保卫细胞中ABA信号调控机制研究进展

脱落酸(ABA)具有调节植物快速响应逆境的重要功能。植物细胞中ABA核心信号通路由ABA受体PYR1/PYLs/ RCARs、A类碱性蛋白磷酸酶PP2Cs和Snf1相关蛋白激酶SnRK2s组成。活性氧(ROS)和Ca²⁺是保卫细胞中的重要第二信使, 调控ABA诱导的气孔关闭。该文对保卫细胞中核心ABA信号蛋白的调控以及ROS和Ca²⁺介导的ABA信号转导等最新研究成果进行综述, 旨在阐明保卫细胞中ABA信号调控机制。     

Analysis of abscisic acid responsive proteins in Brassica napus guard cells by multiplexed isobaric tagging

Guard cells, which form stomata on the leaf epidermis, play important roles in plant gas exchange and defense against pathogens. Abscisic acid (ABA) is a phytohormone that can be induced by drought and leads to stomatal closure. Guard cells have been a premier model system for studying ABA signal transduction. Despite significant progress on the identification of molecular components in the ABA signaling pathway, our knowledge of the protein components is very limited. Here, we employ a recently developed multiplexed isobaric tagging technology to identify ABA-responsive proteins in Brassica napus guard cells. A total of 431 unique proteins were identified with relative quantitative information in control and ABA-treated samples. Proteins involved in stress and defense constituted a major group among the 66 proteins with increased abundance. Thirty-eight proteins were decreased in abundance and fell into several functional groups including metabolism and protein synthesis. Many of the proteins have not been reported as being ABA responsive or involved in stomatal movement. A large percentage of the protein-coding genes contained ABA-responsive elements. This study not only established a comprehensive inventory of ABA-responsive proteins, but also identified new proteins for further investigation of their functions in guard cell ABA signaling.     

Identification of features regulating OST1 kinase activity and OST1 function in guard cells

The phytohormone abscisic acid (ABA) mediates drought responses in plants and, in particular, triggers stomatal closure. Snf1-related kinase 2 (SnRK2) proteins from several plant species have been implicated in ABA-signaling pathways. In Arabidopsis (Arabidopsis thaliana) guard cells, OPEN STOMATA 1 (OST1)/SRK2E/SnRK2-6 is a critical positive regulator of ABA signal transduction. A better understanding of the mechanisms responsible for SnRK2 protein kinase activation is thus a major goal toward understanding ABA signal transduction. Here, we report successful purification of OST1 produced in Escherichia coli: The protein is active and autophosphorylates. Using mass spectrometry, we identified five target residues of autophosphorylation in recombinant OST1. Sequence analysis delineates two conserved boxes located in the carboxy-terminal moiety of OST1 after the catalytic domain: the SnRK2-specific box (glutamine-303 to proline-318) and the ABA-specific box (leucine-333 to methionine-362). Site-directed mutagenesis and serial deletions reveal that serine (Ser)-175 in the activation loop and the SnRK2-specific box are critical for the activity of recombinant OST1 kinase. Targeted expression of variants of OST1 kinase in guard cells uncovered additional features that are critical for OST1 function in ABA signaling, although not required for OST1 kinase activity: Ser-7, Ser-18, and Ser-29 and the ABA-specific box. Ser-7, Ser-18, Ser-29, and Ser-43 represent putative targets for regulatory phosphorylation and the ABA-specific box may be a target for the binding of signaling partners in guard cells.     

Arabidopsis OST1 protein kinase mediates the regulation of stomatal aperture by abscisic acid and acts upstream of reactive oxygen species production

During drought, the plant hormone abscisic acid (ABA) triggers stomatal closure, thus reducing water loss. Using infrared thermography, we isolated two allelic Arabidopsis mutants (ost1-1 and ost1-2) impaired in the ability to limit their transpiration upon drought. These recessive ost1 mutations disrupted ABA induction of stomatal closure as well as ABA inhibition of light-induced stomatal opening. By contrast, the ost1 mutations did not affect stomatal regulation by light or CO(2), suggesting that OST1 is involved specifically in ABA signaling. The OST1 gene was isolated by positional cloning and was found to be expressed in stomatal guard cells and vascular tissue. In-gel assays indicated that OST1 is an ABA-activated protein kinase related to the Vicia faba ABA-activated protein kinase (AAPK). Reactive oxygen species (ROS) were shown recently to be an essential intermediate in guard cell ABA signaling. ABA-induced ROS production was disrupted in ost1 guard cells, whereas applied H(2)O(2) or calcium elicited the same degree of stomatal closure in ost1 as in the wild type. These results suggest that OST1 acts in the interval between ABA perception and ROS production. The relative positions of ost1 and the other ABA-insensitive mutations in the ABA signaling network (abi1-1, abi2-1, and gca2) are discussed.     

