ABSTRACT
The process of apoptotic cell clearance by phagocytes, known as efferocytosis, plays an essential role in maintaining homeostasis. Defects in efferocytosis can lead to inflammatory diseases such as atherosclerosis and autoimmune disorders. Therefore, the maintenance and promotion of efferocytosis are considered crucial for preventing these diseases. In this study, we observed that resveratrol, a representative functional food ingredient, and its glycoside, piceid, promoted efferocytosis in both human THP-1 macrophages differentiated with phorbol 12-myristate 13-acetate and peritoneal macrophages from thioglycolate-elicited mice. Resveratrol and piceid significantly increased mRNA expression and protein secretion of MFG-E8 in THP-1 macrophages. Furthermore, the activation of efferocytosis and the increment in MFG-E8 protein secretion caused by resveratrol or piceid treatment were canceled by MFG-E8 knockdown in THP-1 macrophages. In conclusion, we have demonstrated for the first time that resveratrol and piceid promote efferocytosis through the upregulation of MFG-E8 excretion in human THP-1 macrophages.
Subject(s)
Antigens, Surface , Glucosides , Macrophages , Phagocytosis , Resveratrol , Humans , Resveratrol/pharmacology , Macrophages/metabolism , Macrophages/drug effects , Animals , Phagocytosis/drug effects , THP-1 Cells , Mice , Glucosides/pharmacology , Antigens, Surface/metabolism , Antigens, Surface/genetics , Stilbenes/pharmacology , Apoptosis/drug effects , Efferocytosis , Milk ProteinsABSTRACT
Existing CRISPR/Cas12a-based diagnostic platforms offer accurate and vigorous monitoring of nucleic acid targets, but have the potential to be further optimized for more efficient detection. Here, we profiled 16 Cas12a orthologs, focusing on their trans-cleavage activity and their potential as diagnostic enzymes. We observed the Mb2Cas12a has more robust trans-cleavage activity than other orthologs, especially at lower temperatures. An engineered Mb2Cas12a-RRVRR variant presented robust trans-cleavage activity and looser PAM constraints. Moreover, we found the existing one-pot assay, which simultaneously performed Recombinase Polymerase Amplification (RPA) and Cas12a reaction in one system, resulted in the loss of single-base discrimination during diagnosis. Therefore, we designed a reaction vessel that physically separated the RPA and Cas12a steps while maintaining a closed system. This isolated but closed system made diagnostics more sensitive and specific and effectively prevented contamination. This shelved Mb2Cas12a-RRVRR variant-mediated assay detected various targets in less than 15 min and exhibited equal or greater sensitivity than qPCR when detecting bacterial pathogens, plant RNA viruses and genetically modified crops. Overall, our findings further improved the efficiency of the current CRISPR-based diagnostic system and undoubtedly have great potential for highly sensitive and specific detection of multiple sample types.
Subject(s)
Nucleic Acids , Crops, Agricultural , Plants, Genetically Modified , RNA, Plant , Recombinases/genetics , CRISPR-Cas Systems/geneticsABSTRACT
Many pathogenic fungi exploit stomata as invasion routes, causing destructive diseases of major cereal crops. Intensive interaction is expected to occur between guard cells and fungi. In the present study, we took advantage of well-conserved molecules derived from the fungal cell wall, chitin oligosaccharide (CTOS), and chitosan oligosaccharide (CSOS) to study how guard cells respond to fungal invasion. In Arabidopsis, CTOS induced stomatal closure through a signaling mediated by its receptor CERK1, Ca2+, and a major S-type anion channel, SLAC1. CSOS, which is converted from CTOS by chitin deacetylases from invading fungi, did not induce stomatal closure, suggesting that this conversion is a fungal strategy to evade stomatal closure. At higher concentrations, CSOS but not CTOS induced guard cell death in a manner dependent on Ca2+ but not CERK1. These results suggest that stomatal immunity against fungal invasion comprises not only CTOS-induced stomatal closure but also CSOS-induced guard cell death.
