Project description:Transcription factor WRKY46 regulates osmotic stress responses and stomatal movement tissue specifically and independently in Arabidopsis
Project description:Drought stress can damage crop growth and lead to a decline in yield, thereby affecting food security, especially in regions vulnerable to climate change. SNAC1 (Stress-responsive NAC1), the NAC transcription factor family member, plays a crucial role in stomatal movement regulation. Effective regulation of stomatal movement is essential for protecting plants from water loss during adverse conditions. Our hypothesis revolves around altering HvSNAC1 activity by introducing a point mutation in its encoding gene, thereby influencing stomatal dynamics in barley. Two TILLING mutants, each harboring missense mutations in the NAC domain, exhibited higher stomatal density after drought stress compared to the parent cultivar 'Sebastian'. These mutants also demonstrated distinct patterns of ABA-induced stomatal movement compared to the ’Sebastian’. To delve deeper, we conducted a comprehensive analysis of the transcriptomes of these mutants and the parent cultivar 'Sebastian' under both optimal watering conditions and ten days of drought stress treatment. We identified differentially expressed genes (DEGs) between the mutants and ‘Sebastian’ under control and drought conditions. Furthermore, we pinpointed DEGs specifically expressed in both mutants under drought conditions. Our experiments revealed that the cis-regulatory motif CACG, previously identified in Arabidopsis and rice, is recognized by HvSNAC1 in vitro. Enrichment analysis led to the identification of the cell wall organization category and potential target genes, such as HvEXPA8 (Expansin 8), HvXTH (Xyloglucan endotransglucosylase/hydrolase), and HvPAE9 (Pectin acetylesterase 9), suggesting their regulation by HvSNAC1. These findings suggest that HvSNAC1 may play a role in regulating genes associated with stomatal density, size, and reopening.
Project description:Drought stress can damage crop growth and lead to a decline in yield, thereby affecting food security, especially in regions vulnerable to climate change. SNAC1 (Stress-responsive NAC1), the NAC transcription factor family member, plays a crucial role in stomatal movement regulation. Effective regulation of stomatal movement is essential for protecting plants from water loss during adverse conditions. Our hypothesis revolves around altering HvSNAC1 activity by introducing a point mutation in its encoding gene, thereby influencing stomatal dynamics in barley. Two TILLING mutants, each harboring missense mutations in the NAC domain, exhibited higher stomatal density after drought stress compared to the parent cultivar 'Sebastian'. These mutants also demonstrated distinct patterns of ABA-induced stomatal movement compared to the ’Sebastian’. To delve deeper, we conducted a comprehensive analysis of the transcriptomes of these mutants and the parent cultivar 'Sebastian' under both optimal watering conditions and ten days of drought stress treatment. We identified differentially expressed genes (DEGs) between the mutants and ‘Sebastian’ under control and drought conditions. Furthermore, we pinpointed DEGs specifically expressed in both mutants under drought conditions. Our experiments revealed that the cis-regulatory motif CACG, previously identified in Arabidopsis and rice, is recognized by HvSNAC1 in vitro. Enrichment analysis led to the identification of the cell wall organization category and potential target genes, such as HvEXPA8 (Expansin 8), HvXTH (Xyloglucan endotransglucosylase/hydrolase), and HvPAE9 (Pectin acetylesterase 9), suggesting their regulation by HvSNAC1. These findings suggest that HvSNAC1 may play a role in regulating genes associated with stomatal density, size, and reopening.
Project description:Stomata are highly specialized organs which consist of pairs of guard cells and regulate gas and water vapor exchange in plants. While early stages of guard cell differentiation have been described and were interpreted in analogy to processes of cell type differentiation in animals, the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, scap1 (stomatal carpenter 1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as a K+ channel protein, MYB60 transcription factor, and pectin methyl esterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation. We isolated guard cell protoplasts from 4-week-old WT(Col-0) and scap1 mutant plants and extracted RNA independently. Four biological replicates were performed for each experiment.
Project description:Drought and salt stress severely inhibit plant growth and development. However, the regulatory mechanisms of plants in response to these stresses are not fully understood. Here we find that the expression of a WRKY transcription factor WRKY46 is rapidly induced by drought, salt and oxidative stresses. Mutations of WRKY46 by T-DNA insertion lead to more sensitive to drought and salt stress, whereas, overexpression of WRKY46 exhibits hypersensitive in soil culture with higher water loss rate, but increased tolerance on the agar plates. ABA induced stomatal closing is impaired in the WRKY46 overexpressing line (OV46), which is potentially due to the lower ROS accumulation in the guard cells. Real-time qPCR and GUS activity assay further demonstrate that WRKY46 is expressed in guard cells, but its expression is not affected by dehydration treatment, suggesting different regulatory mechanisms for WRKY46 between guard cells and other WRKY46 expressed tissues. The stomatal movement and conductance assay indicate that WRKY46 is involved in light-dependent stomatal opening. Further microarray analysis reveals that WRKY46 regulates a set of genes involved in cellular osmoprotection and redox homeostasis under dehydration stress. Determinations of ROS and MDA content confirm its role in oxidative detoxification under stress. Furthermore, we find that WRKY46 modulates light-dependent starch metabolism in guard cells via regulating QQS gene expression. Taken together, we demonstrate that WRKY46 plays a role in modulating cellular osmoprotection and redox homeostasis under drought and salt stress, and functions independently in stomatal movement via regulating light-dependent starch metabolism and ROS levels in guard cells. We used microarrays to identify the certain downstream genes regulated by WRKY46 under normal and dehydration conditions. One-week-old Arabidopsis seedlings of wild-type Col-0 (WT) and WRKY46 overexpressing line (46T) with or without dehydration treatment for 1 h were harvested and used for RNA extraction and hybridization to Affymetrix Arabidopsis ATH1 microarrays. The experiment includes 3 biological replicates.