The stomata frontline of plant interaction with the environment-perspectives from hormone regulation

Plants have evolved elaborate mechanisms to perceive and integrate signals from various environmental conditions.On leaf surface,stomata formed by pairs of guard cells mediate gas exchange,water transp...     

NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis

Reactive oxygen species (ROS) have been proposed to function as second messengers in abscisic acid (ABA) signaling in guard cells. However, the question whether ROS production is indeed required for ABA signal transduction in vivo has not yet been addressed, and the molecular mechanisms mediating ROS production during ABA signaling remain unknown. Here, we report identification of two partially redundant Arabidopsis guard cell-expressed NADPH oxidase catalytic subunit genes, AtrbohD and AtrbohF, in which gene disruption impairs ABA signaling. atrbohD/F double mutations impair ABA-induced stomatal closing, ABA promotion of ROS production, ABA-induced cytosolic Ca(2+) increases and ABA- activation of plasma membrane Ca(2+)-permeable channels in guard cells. Exogenous H(2)O(2) rescues both Ca(2+) channel activation and stomatal closing in atrbohD/F. ABA inhibition of seed germination and root elongation are impaired in atrbohD/F, suggesting more general roles for ROS and NADPH oxidases in ABA signaling. These data provide direct molecular genetic and cell biological evidence that ROS are rate-limiting second messengers in ABA signaling, and that the AtrbohD and AtrbohF NADPH oxidases function in guard cell ABA signal transduction.     

Constitutive activation of a plasma membrane H(+)-ATPase prevents abscisic acid-mediated stomatal closure

Light activates proton (H(+))-ATPases in guard cells, to drive hyperpolarization of the plasma membrane to initiate stomatal opening, allowing diffusion of ambient CO(2) to photosynthetic tissues. Light to darkness transition, high CO(2) levels and the stress hormone abscisic acid (ABA) promote stomatal closing. The overall H(+)-ATPase activity is diminished by ABA treatments, but the significance of this phenomenon in relationship to stomatal closure is still debated. We report two dominant mutations in the OPEN STOMATA2 (OST2) locus of Arabidopsis that completely abolish stomatal response to ABA, but importantly, to a much lesser extent the responses to CO(2) and darkness. The OST2 gene encodes the major plasma membrane H(+)-ATPase AHA1, and both mutations cause constitutive activity of this pump, leading to necrotic lesions. H(+)-ATPases have been traditionally assumed to be general endpoints of all signaling pathways affecting membrane polarization and transport. Our results provide evidence that AHA1 is a distinct component of an ABA-directed signaling pathway, and that dynamic downregulation of this pump during drought is an essential step in membrane depolarization to initiate stomatal closure.     

Phospholipase C is required for the control of stomatal aperture by ABA

The calcium-releasing second messenger inositol 1,4,5-trisphosphate is involved in the regulation of stomatal aperture by ABA. In other signalling pathways, inositol 1,4,5-trisphosphate is generated by the action of phospholipase C. We have studied the importance of phospholipase C in guard cell ABA-signalling pathways. Immunolocalisation of a calcium-activated phospholipase C confirmed the presence of phospholipase C in tobacco guard cells. Transgenic tobacco plants with considerably reduced levels of phospholipase C in their guard cells were only partially able to regulate their stomatal apertures in response to ABA. These results suggest that phospholipase C is involved in the amplification of the calcium signal responsible for reductions in stomatal aperture in response to ABA. As full ABA-induced inhibition of stomatal opening was not observed, our results support a role for the action of other calcium-releasing second messengers in the guard cell ABA-signalling pathway. It is not known whether these different calcium-releasing second messengers act in the same or parallel ABA-signalling pathways.     

