A plasma membrane receptor kinase, GHR1, mediates abscisic acid- and hydrogen peroxide-regulated stomatal movement in Arabidopsis

The plant hormone abscisic acid (ABA) regulates stomatal movement under drought stress, and this regulation requires hydrogen peroxide (H2O2). We isolated GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), which encodes a receptor-like kinase localized on the plasma membrane in Arabidopsis thaliana. ghr1 mutants were defective ABA and H2O2 induction of stomatal closure. Genetic analysis indicates that GHR1 is a critical early component in ABA signaling. The ghr1 mutation impaired ABA- and H2O2-regulated activation of S-type anion currents in guard cells. Furthermore, GHR1 physically interacted with, phosphorylated, and activated the S-type anion channel SLOW ANION CHANNEL-ASSOCIATED1 when coexpressed in Xenopus laevis oocytes, and this activation was inhibited by ABA-INSENSITIVE2 (ABI2) but not ABI1. Our study identifies a critical component in ABA and H2O2 signaling that is involved in stomatal movement and resolves a long-standing mystery about the differential functions of ABI1 and ABI2 in this process.     

Hydrogen peroxide is a regulator of ABI1, a protein phosphatase 2C from Arabidopsis.

Protein phosphatases 2C (PP2Cs) exhibit diverse regulatory functions in signalling pathways of animals, yeast and plants. ABI1 is a PP2C of Arabidopsis that exerts negative control on signalling of the phytohormone abscissic acid (ABA). Characterisation of the redox sensitivity of ABI1 revealed a strong enzymatic inactivation by hydrogen peroxide (H2O2) which has recently been implicated as a secondary messenger of ABA signalling. H2O2 reversibly inhibited ABI1 activity in vitro with an IC(50) of approximately 140 microM in the presence of physiological concentrations of glutathione. In addition, ABI1 was highly susceptible to inactivation by phenylarsine oxide (IC(50)=3-4 microM) indicative for the facile oxidation of vicinal cysteine residues. Thus, H2O2 generated during ABA signalling seems to inactivate the negative regulator of the ABA response.     

The sensitivity of ABI2 to hydrogen peroxide links the abscisic acid-response regulator to redox signalling

ABI1 and ABI2 are two protein serine/threonine phosphatases of type 2C (EC 3.1.3.16) that act as key regulators in the responses of Arabidopsis thaliana (L.) Heynh. to abscisic acid (ABA). They are involved in the control of ABA-mediated seed dormancy, stomatal closure and vegetative growth inhibition. Analysis of the enzymatic properties of ABI2 revealed high sensitivities towards protons and unsaturated fatty acids. Furthermore, the protein phosphatase activity of ABI2 is very sensitive to H2O2, which has recently emerged as a secondary messenger of ABA signalling. Upon H2O2 challenge, ABI2 is rapidly inactivated with an IC50 value of 50 microM in the presence of reduced glutathione. Inhibitor studies with phenylarsine oxide and manipulation of the redox status of ABI2 in vitro indicate that oxidation of critical cysteine residue(s) is responsible for inactivation. The levels of the major cellular thiol compounds cysteine and glutathione in leaves and seedlings of A. thaliana are compatible with a physiological role of H2O2 in regulating ABI2 activity. ABI2 is considered to exert negative regulation on ABA action. Thus, transient inactivation of this protein phosphatase by H2O2 would allow or enhance the ABA-dependent signalling process. In conclusion, ABI2 represents a likely target for redox-regulation of a hormonal signalling pathway in higher plants.     