Subject(s)
Chitin/metabolism , Plant Stomata/immunology , Plant Stomata/metabolism , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/physiology , Calcium/metabolism , Cell Death/drug effects , Chitin/physiology , Chitosan/metabolism , Fungi/metabolism , Protein Kinases/metabolism , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/physiology , Signal Transduction/drug effectsABSTRACT
The oomycete pathogen Hyaloperonospora arabidopsidis delivers diverse effector proteins into host plant cells to suppress the plant's innate immunity. In this study, we investigate the mechanism of action of a conserved RxLR effector, HaRxLL470, in suppressing plant immunity. Genomic, molecular and biochemical analyses were performed to investigate the function of HaRxLL470 and the mechanism of the interaction between HaRxLL470 and the target host protein during H. arabidopsidis infection. We report that HaRxLL470 enhances plant susceptibility to H. arabidopsidis isolate Noco2 by interacting with the host photomorphogenesis regulator protein HY5. Our results demonstrate that HY5 is not only an important component in the regulation of light signalling, but also positively regulates host plant immunity against H. arabidopsidis by transcriptional activation of defense-related genes. We show that the interaction between HaRxLL470 and HY5 compromises the function of HY5 as a transcription factor by attenuating its DNA-binding activity. The present study demonstrates that HY5 positively regulates host plant defense against H. arabidopsidis whereas HaRxLL470, a conserved RxLR effector across oomycete pathogens, enhances pathogenicity by interacting with HY5 and suppressing transcriptional activation of defense-related genes.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Oomycetes , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Basic-Leucine Zipper Transcription Factors , DNA , Gene Expression Regulation, Plant , Host-Pathogen Interactions , Oomycetes/metabolism , Plant Diseases , Plant ImmunityABSTRACT
KEY MESSAGE: 5-aminolevulinic acid (ALA) modulates various defense systems in plants and confers abiotic stress tolerance. Enhancement of crop production is a challenge due to numerous abiotic stresses such as, salinity, drought, temperature, heavy metals, and UV. Plants often face one or more abiotic stresses in their life cycle because of the challenging growing environment which results in reduction of growth and yield. Diverse studies have been conducted to discern suitable mitigation strategies to enhance crop production by minimizing abiotic stress. Exogenous application of different plant growth regulators is a well-renowned approach to ameliorate adverse effects of abiotic stresses on crop plants. Among the numerous plant growth regulators, 5-aminolevulinic acid (ALA) is a novel plant growth regulator, also well-known to alleviate the injurious effects of abiotic stresses in plants. ALA enhances abiotic stress tolerance as well as growth and yield by regulating photosynthetic and antioxidant machineries and nutrient uptake in plants. However, the regulatory roles of ALA in plants under different stresses have not been studied and assembled systematically. Also, ALA-mediated abiotic stress tolerance mechanisms have not been fully elucidated yet. Therefore, this review discusses the role of ALA in crop growth enhancement as well as its ameliorative role in abiotic stress mitigation and also discusses the ALA-mediated abiotic stress tolerance mechanisms and its limitation and future promises for sustainable crop production.
Subject(s)
Aminolevulinic Acid/metabolism , Plant Physiological Phenomena , Stress, Physiological/physiology , Aminolevulinic Acid/pharmacology , Crops, Agricultural/physiology , Droughts , Metals, Heavy/toxicity , Plant Growth Regulators/metabolism , Salinity , Soil Pollutants/toxicity , Stress, Physiological/drug effectsABSTRACT
Production of reactive oxygen species (ROS) is a key signal event for methyl jasmonate (MeJA)- and abscisic acid (ABA)-induced stomatal closure. We recently showed that reactive carbonyl species (RCS) stimulates stomatal closure as an intermediate downstream of hydrogen peroxide (H2O2) production in the ABA signaling pathway in guard cells of Nicotiana tabacum and Arabidopsis thaliana. In this study, we examined whether RCS functions as an intermediate downstream of H2O2 production in MeJA signaling in guard cells using transgenic tobacco plants overexpressing A. thaliana 2-alkenal reductase (n-alkanal + NAD(P)+ â 2-alkenal + NAD(P)H + H+) (AER-OE tobacco) and Arabidopsis plants. The stomatal closure induced by MeJA was impaired in the AER-OE tobacco and was inhibited by RCS scavengers, carnosine and pyridoxamine, in the wild-type (WT) tobacco plants and Arabidopsis plants. Application of MeJA significantly induced the accumulation of RCS, including acrolein and 4-hydroxy-(E)-2-nonenal, in the WT tobacco but not in the AER-OE plants. Application of MeJA induced H2O2 production in the WT tobacco and the AER-OE plants and the H2O2 production was not inhibited by the RCS scavengers. These results suggest that RCS functions as an intermediate downstream of ROS production in MeJA signaling and in ABA signaling in guard cells.