ZmOST1 mediates abscisic acid regulation of guard cell ion channels and drought stress responses

The phytohormone abscisic acid (ABA) is an important mediator in the drought response, participating in, among other processes, stomatal movements. In Arabidopsis thaliana, the serine/threonine protein kinase, OST1, regulates this response, but the function of its maize homolog has yet to be established. Here, we isolated ZmOST1 and show that its encoded protein indeed acts to regulate guard cell movement. ZmOST1 was ubiquitously expressed throughout the plant, being highly expressed in guard cells, and inducible both by exogenous ABA and water stress. Transient expression of a ZmOST1-GFP fusion protein, in maize mesophyll protoplasts, indicated its subcellular localization in the cytoplasm and nucleus. A Zmost1 loss-of-function mutant exhibited reduced sensitivity to ABA-activated slow anion channels in maize guard cells, and reduced drought tolerance. Constitutive expression of ZmOST1, in an A. thaliana ost1-1 mutant rescued the phenotype with respect both to the sensitivity of guard cell slow anion currents to ABA treatment and stomatal closure. Our findings indicate a positive regulatory role for ZmOST1 in guard cell ABA signaling and drought response in maize plants.     

Cytoplasmic alkalization precedes reactive oxygen species production during methyl jasmonate- and abscisic acid-induced stomatal closure

Signaling events during abscisic acid (ABA) or methyl jasmonate (MJ)-induced stomatal closure were examined in Arabidopsis wild type, ABA-insensitive (ost1-2), and MJ-insensitive mutants (jar1-1) in order to examine a crosstalk between ABA and MJ signal transduction. Some of the experiments were performed on epidermal strips of Pisum sativum. Stomata of jar1-1 mutant plants are insensitive to MJ but are able to close in response to ABA. However, their sensitivity to ABA is less than that of wild-type plants. Reciprocally, the stomata of ost1-2 are insensitive to ABA but are able to close in response to MJ to a lesser extent compared to wild-type plants. Both MJ and ABA promote H(2)O(2) production in wild-type guard cells, while exogenous application of diphenylene iodonium (DPI) chloride, an inhibitor of NAD(P)H oxidases, results in the suppression of ABA- and MJ-induced stomatal closure. ABA elevates H(2)O(2) production in wild-type and jar1-1 guard cells but not in ost1-2, whereas MJ induces H(2)O(2) production in both wild-type and ost1-2 guard cells, but not in jar1-1. MJ-induced stomatal closing is suppressed in the NAD(P)H oxidase double mutant atrbohD/F and in the outward potassium channel mutant gork1. Furthermore, MJ induces alkalization in guard cell cytosol, and MJ-induced stomatal closing is inhibited by butyrate. Analyses of the kinetics of cytosolic pH changes and reactive oxygen species (ROS) production show that the alkalization of cytoplasm precedes ROS production during the stomatal response to both ABA and MJ. Our results further indicate that JAR1, as OST1, functions upstream of ROS produced by NAD(P)H oxidases and that the cytoplasmic alkalization precedes ROS production during MJ or ABA signal transduction in guard cells.     

The coronatine-insensitive 1 mutation reveals the hormonal signaling interaction between abscisic acid and methyl jasmonate in Arabidopsis guard cells. Specific impairment of ion channel activation and second messenger production

Methyl jasmonate (MeJA) elicits stomatal closing similar to abscisic acid (ABA), but whether the two compounds use similar or different signaling mechanisms in guard cells remains to be clarified. We investigated the effects of MeJA and ABA on second messenger production and ion channel activation in guard cells of wild-type Arabidopsis (Arabidopsis thaliana) and MeJA-insensitive coronatine-insensitive 1 (coi1) mutants. The coi1 mutation impaired MeJA-induced stomatal closing but not ABA-induced stomatal closing. MeJA as well as ABA induced production of reactive oxygen species (ROS) and nitric oxide (NO) in wild-type guard cells, whereas MeJA did not induce production of ROS and NO in coi1 guard cells. The experiments using an inhibitor and scavengers demonstrated that both ROS and NO are involved in MeJA-induced stomatal closing as well as ABA-induced stomatal closing. Not only ABA but also MeJA activated slow anion channels and Ca(2+) permeable cation channels in the plasma membrane of wild-type guard cell protoplasts. However, in coi1 guard cell protoplasts, MeJA did not elicit either slow anion currents or Ca(2+) permeable cation currents, but ABA activated both types of ion channels. Furthermore, to elucidate signaling interaction between ABA and MeJA in guard cells, we examined MeJA signaling in ABA-insensitive mutant ABA-insensitive 2 (abi2-1), whose ABA signal transduction cascade has some disruption downstream of ROS production and NO production. MeJA also did not induce stomatal closing but stimulated production of ROS and NO in abi2-1. These results suggest that MeJA triggers stomatal closing via a receptor distinct from the ABA receptor and that the coi1 mutation disrupts MeJA signaling upstream of the blanch point of ABA signaling and MeJA signaling in Arabidopsis guard cells.     