Enhancement of abscisic acid sensitivity and reduction of water consumption in Arabidopsis by combined inactivation of the protein phosphatases type 2C ABI1 and HAB1

Abscisic acid (ABA) plays a key role in plant responses to abiotic stress, particularly drought stress. A wide number of ABA-hypersensitive mutants is known, however, only a few of them resist/avoid drought stress. In this work we have generated ABA-hypersensitive drought-avoidant mutants by simultaneous inactivation of two negative regulators of ABA signaling, i.e. the protein phosphatases type 2C (PP2Cs) ABA-INSENSITIVE1 (ABI1) and HYPERSENSITIVE TO ABA1 (HAB1). Two new recessive loss-of-function alleles of ABI1, abi1-2 and abi1-3, were identified in an Arabidopsis (Arabidopsis thaliana) T-DNA collection. These mutants showed enhanced responses to ABA both in seed and vegetative tissues, but only a limited effect on plant drought avoidance. In contrast, generation of double hab1-1 abi1-2 and hab1-1 abi1-3 mutants strongly increased plant responsiveness to ABA. Thus, both hab1-1 abi1-2 and hab1-1 abi1-3 were particularly sensitive to ABA-mediated inhibition of seed germination. Additionally, vegetative responses to ABA were reinforced in the double mutants, which showed a strong hypersensitivity to ABA in growth assays, stomatal closure, and induction of ABA-responsive genes. Transpirational water loss under drought conditions was noticeably reduced in the double mutants as compared to single parental mutants, which resulted in reduced water consumption of whole plants. Taken together, these results reveal cooperative negative regulation of ABA signaling by ABI1 and HAB1 and suggest that fine tuning of ABA signaling can be attained through combined action of PP2Cs. Finally, these results suggest that combined inactivation of specific PP2Cs involved in ABA signaling could provide an approach for improving crop performance under drought stress conditions.     

小麦TaLCD基因的克隆及其对渗透胁迫的调节作用

半胱氨酸脱巯基酶(CDes)可催化降解半胱氨酸(Cys)生成硫化氢(H₂S)。通过克隆小麦(Triticum aestivum)中的L-半胱氨酸脱巯基酶基因TaLCD, 并将其在拟南芥(Arabidopsis thaliana)中过表达, 探讨TaLCD对渗透胁迫条件下种子萌发和根系生长的影响, 并分析其对干旱胁迫的调节作用。结果显示, 盐胁迫条件下, TaLCD过表达植株种子萌发率显著高于野生型; 甘露醇处理条件下, TaLCD过表达植株的根长也显著高于野生型, 且TaLCD过表达显著提高植株抗旱性。此外, TaLCD过表达植株对ABA更加敏感, ABA处理下TaLCD过表达植株的种子萌发率及根长均显著低于野生型。干旱胁迫下, TaLCD过表达植株胁迫响应基因(COR47、RD29A、RAB18和RD22)及ABA信号途径相关基因(NCED3、HAB1、HAB2、ABI1、ABI2和ABF2)的表达水平均显著高于野生型。因此推测, TaLCD增强植株抗旱和抗盐能力可能依赖于ABA信号途径。     

KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling

Analysis of the Arabidopsis thaliana RING-ANK (for Really Interesting New Gene-Ankyrin) family, a subgroup of RING-type E3 ligases, identified KEEP ON GOING (KEG) as essential for growth and development. In addition to the RING-HCa and ankyrin repeats, KEG contains a kinase domain and 12 HERC2-like repeats. The RING-HCa and kinase domains were functional in in vitro ubiquitylation and phosphorylation assays, respectively. Seedlings homozygous for T-DNA insertions in KEG undergo growth arrest immediately after germination, suggestive of increased abscisic acid (ABA) signaling, a major phytohormone that plays a key role in plant development and survival under unfavorable conditions. Here, we show that KEG is a negative regulator of ABA signaling. keg roots are extremely sensitive to the inhibitory effects of ABA and exhibit hypersensitivity to exogenous glucose, consistent with the known interaction between glucose and ABA signaling. The observations that KEG accumulates high levels of ABSCISIC ACID-INSENSITIVE5 (ABI5) without exogenous ABA, interacts with ABI5 in vitro, and that loss of ABI5 rescues the growth-arrest phenotype of keg mutant seedlings indicate that KEG is required for ABI5 degradation. In this capacity, KEG is central to ABA signaling by maintaining low levels of ABI5 in the absence of stress.     

ABI1 protein phosphatase 2C is a negative regulator of abscisic acid signaling.