Subject(s)
Acetates/metabolism , Cyclopentanes/metabolism , Oxylipins/metabolism , Plant Growth Regulators/physiology , Plant Stomata/physiology , Abscisic Acid/metabolism , Arabidopsis/metabolism , Arabidopsis/physiology , Hydrogen Peroxide/metabolism , Plant Growth Regulators/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Nicotiana/metabolism , Nicotiana/physiologyABSTRACT
The glucosinolate-myrosinase system is a well-known defense system that has been shown to induce stomatal closure in Brassicales. Isothiocyanates are highly reactive hydrolysates of glucosinolates, and an isothiocyanate, allyl isothiocyanate (AITC), induces stomatal closure accompanied by elevation of free cytosolic Ca2+ concentration ([Ca2+]cyt) in Arabidopsis. It remains unknown whether AITC inhibits light-induced stomatal opening. This study investigated the role of Ca2+ in AITC-induced stomatal closure and inhibition of light-induced stomatal opening. AITC induced stomatal closure and inhibited light-induced stomatal opening in a dose-dependent manner. A Ca2+ channel inhibitor, La3+, a Ca2+chelator, EGTA, and an inhibitor of Ca2+ release from internal stores, nicotinamide, inhibited AITC-induced [Ca2+]cyt elevation and stomatal closure, but did not affect inhibition of light-induced stomatal opening. AITC activated non-selective Ca2+-permeable cation channels and inhibited inward-rectifying K+ (K+in) channels in a Ca2+-independent manner. AITC also inhibited stomatal opening induced by fusicoccin, a plasma membrane H+-ATPase activator, but had no significant effect on fusicoccin-induced phosphorylation of the penultimate threonine of H+-ATPase. Taken together, these results suggest that AITC induces Ca2+ influx and Ca2+ release to elevate [Ca2+]cyt, which is essential for AITC-induced stomatal closure but not for inhibition of K+in channels and light-induced stomatal opening.
Subject(s)
Arabidopsis , Plant Stomata , Calcium , Isothiocyanates/pharmacologyABSTRACT
We have demonstrated that reactive carbonyl species (RCS) function as an intermediate downstream of hydrogen peroxide (H2O2) production in abscisic acid (ABA) signaling for stomatal closure in guard cells using transgenic tobacco plants overexpressing alkenal reductase. We investigated the conversion of the RCS production into downstream signaling events in the guard cells. Both ABA and H2O2 induced production of the RCS, such as acrolein and 4-hydroxy-(E)-2-nonenal (HNE), in epidermal tissues of wild-type Arabidopsis thaliana plants. Application of the RCS scavengers, carnosine and pyridoxamine, did not affect the ABA-induced H2O2 production but inhibited the ABA- and H2O2-induced stomatal closure. Both acrolein and HNE induced stomatal closure in a plasma membrane NAD(P)H oxidase mutant atrbohD atrbohF as well as in the wild type, but not in a calcium-dependent kinase mutant cpk6. Acrolein activated plasma membrane Ca2+-permeable cation channels, triggered cytosolic free Ca2+ concentration ([Ca2+]cyt) elevation, and induced stomatal closure accompanied by depletion of glutathione in the guard cells. These results suggest that RCS production is a signaling event between the ROS production and [Ca2+]cyt elevation during guard cell ABA signaling.