Use of infrared thermal imaging to isolate Arabidopsis mutants defective in stomatal regulation

In response to drought, plants synthesise the hormone abscisic acid (ABA), which triggers closure of the stomatal pores. This process is vital for plants to conserve water by reducing transpirational water loss. Moreover, recent studies have demonstrated the advantages of the Arabidopsis stomatal guard cell for combining genetic, molecular and biophysical approaches to characterise ABA action. However, genetic dissection of stomatal regulation has been limited by the difficulty of identifying a reliable phenotype for mutant screening. Leaf temperature can be used as an indicator to detect mutants with altered stomatal control, since transpiration causes leaf cooling. In this study, we optimised experimental conditions under which individual Arabidopsis plants with altered stomatal responses to drought can be identified by infrared thermography. These conditions were then used to perform a pilot screen for mutants that displayed a reduced ability to close their stomata and hence appeared colder than the wild type. Some of the mutants recovered were deficient in ABA accumulation, and corresponded to alleles of the ABA biosynthesis loci ABA1, ABA2 and ABA3. Interestingly, two of these novel aba2 alleles were able to intragenically complement the aba2-1 mutation. The remaining mutants showed reduced ABA responsiveness in guard cells. In addition to the previously known abi1-1 mutation, we isolated mutations at two novel loci designated as OST1 (OPEN STOMATA 1) and OST2. Remarkably, ost1 and ost2 represent, to our knowledge, the first Arabidopsis mutations altering ABA responsiveness in stomata and not in seeds.     

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.     

DOR: A link between an F-box protein and guard cell ABA signaling

Guard cells are a model system for studying signal transduction. F-box proteins, representing one of the largest gene families in Arabidopsis, have been shown to be involved in many developmental and physiological processes, including stress responses. However, it is unclear if there is a direct link between an F-box protein and the guard cell ABA signaling. DOR is a guard cell-preferential F-box protein, and our results suggested that it likely forms two negative feedback regulatory loops for the ABA-induced stomatal closure under drought conditions in Arabidopsis. These findings have a potential impact on genetically modifying drought stress responses in plants.Key words: DOR, F-box protein, guard cell, ABA signaling     

Thiol‐based redox proteins in abscisic acid and methyl jasmonate signaling in Brassica napus guard cells

Reversibly oxidized cysteine sulfhydryl groups serve as redox sensors or targets of redox sensing that are important in various physiological processes. However, little is known about redox‐sensitive proteins in guard cells and how they function in stomatal signaling. In this study, Brassica napus guard‐cell proteins altered by redox in response to abscisic acid (ABA) or methyl jasmonate (MeJA) were identified by complementary proteomics approaches, saturation differential in‐gel electrophoresis and isotope‐coded affinity tagging. In total, 65 and 118 potential redox‐responsive proteins were identified in ABA‐ and MeJA‐treated guard cells, respectively. All the proteins contain at least one cysteine, and over half of them are predicted to form intra‐molecular disulfide bonds. Most of the proteins fall into the functional groups of ‘energy’, ‘stress and defense’ and ‘metabolism’. Based on the peptide sequences identified by mass spectrometry, 30 proteins were common to ABA‐ and MeJA‐treated samples. A total of 44 cysteines were mapped in the identified proteins, and their levels of redox sensitivity were quantified. Two of the proteins, a sucrose non‐fermenting 1‐related protein kinase and an isopropylmalate dehydrogenase, were confirmed to be redox‐regulated and involved in stomatal movement. This study creates an inventory of potential redox switches, and highlights a protein redox regulatory mechanism in ABA and MeJA signal transduction in guard cells.     