The plant hormone abscisic acid (ABA) is a key regulator of seed maturation and germination and mediates adaptive responses to environmental stress. In Arabidopsis, the ABI1 gene encodes a member of the 2C class of protein serine/threonine phosphatases (PP2C), and the abi1-1 mutation markedly reduces ABA responsiveness in both seeds and vegetative tissues. However, this mutation is dominant and has been the only mutant allele available for the ABI1 gene. Hence, it remained unclear whether ABI1 contributes to ABA signaling, and in case ABI1 does regulate ABA responsiveness, whether it is a positive or negative regulator of ABA action. In this study, we isolated seven novel alleles of the ABI1 gene as intragenic revertants of the abi1-1 mutant. In contrast to the ABA-resistant abi1-1 mutant, these revertants were more sensitive than the wild type to the inhibition of seed germination and seedling root growth by applied ABA. They also displayed increases in seed dormancy and drought adaptive responses that are indicative of a higher responsiveness to endogenous ABA. The revertant alleles were recessive to the wild-type ABI1 allele in enhancing ABA sensitivity, indicating that this ABA-supersensitive phenotype results from a loss of function in ABI1. The seven suppressor mutations are missense mutations in conserved regions of the PP2C domain of ABI1, and each of the corresponding revertant alleles encodes an ABI1 protein that lacked any detectable PP2C activity in an in vitro enzymatic assay. These results indicate that a loss of ABI1 PP2C activity leads to an enhanced responsiveness to ABA. Thus, the wild-type ABI1 phosphatase is a negative regulator of ABA responses.     

干旱与氧化胁迫对小麦根氧化还原状态和叶片ABA积累的影响

通过分析一氧化氮(nitric oxide,NO)、活性氧(reactive oxygen species,ROS)和干旱胁迫对小麦根氧化还原状态和叶片脱落酸(abscisic acid,ABA)积累的影响,探讨了干旱胁迫下NO和H2O2调节ABA合成的可能机制。结果表明:干旱胁迫处理初期小麦根还原型谷胱甘肽含量降低、抗氧化酶活性发生振荡变化,细胞氧化还原状态向氧化型转变。NO和H2O2能模拟干旱胁迫的作用使细胞状态向氧化型转变,还可以使小麦叶片ABA积累量上升。干旱胁迫下NO和H2O2对ABA合成的调节作用可能是通过调节细胞氧化还原状态进行。     

Protein phosphorylation and binding of a 14-3-3 protein in Vicia guard cells in response to ABA

Under drought stress, ABA promotes stomatal closure to prevent water loss. Although protein phosphorylation plays an important role in ABA signaling, little is known about these processes at the biochemical level. In this study, we searched for substrates of protein kinases in ABA signaling through the binding of a 14-3-3 protein to phosphorylated proteins using Vicia guard cell protoplasts. ABA induced binding of a 14-3-3 protein to proteins with molecular masses of 61, 43 and 39 kDa, with the most remarkable signal for the 61 kDa protein. The ABA-induced binding to the 61 kDa protein occurred only in guard cells, and reached a maximum within 3 min at 1 microM ABA. The 61 kDa protein localized in the cytosol. ABA induced the binding of endogenous vf14-3-3a to the 61 kDa protein in guard cells. Autophosphorylation of ABA-activated protein kinase (AAPK), which mediates anion channel activation, and ABA-induced phosphorylation of the 61 kDa protein showed similar time courses and similar sensitivities to the protein kinase inhibitor K-252a. AAPK elicits the binding of the 14-3-3 protein to the 61 kDa protein in vitro when AAPK in guard cells was activated by ABA. The phosphorylation of the 61 kDa protein by ABA was not affected by the NADPH oxidase inhibitor, H(2)O(2), W-7 or EGTA. From these results, we conclude that the 61 kDa protein may be a substrate for AAPK and that the 61 kDa protein is located upstream of H(2)O(2) and Ca(2+), or on Ca(2+)-independent signaling pathways in guard cells.     