Subject(s)
Abscisic Acid/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Hydrogen Peroxide/metabolism , Phytochrome/metabolism , Signal TransductionABSTRACT
Salicylic acid (SA) induces stomatal closure sharing several components with abscisic acid (ABA) and methyl jasmonate (MeJA) signaling. We have previously shown that two guard cell-preferential mitogen-activated protein kinases (MAPKs), MPK9 and MPK12, positively regulate ABA signaling and MeJA signaling in Arabidopsis thaliana. In this study, we examined whether these two MAPKs are involved in SA-induced stomatal closure using genetic mutants and a pharmacological, MAPKK inhibitor. Salicylic acid induced stomatal closure in mpk9 and mpk12 single mutants but not in mpk9 mpk12 double mutants. The MAPKK inhibitor PD98059 inhibited SA-induced stomatal closure in wild-type plants. Salicylic acid induced extracellular reactive oxygen species (ROS) production, intracellular ROS accumulation, and cytosolic alkalization in the mpk9, mpk12, and mpk9 mpk12 mutants. Moreover, SA-activated S-type anion channels in guard cells of wild-type plants but not in guard cells of mpk9 mpk12 double mutants. These results imply that MPK9 and MPK12 are positive regulators of SA signaling in Arabidopsis guard cells.
Subject(s)
Arabidopsis/drug effects , Gene Expression Regulation, Plant , Plant Stomata/drug effects , Salicylic Acid/pharmacology , Abscisic Acid/metabolism , Abscisic Acid/pharmacology , Acetates/metabolism , Acetates/pharmacology , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cyclopentanes/metabolism , Cyclopentanes/pharmacology , Flavonoids/pharmacology , Hydrogen-Ion Concentration , Membrane Potentials/drug effects , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Mutation , Oxylipins/metabolism , Oxylipins/pharmacology , Plant Growth Regulators/metabolism , Plant Growth Regulators/pharmacology , Plant Stomata/genetics , Plant Stomata/metabolism , Protein Kinase Inhibitors/pharmacology , Reactive Oxygen Species/agonists , Reactive Oxygen Species/metabolism , Salicylic Acid/metabolism , Signal Transduction , Voltage-Dependent Anion Channels/genetics , Voltage-Dependent Anion Channels/metabolismABSTRACT
Drought is responsible for a massive reduction in crop yields. In response to drought, plants synthesize the hormone ABA, which induces stomatal closure, thus reducing water loss. In guard cells, ABA triggers production of reactive oxygen species (ROS), which is mediated by NAD(P)H oxidases. The production of ROS is a key factor for ABA-induced stomatal closure, but it remains to be clarified how the production of ROS is transduced into downstream signaling components in guard cells. We investigated roles of reactive carbonyl species (RCS) in ABA-induced stomatal closure using transgenic tobacco (Nicotiana tabacum) overexpressing Arabidopsis 2-alkenal reductase (AER-OE), which scavenges RCS. ABA and hydrogen peroxide (H2O2) induced accumulation of RCS including acrolein and 4-hydroxy-(E)-2-nonenal in wild-type tobacco but not in AER-OE. Stomatal closure and RCS accumulation in response to ABA and H2O2 were inhibited in AER-OE unlike in the wild type, while ABA-induced H2O2 production in guard cells was observed in AER-OE as well as in the wild type. Moreover, ABA inhibited inward-rectifying K+ channels in wild-type guard cells but not in AER-OE guard cells. These results suggest that RCS is involved in ABA-induced stomatal closure and functions downstream of H2O2 production in the ABA signaling pathway in guard cells.
