phosphate phosphatase (TPP) family genes have been reported to participate in adjustment of stomatal aperture. However, there have been no reports of the trehalose metabolism pathway genes improving WUE, and the upstream signalling pathway modulating these genes is not clear. Here, we demonstrate that a member of the TPP gene family, AtTPPI , confers drought resistance and improves WUE by decreasing stomatal apertures and improving root architecture. The reduced expression of AtTPPI caused a drought-sensitive phenotype, while its overexpression significantly increased drought tolerance. Abscisic acid (ABA)-induced stomatal closure experiments confirmed that AtTPPI mutation increased the stomatal aperture compared with that of wild-type plants; in contrast, overexpression plants had smaller stomatal apertures than those of wild-type plants. Moreover, AtTPPI mutation also caused stunted primary root length and compromised auxin transport, while overexpression plants had longer primary root lengths. Yeast one-hybrid assays showed that ABA-responsive element-binding factor1 (ABF1), ABF2, and ABF4 directly regulated AtTPPI expression. In summary, the way in which AtTPPI responds to drought stress suggests that AtTPPI -mediated stomatal regulation is an important mechanism to cope with drought stress and improve WUE. [ABSTRACT FROM AUTHOR]

Transcription factors (TFs) help plants respond to environmental stresses by regulating gene expression. Up till now, studies on the MYB family of TFs have mainly focused on the highly abundant R2R3-subtype. While the less well-known 1R-subtype has been generally shown to enhance abscisic acid (ABA) sensitivity by acting as transcriptional activators, the mechanisms of their functions are unclear. Here we identified an ABA sensitivity-associated gene from soybean, ABA-Sensitive 1 (GmABAS1), of the 1R-subtype of MYB. Using the GFP-GmABAS1 fusion protein, we demonstrated that GmABAS1 is localized in the nucleus, and with yeast reporter systems, we showed that it is a transcriptional repressor. We then identified the target gene of GmABAS1 to be Glyma.01G060300 , an annotated ABI five-binding protein 3 and showed that GmABAS1 binds to the promoter of Glyma.01G060300 both in vitro and in vivo. Furthermore, Glyma.01G060300 and GmABAS1 exhibited reciprocal expression patterns under osmotic stress, inferring that GmABAS1 is a transcriptional repressor of Glyma.01G060300. As a further confirmation, AtAFP2 , an orthologue of Glyma.01G060300 , was down-regulated in GmABAS1 -transgenic Arabidopsis thaliana , enhancing the plant's sensitivity to ABA. This is the first time a 1R-subtype of MYB from soybean has been reported to enhance ABA sensitivity by acting as a transcriptional repressor. [ABSTRACT FROM AUTHOR]

Stomata control gas exchange and water transpiration and are one of the most important physiological apparatuses in higher plants. The regulation of stomatal aperture is closely coordinated with photosynthesis, nutrient uptake, plant growth, development, and so on. With advances in scanning electron microscopy (SEM), high-resolution images of plant stomata and cell surfaces can be obtained from detached plant tissues. However, this method does not allow for rapid analysis of the dynamic variation of plant stomata and cell surfaces in situ under nondestructive conditions. In this study, we demonstrated a novel plant surface impression technique (PSIT, Silagum-Light as correction impression material based on A-silicones for all two-phase impression techniques) that allows for precise analysis of plant stomata aperture and cell surfaces. Using this method, we successfully monitored the dynamic variation of stomata and observed the nanoscale microstructure of soybean leaf trichomes and dragonfly wings. Additionally, compared with the analytical precision and the time used for preparing the observation samples between PSIT and traditional SEM, the results suggested that the analytical precision of PSIT was the same to traditional SEM, but the PSIT was more easy to operate. Thus, our results indicated that PSIT can be widely applied to the plant science field.

Stomata control gas exchange and water transpiration and are one of the most important physiological apparatuses in higher plants. The regulation of stomatal aperture is closely coordinated with photosynthesis, nutrient uptake, plant growth, development, and so on. With advances in scanning electron microscopy (SEM), high-resolution images of plant stomata and cell surfaces can be obtained from detached plant tissues. However, this method does not allow for rapid analysis of the dynamic variation of plant stomata and cell surfaces in situ under nondestructive conditions. In this study, we demonstrated a novel plant surface impression technique (PSIT, Silagum-Light as correction impression material based on A-silicones for all two-phase impression techniques) that allows for precise analysis of plant stomata aperture and cell surfaces. Using this method, we successfully monitored the dynamic variation of stomata and observed the nanoscale microstructure of soybean leaf trichomes and dragonfly wings. Additionally, compared with the analytical precision and the time used for preparing the observation samples between PSIT and traditional SEM, the results suggested that the analytical precision of PSIT was the same to traditional SEM, but the PSIT was more easy to operate. Thus, our results indicated that PSIT can be widely applied to the plant science field. [ABSTRACT FROM AUTHOR]