Integration of C/N-nutrient and multiple environmental signals into the ABA signaling cascade

Due to their immobility, plants have developed sophisticated mechanisms to robustly monitor and appropriately respond to dynamic changes in nutrient availability. Carbon (C) and nitrogen (N) are especially important in regulating plant metabolism and development, thereby affecting crop productivity. In addition to their independent utilization, the ratio of C to N metabolites in the cell, referred to as the “C/N balance”, is important for the regulation of plant growth, although molecular mechanisms mediating C/N signaling remain unclear. Recently ABI1, a protein phosphatase type 2C (PP2C), was shown to be a regulator of C/N response in Arabidopsis plants. ABI1 functions as a negative regulator of abscisic acid (ABA) signal transduction. ABA is versatile phytohormone that regulates multiple aspects of plant growth and adaptation to environmental stress. This review highlights the regulation of the C/N response mediated by a non-canonical ABA signaling pathway that is independent of ABA biosynthesis, as well as recent findings on the direct crosstalk between multiple cellular signals and the ABA signaling cascade.     

Action of natural abscisic acid precursors and catabolites on abscisic acid receptor complexes

The phytohormone abscisic acid (ABA) regulates stress responses and controls numerous aspects of plant growth and development. Biosynthetic precursors and catabolites of ABA have been shown to trigger ABA responses in physiological assays, but it is not clear whether these are intrinsically active or whether they are converted into ABA in planta. In this study, we analyzed the effect of ABA precursors, conjugates, and catabolites on hormone signaling in Arabidopsis (Arabidopsis thaliana). The compounds were also tested in vitro for their ability to regulate the phosphatase moiety of ABA receptor complexes consisting of the protein phosphatase 2C ABI2 and the coreceptors RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. Using mutants defective in ABA biosynthesis, we show that the physiological activity associated with ABA precursors derives predominantly from their bioconversion to ABA. The ABA glucose ester conjugate, which is the most widespread storage form of ABA, showed weak ABA-like activity in germination assays and in triggering ABA signaling in protoplasts. The ABA conjugate and precursors showed negligible activity as a regulatory ligand of the ABI2/RCAR receptor complexes. The majority of ABA catabolites were inactive in our assays. To analyze the chemically unstable 8'- and 9'-hydroxylated ABA catabolites, we used stable tetralone derivatives of these compounds, which did trigger selective ABA responses. ABA synthetic analogs exhibited differential activity as regulatory ligands of different ABA receptor complexes in vitro. The data show that ABA precursors, catabolites, and conjugates have limited intrinsic bioactivity and that both natural and synthetic ABA-related compounds can be used to probe the structural requirements of ABA ligand-receptor interactions.     

Two vicinal cysteines confer a peculiar redox regulation to low molecular weight protein tyrosine phosphatase in response to platelet-derived growth factor receptor stimulation.

Low molecular weight protein tyrosine phosphatase (LMW-PTP) is an enzyme involved in platelet-derived growth factor (PDGF)-induced mitogenesis and cytoskeleton rearrangement because it is able to bind and dephosphorylate the activated receptor. LMW-PTP presents two cysteines in positions 12 and 17, both belonging to the catalytic pocket; this is a unique feature of LMW-PTP among all protein tyrosine phosphatases. Our previous results demonstrated that in vitro LMW-PTP is oxidized by either H(2)O(2) or nitric oxide with the formation of a disulfide bond between Cys-12 and Cys-17. This oxidation leads to reversible enzyme inactivation because treatment with reductants permits catalytic activity rescue. In the present study we investigated the in vivo inactivation of LMW-PTP by either extracellularly or intracellularly generated H(2)O(2), evaluating its action directly on its natural substrate, PDGF receptor. LMW-PTP is oxidized and inactivated by exogenous oxidative stress and recovers its activity after oxidant removal. LMW-PTP is oxidized also during PDGF signaling, very likely upon PDGF-induced H(2)O(2) production, and recovers its activity within 40 min. Our results strongly suggest that reversibility of in vivo LMW-PTP oxidation is glutathione-dependent. In addition, we propose an intriguing and peculiar role of Cys-17 in the formation of a S-S intramolecular bond, which protects the catalytic Cys-12 from further and irreversible oxidation. On the basis of our results we propose that the presence of an additional cysteine near the catalytic cysteine could confer to LMW-PTP the ability to rapidly recover its activity and finely regulate PDGF receptor activation during both extracellularly and intracellularly generated oxidative stress.     