Subject(s)
Abscisic Acid/metabolism , Arabidopsis/genetics , Free Radicals/metabolism , Nicotiana/physiology , Plant Growth Regulators/metabolism , Signal Transduction , Arabidopsis Proteins/genetics , Droughts , Free Radicals/analysis , Gene Expression , Hydrogen Peroxide/analysis , Hydrogen Peroxide/metabolism , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Plant Stomata/genetics , Plant Stomata/physiology , Plants, Genetically Modified , Reactive Oxygen Species/analysis , Reactive Oxygen Species/metabolism , Nicotiana/enzymology , Nicotiana/geneticsABSTRACT
We recently demonstrated that yeast elicitor (YEL)-induced stomatal closure requires a Ca(2+)-dependent kinase, CPK6. A Ca(2+)-independent kinase, Open Stomata 1 (OST1), is involved in stomatal closure induced by various stimuli including ABA. In the present study, we investigated the role of OST1 in YEL-induced stomatal closure in Arabidopsis using a knock-out mutant, ost1-3, and a kinase-deficient mutant, ost1-2. YEL did not induce stomatal closure or activation of guard cell S-type anion channels in the ost1 mutants unlike in wild-type plants. However, YEL did not increase OST1 kinase activity in wild-type guard cells. The YEL-induced stomatal closure and activation of S-type anion channels were also impaired in a gain-of-function mutant of a clade A type 2C protein phosphatase (ABA INSENSITIVE 1), abi1-1C. In the ost1 mutants like in the wild type, YEL induced H2O2 accumulation, activation of non-selective Ca(2+)-permeable cation (ICa) channels and transient elevations in cytosolic free Ca(2+) concentration ([Ca(2+)]cyt) in guard cells. These results suggest that OST1 kinase is essential for stomatal closure and activation of S-type anion channels induced by YEL and that OST1 is not involved in H2O2 accumulation, ICa channel activation or [Ca(2+)]cyt elevations in guard cells induced by YEL.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Arabidopsis/physiology , Plant Stomata/physiology , Protein Kinases/metabolism , Saccharomyces cerevisiae/metabolism , Anions , Arabidopsis/genetics , Cold Shock Proteins and Peptides/metabolism , Gene Expression Regulation, Plant , Hydrogen Peroxide/metabolism , Ion Channel Gating , Mutation , Plant Stomata/cytology , Plant Stomata/enzymology , Plant Stomata/genetics , Protoplasts/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolismABSTRACT
Isothiocyanates are enzymatically produced from glucosinolates in plants, and allyl isothiocyanate (AITC) induces stomatal closure in Arabidopsis thaliana. In this study, we investigated stomatal responses to AITC in Vicia faba. AITC-induced stomatal closure accompanied by reactive oxygen species (ROS) and NO production, cytosolic alkalization and glutathione (GSH) depletion in V. faba. GSH monoethyl ester induced stomatal reopening and suppressed AITC-induced GSH depletion in guard cells. Exogenous catalase and a peroxidase inhibitor, salicylhydroxamic acid, inhibited AITC-induced stomatal closure, unlike an NAD(P)H oxidase inhibitor, diphenylene iodonium chloride. The peroxidase inhibitor also abolished the AITC-induced ROS production, NO production, and cytosolic alkalization. AITC-induced stomatal closure was suppressed by an NO scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and an agent to acidify cytosol, butyrate. These results indicate that AITC-induced stomatal closure in V. faba as well as in A. thaliana and suggest that AITC signaling in guard cells is conserved in both plants.
Subject(s)
Isothiocyanates/pharmacology , Plant Stomata/drug effects , Signal Transduction , Vicia faba/drug effects , Arabidopsis/drug effects , Arabidopsis/metabolism , Benzoates/pharmacology , Butyric Acid/pharmacology , Catalase/antagonists & inhibitors , Catalase/metabolism , Cytosol/drug effects , Cytosol/metabolism , Free Radical Scavengers/pharmacology , Glutathione/analogs & derivatives , Glutathione/metabolism , Glutathione/pharmacology , Imidazoles/pharmacology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/metabolism , Onium Compounds/pharmacology , Peroxidase/antagonists & inhibitors , Peroxidase/metabolism , Plant Stomata/metabolism , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism , Salicylamides/pharmacology , Vicia faba/metabolismABSTRACT
Acrolein is a reactive α,ß-unsaturated aldehyde derived from lipid peroxides, which are produced in plants under a variety of stress. We investigated effects of acrolein on light-induced stomatal opening using Arabidopsis thaliana. Acrolein inhibited light-induced stomatal opening in a dose-dependent manner. Acrolein at 100 µM inhibited plasma membrane inward-rectifying potassium (Kin) channels in guard cells. Acrolein at 100 µM inhibited Kin channel KAT1 expressed in a heterologous system using Xenopus leaves oocytes. These results suggest that acrolein inhibits light-induced stomatal opening through inhibition of Kin channels in guard cells.