• Common buckthorn assimilates carbon faster than glossy in high light intensities. • Hawthorn assimilated carbon faster than both buckthorn species in moderate light. • Shade acclimated hawthorn was inferior to buckthorn at higher light intensities. • Gray dogwood assimilated carbon and used water less efficiently than buckthorn. Common buckthorn (Rhamnus cathartica L.) and glossy buckthorn (Frangula alnus Mill.; or, Rhamnus frangula L.) are non-native, invasive plant species invading U.S. Midwest natural areas. Both are ubiquitous on the Calvin College campus in Southwest Michigan where they compete with native plants for light, water, and nutrients exceptionally well. We hypothesize that common buckthorn will assimilate carbon faster and use water more efficiently than glossy buckthorn and native co-habiting shrubs in relatively dry and bright edge and meadow habitats whereas glossy buckthorn will thrive in moist and thin forest canopies. These are habitats in which each buckthorn species is abundant and colonizes rapidly. The objective of this project was to compare light use efficiency (LUE), water use efficiency (WUE), chlorophyll content and stomatal characteristics of both buckthorn species with two locally common co-habiting native species, hawthorn (Crataegus spp.) and gray dogwood (Cornus racemosa Lam.), acclimated to forest edge and understory environments on the Calvin campus in 2016 and 2017. Between 380 and 480 light use efficiency curves were measured with a LI-6400XT Portable Photosynthesis System using common and glossy buckthorn, hawthorn and gray dogwood leaves during two growing seasons from plants acclimated to edge and understory habitats. Common buckthorn assimilated carbon faster than glossy buckthorn when leaves were exposed to moderate or high light intensities. Water use efficiency was advantageous to glossy buckthorn at low to moderate light intensities, potentially enabled by the high density of stomates on lower leaf surfaces. Shade-acclimated common buckthorn displayed only minor decreases in photosynthesis rate relative to leaves in sun-acclimated plants after exposure to a wide range of light intensities possibly because of its higher leaf chlorophyll content. Sun-acclimated hawthorn grew faster than both buckthorn species when exposed to higher light intensities and produced comparable growth rates to buckthorn species in the shaded understory. Gray dogwood produced the slowest growth rate of the four species in all environments. Restoration of buckthorn infested areas is problematic in both sunny edge and shaded understory habitats and will require continuous mitigation of common and glossy buckthorn along with the concomitant establishment of more competitive native species like hawthorn. [ABSTRACT FROM AUTHOR]

Abstract MYB proteins are major transcription factors that play significant roles in plant defenses against various stresses. However, available information regarding stress-related MYB genes in maize is minimal. Herein, a maize MYB gene, ZmMYB3R , was cloned and functionally characterized. Subcellular localisation analysis showed that ZmMYB3R is localised to the nucleus. Yeast one-hybrid results revealed that ZmMYB3R has trans-activation activity, and a minimal activation domain at the C-terminus spanning residues 217–563. Gene expression analysis suggested that ZmMYB3 R was induced by drought, salt and abscisic acid (ABA). Transgenic Arabidopsis plants overexpressing ZmMYB3R displayed enhanced growth performance and higher survival rates, elevated catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) enzyme activities, increased sensitivity to ABA, and regulation of the stomatal aperture, suggesting that ZmMYB3R enhances tolerance to drought and salt stress. qRT-PCR assays revealed elevated expression levels of stress/ABA genes in transgenic plants following stress treatments. Moreover, transgenic plants accumulated higher ABA content than wild-type plants under drought and salt stress conditions. Collectively, these results indicate that ZmMYB3R is a positive transcription factor that enhances tolerance to drought and salt stress via an ABA-dependent pathway. The findings may prove useful for engineering economically important crops. Highlights •. A novel maize MYB-3R gene was cloned and characterized. •. ZmMYB3R increases tolerance to drought and salt stress in transgenic plants. • ZmMYB3R is a positive transcription factor for stress responses. •. ZmMYB3R functions via an ABA-dependent pathway. [ABSTRACT FROM AUTHOR]

Flavonols, the main flavonoids in plant leaves, have newly been proposed as a H2O2 scavenger in guard cells. However, whether 5-aminolevulinic acid (5-ALA) is involved in regulating stomatal movement of apple (Malus × domestica Borkh. ‘Fuji’) leaves through flavonol accumulation remains unclear. In this study, using diphenylboric acid 2-amino ethyl ester (DPBA, a flavonol fluorescent dye) and a laser scanning confocal microscope, we observed that 5-ALA pretreatment significantly improved flavonol accumulation in guard cells of apple leaves, especially around the nucleus. Then we demonstrated that 5-ALA pretreatment inhibited ABA-induced stomatal closure via decreasing reaction oxygen species (ROS) accumulation in guard cells. Pretreatment with quercetin or kaempferol, two main kinds of flavonols in plants, also inhibited ABA-induced stomatal closure by decreasing ROS content in the guard cells. Furthermore, exogenous flavonols could suppress H2O2-induced stomatal closure in apple leaves. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is involved in the inhibitory effect of ALA on ABA-induced ROS accumulation and stomatal closure in apple leaves.

Stomata play a critical role in the regulation of gas exchange between the interior of the leaf and the exterior environment and are affected by environmental and endogenous stimuli. This study aimed to evaluate the effect of the arbuscular mycorrhizal (AM) fungus, Rhizophagus irregularis, on the stomatal behavior of wheat (Triticum aestivum L.) plants under combination with elevated CO2 and NaCl stress. Wheat seedlings were exposed to ambient (400 ppm) or elevated (700 ppm) CO2 concentrations and 0, 1, and 2 g kg−1 dry soil NaCl treatments for 10 weeks. AM symbiosis increased the leaf area and stomatal density (SD) of the abaxial surface. Stomatal size and the aperture of adaxial and abaxial leaf surfaces were higher in the AM than non-AM plants under elevated CO2 and salinity stress. AM plants showed higher stomatal conductance (gs) and maximum rate of gs to water vapor (gsmax) compared with non-AM plants. Moreover, leaf water potential (Ψ) was increased and carbon isotope discrimination (Δ13C) was decreased by AM colonization, and both were significantly associated with stomatal conductance. The results suggest that AM symbiosis alters stomatal morphology by changing SD and the size of the guard cells and stomatal pores, thereby improving the stomatal conductance and water relations of wheat leaves under combined elevated CO2 and salinity stress.