Role of abscissic acid in water stress-induced antioxidant defense in leaves of maize seedlings

Roles of abscisic acid (ABA) in water stress-induced oxidative stress were investigated in leaves of maize ( Zea mays L.) seedlings exposed to water stress induced by polyethylene glycol (PEG 6000). Treatment with PEG at -0.7 MPa for 12 and 24 h led to a reduction in leaf relative water content (RWC) by 7.8 and 14.1%, respectively. Duration of the osmotic treatments is considered as mild and moderate water stress. The mild water stress caused significant increases in the generation of superoxide radical ( O 2 - ) and hydrogen peroxide (H 2 O 2 ), the activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) and the contents of ascorbate (ASC), reduced glutathione (GSH). The moderate water stress failed to further enhance the capacity of antioxidant defense systems, as compared to the mild water stress. The contents of catalytic Fe, which is critical for H 2 O 2 -dependent hydroxyl radical ( •OH) production, and the oxidized forms of ascorbate and glutathione pools, dehydroascorbate (DHA) and oxidized glutathione (GSSG), markedly increased, a significant oxidative damage to lipids and proteins took place under the moderate water stress. Pretreatment with ABA caused an obvious reduction in the content of catalytic Fe and significant increases in the activities of antioxidant enzymes and the contents of non-enzymatic antioxidants, and then significantly reduced the contents of DHA and GSSG and the degrees of oxidative damage in leaves exposed to the moderate water stress. Pretreatment with an ABA biosynthesis inhibitor, tungstate, significantly suppressed the accumulation of ABA induced by water stress, reduced the enhancement in the capacity of antioxidant defense systems, and resulted in an increase in catalytic Fe, DHA and GSSG, and oxidative damage in the water-stressed leaves. These effects were completely prevented by addition of ABA, which raised the internal ABA content. Our data indicate that ABA plays an important role in water stress-induced antioxidant defense against oxidative stress.     

AtMEK1 mediates stress-induced gene expression of CAT1 catalase by triggering H2O2 production in Arabidopsis

Catalase and hydrogen peroxide (H(2)O(2)) have been extensively studied for their roles in various stress responses. However, little is known about the triggering mechanisms for stress-induced catalase gene expression or about H(2)O(2) production as a stress signal. It is reported here that ABA-, drought-, and salt stress-induced gene expression of CAT1 catalase is mediated by AtMEK1, an Arabidopsis MAPK kinase, by triggering H(2)O(2) signal production. Both CAT1 expression and AtMEK1 activity were activated by ABA, drought, and salt stresses. The mek1 mutant totally blocked stress-induced CAT1 expression and, interestingly, stress-induced H(2)O(2) production was also blocked. Over-expression of AtMEK1 significantly promoted stress-induced CAT1 expression, and also promoted H(2)O(2) production. These results conclusively indicate that stress-induced CAT1 expression is mediated by AtMEK1 and, furthermore, that the triggering of H(2)O(2) production might be involved in this process, as further proved by the observation that CAT1 expression was induced by applied H(2)O(2.) Surprisingly, the signalling mechanisms for stress-induced gene expression of CAT2 and CAT3 were very different from that of CAT1. Except for drought stress, expression of CAT2 or CAT3 was also activated by salt stress or ABA treatment, and AtMEK1 was not proved to be involved in the drought-induced expression of CAT2 or CAT3. Further studies showed that stomatal movement was much less sensitive to ABA in AtMEK1 mutant (mek1), and over-expression of AtMEK1 in Arabidopsis increased plant resistance to drought or salt stress, which further demonstrated that AtMEK1 is a crucial mediator in plant stress signal transduction.