Subject(s)
Acrolein/pharmacology , Arabidopsis Proteins/antagonists & inhibitors , Arabidopsis/drug effects , Plant Cells/drug effects , Plant Stomata/drug effects , Potassium Channels, Inwardly Rectifying/antagonists & inhibitors , Potassium/metabolism , Acrolein/metabolism , Animals , Arabidopsis/metabolism , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Dose-Response Relationship, Drug , Gene Expression , Kinetics , Light , Membrane Potentials/drug effects , Oocytes/cytology , Oocytes/drug effects , Oocytes/metabolism , Patch-Clamp Techniques , Plant Cells/metabolism , Plant Cells/radiation effects , Plant Stomata/metabolism , Plant Stomata/radiation effects , Potassium Channels, Inwardly Rectifying/genetics , Potassium Channels, Inwardly Rectifying/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Xenopus laevisABSTRACT
Abscisic acid (ABA) induces stomatal closure and inhibits light-induced stomatal opening. The mechanisms in these two processes are not necessarily the same. It has been postulated that the ABA receptors involved in opening inhibition are different from those involved in closure induction. Here, we provide evidence that four recently identified ABA receptors (PYRABACTIN RESISTANCE1 [PYR1], PYRABACTIN RESISTANCE-LIKE1 [PYL1], PYL2, and PYL4) are not sufficient for opening inhibition in Arabidopsis (Arabidopsis thaliana). ABA-induced stomatal closure was impaired in the pyr1/pyl1/pyl2/pyl4 quadruple ABA receptor mutant. ABA inhibition of the opening of the mutant's stomata remained intact. ABA did not induce either the production of reactive oxygen species and nitric oxide or the alkalization of the cytosol in the quadruple mutant, in accordance with the closure phenotype. Whole cell patch-clamp analysis of inward-rectifying K(+) current in guard cells showed a partial inhibition by ABA, indicating that the ABA sensitivity of the mutant was not fully impaired. ABA substantially inhibited blue light-induced phosphorylation of H(+)-ATPase in guard cells in both the mutant and the wild type. On the other hand, in a knockout mutant of the SNF1-related protein kinase, srk2e, stomatal opening and closure, reactive oxygen species and nitric oxide production, cytosolic alkalization, inward-rectifying K(+) current inactivation, and H(+)-ATPase phosphorylation were not sensitive to ABA.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis/physiology , Plant Stomata/drug effects , Plant Stomata/physiology , Signal Transduction/drug effects , Alkalies/metabolism , Arabidopsis/drug effects , Arabidopsis/enzymology , Arabidopsis/radiation effects , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Cell Membrane/drug effects , Cell Membrane/enzymology , Cell Membrane/radiation effects , Cytosol/metabolism , Ion Channel Gating/drug effects , Ion Channel Gating/radiation effects , Light , Mutation/genetics , Nitric Oxide/metabolism , Phosphorylation/drug effects , Phosphorylation/radiation effects , Plant Stomata/cytology , Plant Stomata/radiation effects , Proton-Translocating ATPases/metabolism , Protoplasts/drug effects , Protoplasts/metabolism , Protoplasts/radiation effects , Reactive Oxygen Species/metabolismABSTRACT
Yeast elicitor (YEL) induces stomatal closure that is mediated by a Ca(2+)-dependent signaling pathway. A Ca(2+)-dependent protein kinase, CPK6, positively regulates activation of ion channels in abscisic acid and methyl jasmonate signaling, leading to stomatal closure in Arabidopsis (Arabidopsis thaliana). YEL also inhibits light-induced stomatal opening. However, it remains unknown whether CPK6 is involved in induction by YEL of stomatal closure or in inhibition by YEL of light-induced stomatal opening. In this study, we investigated the roles of CPK6 in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis. Disruption of CPK6 gene impaired induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening. Activation by YEL of nonselective Ca(2+)-permeable cation channels was impaired in cpk6-2 guard cells, and transient elevations elicited by YEL in cytosolic-free Ca(2+) concentration