CDPKs CPK6 and CPK3 Function in ABA Regulation of Guard Cell S-Type Anion- and Ca2+- Permeable Channels and Stomatal Closure

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca²⁺ in guard cell ion channel regulation. However, genetic mutants in Ca²⁺ sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca²⁺-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell–expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca²⁺ activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca²⁺-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca²⁺-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca²⁺ oscillation experiments revealed that Ca²⁺-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca²⁺-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca²⁺-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.     

Leucine-rich repeat receptor-like kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis

Abscisic acid (ABA) is important in seed maturation, seed dormancy, stomatal closure, and stress response. Many genes that function in ABA signal transduction pathways have been identified. However, most important signaling molecules involved in the perception of the ABA signal or with ABA receptors have not been identified yet. Receptor-like kinase1 (RPK1), a Leu-rich repeat (LRR) receptor kinase in the plasma membrane, is upregulated by ABA in Arabidopsis thaliana. Here, we show the phenotypes of T-DNA insertion mutants and RPK1-antisense plants. Repression of RPK1 expression in Arabidopsis decreased sensitivity to ABA during germination, growth, and stomatal closure; microarray and RNA gel analysis showed that many ABA-inducible genes are downregulated in these plants. Furthermore, overexpression of the RPK1 LRR domain alone or fused with the Brassinosteroid-insensitive1 kinase domain in plants resulted in phenotypes indicating ABA sensitivity. RPK1 is involved in the main ABA signaling pathway and in early ABA perception in Arabidopsis.     

An Abscisic Acid-Activated and Calcium-Independent Protein Kinase from Guard Cells of Fava Bean

Abscisic acid (ABA) regulation of stomatal aperture is known to involve both Ca2+-dependent and Ca2+-independent signal transduction pathways. Electrophysiological studies suggest that protein phosphorylation is involved in ABA action in guard cells. Using biochemical approaches, we identified an ABA-activated and Ca2+- independent protein kinase (AAPK) from guard cell protoplasts of fava bean. Autophosphorylation of AAPK was rapidly (~1 min) activated by ABA in a Ca2+- independent manner. ABA-activated autophosphorylation of AAPK occurred on serine but not on tyrosine residues and appeared to be guard cell specific. AAPK phosphorylated histone type III-S on serine and threonine residues, and its activity toward histone type III-S was markedly stimulated in ABA-treated guard cell protoplasts. Our results suggest that AAPK may play an important role in the Ca2+-independent ABA signaling pathways of guard cells.     

Regulatory mechanism controlling stomatal behavior conserved across 400 million years of land plant evolution

Stomatal pores evolved more than 410 million years ago [1,?2]?and allowed vascular plants to regulate transpirational water loss during the uptake of CO(2) for photosynthesis [3]. Here, we show that stomata on the sporophytes of the moss Physcomitrella patens [2] respond to environmental signals in a similar way to those of flowering plants [4] and that a homolog of a key signaling component in the vascular plant drought hormone abscisic acid (ABA) response [5] is?involved in stomatal control in mosses. Cross-species complementation experiments reveal that the stomatal ABA response of a flowering plant (Arabidopsis thaliana) mutant, lacking the ABA-regulatory protein kinase OPEN STOMATA 1 (OST1) [6], is rescued by substitution with the moss P.?patens homolog, PpOST1-1, which evolved more than 400 million years earlier. We further demonstrate through the targeted knockout of the PpOST1-1 gene in P.?patens that its role in guard cell closure is conserved, with stomata of mutant mosses exhibiting a significantly attenuated ABA response. Our analyses indicate that core regulatory components involved in guard cell ABA signaling of flowering plants are operational in mosses and likely originated in the last common ancestor of these lineages more than 400 million years ago [7], prior to the evolution of ferns [8, 9].     

ABA-regulated promoter activity in stomatal guard cells

CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum . The CDeT6-19 gene promoter has been fused to the β- glucuronidase reporter gene ( GUS ) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum . This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em , or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca²⁺ and other second messengers in ABA-induced gene expression.