Project description:Environmental stimuli-triggered stomatal movement is a key physiological process that regulates CO<sub>2</sub> uptake and water loss in plants. Stomata are defined by pairs of guard cells that perceive and transduce external signals, leading to cellular volume changes and consequent stomatal aperture change. Within the visible light spectrum, red light induces stomatal opening in intact leaves. However, there has been debate regarding the extent to which red-light-induced stomatal opening arises from direct guard cell sensing of red light versus indirect responses as a result of red light influences on mesophyll photosynthesis. Here we identify conditions that result in red-light-stimulated stomatal opening in isolated epidermal peels and enlargement of protoplasts, firmly establishing a direct guard cell response to red light. We then employ metabolomics workflows utilizing gas chromatography mass spectrometry and liquid chromatography mass spectrometry for metabolite profiling and identification of Arabidopsis guard cell metabolic signatures in response to red light in the absence of the mesophyll. We quantified 223 metabolites in Arabidopsis guard cells, with 104 found to be red light responsive. These red-light-modulated metabolites participate in the tricarboxylic acid cycle, carbon balance, phytohormone biosynthesis and redox homeostasis. We next analyzed selected Arabidopsis mutants, and discovered that stomatal opening response to red light is correlated with a decrease in guard cell abscisic acid content and an increase in jasmonic acid content. The red-light-modulated guard cell metabolome reported here provides fundamental information concerning autonomous red light signaling pathways in guard cells.
Project description:Regulation of stomatal movement is one of the effective strategies for developing resistant crops to air pollutant because stomata allows absorption of various air pollutants, such as ozone and sulfur dioxide. Transcription factor (TF) is a fascinating target of genetic manipulation for this end because TF regulates many genes simultaneously and methods of genetic manipulation are universally established. Here, we have screened transgenic Arabidopsis lines expressing chimeric repressor of TFs in high-concentration of ozone and found that the chimeric repressors of GOLDEN-LIKE1 (GLK1) and GLK2 (GLK1-SRDX and GLK2-SRDX) conferred strong tolerance to ozone. These 35S:GLK1/2-SRDX plants also showed sulfur dioxide tolerance. Their leaves showed lower rate of transpiration than wild type and a remarkable closed-stomata phenotype. The expression of the genes encoding K+ and water channels, including KAT1 and AKT1, which are involved in stomatal opening, was downregulated in 35S:GLK1-SRDX plants. Consistently, expression of GLK1-SRDX driven by the GC1 promoter, which has an activity only in guard cell, also induced closed-stomata and an ozone tolerant phenotype. On the contrary, 35S:GLK1/2 plants showed hypersensitivity to ozone and an opened-stomata phenotype. These data suggested that GLK1 and GLK2 have an ability to induce transcriptional change in guard cell and regulate stomatal movement. Our findings provide an effective tool to confer resistance to air pollutant by regulating stomatal aperture and improve crop productivity in future.
Project description:Stomata are highly specialized organs which consist of pairs of guard cells and regulate gas and water vapor exchange in plants. While early stages of guard cell differentiation have been described and were interpreted in analogy to processes of cell type differentiation in animals, the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, scap1 (stomatal carpenter 1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as a K+ channel protein, MYB60 transcription factor, and pectin methyl esterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation.
Project description:Flavonols are a class of flavonoids. Unlike other classes of flavonoids, flavonols strongly accumulate in guard cells, but the role of their accumulation in guard cells is still unclear so far.The overexpression of TaFLS1, a flavonol synthase gene from wheat, improves flavonol content in guard cells of Arabidopsis, while the mutant fls1-3 of Arabidopsis AtFLS1 reduces the content. Both the increase of flavonols by TaFLS1 overexpression and the decrease in fls1-3 change stomatal apeture, showing the association between flavonols and stomatal movements. We used microarrays to detail the global programme of gene expression underlying both the increase and decrease of flavonol content and identified possible mechanism of flavonols-mediated stomatal movement.
Project description:Potassium is one of the essential macronutrients required for plant growth and development. It plays a major role in different physiological processes like cell elongation, stomatal movement, turgor regulation, osmotic adjustment, and signal transduction by acting as a major osmolyte and component of the ionic environment in the cytosol and subcellular organelles. We used whole genome microarrays to determine the transcriptomic profile of rice seedlings exposed to short-term K+ deficiency followed by K+ resupply.