were suppressed in cpk6-2 and cpk6-1 guard cells. YEL activated slow anion channels in wild-type guard cells but not in cpk6-2 or cpk6-1 and inhibited inward-rectifying K(+) channels in wild-type guard cells but not in cpk6-2 or cpk6-1. The cpk6-2 and cpk6-1 mutations inhibited YEL-induced hydrogen peroxide accumulation in guard cells and apoplast of rosette leaves but did not affect YEL-induced hydrogen peroxide production in the apoplast of rosette leaves. These results suggest that CPK6 positively functions in induction by YEL of stomatal closure and inhibition by YEL of light-induced stomatal opening in Arabidopsis and is a convergent point of signaling pathways for stomatal closure in response to abiotic and biotic stress.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/physiology , Arabidopsis/radiation effects , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Light , Plant Stomata/physiology , Plant Stomata/radiation effects , Saccharomyces cerevisiae/metabolism , Arabidopsis/enzymology , Arabidopsis/microbiology , Hydrogen Peroxide/metabolism , Ion Channel Gating/radiation effects , Mutation/genetics , Plant Stomata/cytology , Plant Stomata/microbiology , Protoplasts/metabolism , Protoplasts/radiation effectsABSTRACT
Plants possess diverse cell types and intricate regulatory mechanisms to adapt to the ever-changing environment of nature. Various strategies have been employed to study cell types and their developmental progressions, including single-cell sequencing methods which provide high-dimensional catalogs to address biological concerns. In recent years, single-cell sequencing technologies in transcriptomics, epigenomics, proteomics, metabolomics, and spatial transcriptomics have been increasingly used in plant science to reveal intricate biological relationships at the single-cell level. However, the application of single-cell technologies to plants is more limited due to the challenges posed by cell structure. This review outlines the advancements in single-cell omics technologies, their implications in plant systems, future research applications, and the challenges of single-cell omics in plant systems.
Subject(s)
Genomics , Metabolomics , Plants , Proteomics , Single-Cell Analysis , Single-Cell Analysis/methods , Plants/genetics , Plants/metabolism , Metabolomics/methods , Proteomics/methods , Genomics/methods , Epigenomics/methods , Transcriptome/geneticsABSTRACT
Phospholipase D (PLD) is involved in responses to abiotic stress and abscisic acid (ABA) signaling. To investigate the roles of two Arabidopsis (Arabidopsis thaliana) PLDs, PLDα1 and PLDδ, in ABA signaling in guard cells, we analyzed ABA responses in guard cells using Arabidopsis wild type, pldα1 and pldδ single mutants, and a pldα1 pldδ double mutant. ABA-induced stomatal closure was suppressed in the pldα1 pldδ double mutant but not in the pld single mutants. The pldα1 and pldδ mutations reduced ABA-induced phosphatidic acid production in epidermal tissues. Expression of either PLDα1 or PLDδ complemented the double mutant stomatal phenotype. ABA-induced stomatal closure in both pldα1 and pldδ single mutants was inhibited by a PLD inhibitor (1-butanol ), suggesting that both PLDα1 and PLDδ function in ABA-induced stomatal closure. During ABA-induced stomatal closure, wild-type guard cells accumulate reactive oxygen species and nitric oxide and undergo cytosolic alkalization, but these changes are reduced in guard cells of the pldα1 pldδ double mutant. Inward-rectifying K(+) channel currents of guard cells were inhibited by ABA in the wild type but not in the pldα1 pldδ double mutant. ABA inhibited stomatal opening in the wild type and the pldδ mutant but not in the pldα1 mutant. In wild-type rosette leaves, ABA significantly increased PLDδ transcript levels but did not change PLDα1 transcript levels. Furthermore, the pldα1 and pldδ mutations mitigated ABA inhibition of seed germination. These results suggest that PLDα1 and PLDδ cooperate in ABA signaling in guard cells but that their functions do not completely overlap.
Subject(s)
Abscisic Acid/pharmacology , Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Phospholipase D/metabolism , Plant Stomata/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Cytosol/drug effects , Cytosol/metabolism , Genetic Complementation Test , Germination/drug effects , Mutation , Nitric Oxide/metabolism , Phenotype , Plant Leaves/drug effects , Plant Leaves/metabolism , Plant Stomata/drug effects , Potassium Channels/drug effects , Reactive Oxygen Species/metabolism , Seeds/drug effects , Seeds/metabolism , Signal Transduction , Transcription, GeneticABSTRACT
Isothiocyanates, nitriles, and thiocyanates are degradation products of glucosinolates in crucifer plants. In this study, we investigated the stomatal response to allyl isothiocyanate (AITC), 3-butenenitrile (3BN), and ethyl thiocyanate (ESCN) in Arabidopsis. AITC, 3BN, and ESCN induced stomatal closure in the wild type and the atrbohD atrbohF mutant. Stomatal closure was inhibited by catalase and salicylhydroxamic acid (SHAM). The degradation products induced extracellular reactive oxygen species (ROS) production in the rosette leaves, and intracellular ROS accumulation, NO production, and cytosolic free calcium concentration ([Ca(2+)]cyt) oscillations in guard cells, which were inhibited by SHAM. These results suggest that glucosinolate degradation products induce stomatal closure accompanied by extracellular ROS production mediated by SHAM-sensitive peroxidases, intracellular ROS accumulation, and [Ca(2+)]cyt oscillation in Arabidopsis.
Subject(s)
Arabidopsis/drug effects , Glucosinolates/metabolism , Isothiocyanates/pharmacology , Nitriles/pharmacology , Plant Stomata/drug effects , Reactive Oxygen Species/metabolism , Thiocyanates/pharmacology , Arabidopsis/anatomy & histology , Arabidopsis/cytology , Arabidopsis/metabolism , Calcium/metabolism , Cytosol/drug effects , Cytosol/metabolism , Isothiocyanates/metabolism , Nitric Oxide/biosynthesis , Nitriles/metabolism , Peroxidase/metabolism , Plant Leaves/cytology , Plant Leaves/drug effects , Plant Stomata/anatomy & histology , Thiocyanates/metabolismABSTRACT
Stomatal pores in the plant epidermis open and close to regulate gas exchange between leaves and the atmosphere. Upon light stimulation, the plasma membrane (PM) H+-ATPase is phosphorylated and activated via an intracellular signal transduction pathway in stomatal guard cells, providing a primary driving force for the opening movement. To uncover and manipulate this stomatal opening pathway, we screened a chemical library and identified benzyl isothiocyanate (BITC), a Brassicales-specific metabolite, as a potent stomatal-opening inhibitor that suppresses PM H+-ATPase phosphorylation. We further developed BITC derivatives with multiple isothiocyanate groups (multi-ITCs), which demonstrate inhibitory activity on stomatal opening up to 66 times stronger, as well as a longer duration of the effect and negligible toxicity. The multi-ITC treatment inhibits plant leaf wilting in both short (1.5 h) and long-term (24 h) periods. Our research elucidates the biological function of BITC and its use as an agrochemical that confers drought tolerance on plants by suppressing stomatal opening.
Subject(s)
Arabidopsis Proteins , Plant Stomata , Plant Stomata/metabolism , Light , Drought Resistance , Proton-Translocating ATPases/metabolism , Isothiocyanates/pharmacology , Isothiocyanates/metabolism , Arabidopsis Proteins/metabolismABSTRACT
Glutathione (GSH) is involved in abscisic acid (ABA)- and methyl jasmonate (MeJA)-induced stomatal closure in Arabidopsis thaliana. In this study, we examined the effects of GSH-decreasing chemicals, p-nitrobenzyl chloride (PNBC), iodomethane (IDM), and ethacrynic acid (EA), on ABA- and MeJA-induced stomatal closure in Arabidopsis. Treatments with PNBC, IDM, and EA decreased GSH contents in guard cells. Depletion of GSH by PNBC and IDM enhanced ABA- and MeJA-induced stomatal closure and inhibition of light-induced stomatal opening by ABA, whereas EA did not enhance either ABA- and MeJA-induced stomatal closure or inhibition of light-induced stomatal opening by ABA. Depletion of GSH did not significantly increase the production of the reactive oxygen species (ROS), cytosolic alkalization, or cytosolic Ca(2+) oscillation induced by ABA and MeJA. These results indicate that depletion of GSH enhances ABA- and MeJA-induced stomatal closure without affecting ROS production, cytosolic alkalization, or cytosolic Ca(2+) oscillation in guard cells of Arabidopsis.