Role of PP2C-mediated ABA signaling in the moss Physcomitrella patens

Plant hormone abscisic acid (ABA) is found in a wide range of land plants, from mosses to angiosperms. However, our knowledge concerning the function of ABA is limited to some angiosperm plant species. We have shown that the basal land plant Physcomitrella patens and the model plant Arabidopsis thaliana share a conserved abscisic acid (ABA) signaling pathway mediated through ABI1-related type 2C protein phosphatases (PP2Cs). Ectopic expression of Arabidopsis abi1-1, a dominant allele of ABI1 that functions as a negative regulator of ABA signaling, or targeted disruption of Physcomitrella ABI1-related gene (PpABI1A) resulted in altered ABA sensitivity and abiotic stress tolerance of Physcomitrella, as demonstrated by osmostress and freezing stress. Moreover, transgenic Physcomitrella overexpressing abi1-1 showed altered morphogenesis. These trangenic plants had longer stem lengths compared to the wild type, and continuous growth of archegonia (female organ) with few sporophytes under non-stress conditions. Our results suggest that PP2C-mediated ABA signaling is involved in both the abiotic stress responses and developmental regulation of Physcomitrella.Key words: ABA, ABI1, Physcomitrella patens, PP2C, signaling     

H2O2 Inhibits ABA-Signaling Protein Phosphatase HAB1

Due to its ability to be rapidly generated and propagated over long distances, H₂O₂ is an important second messenger for biotic and abiotic stress signaling in plants. In response to low water potential and high salt concentrations sensed in the roots of plants, the stress hormone abscisic acid (ABA) activates NADPH oxidase to generate H₂O₂, which is propagated in guard cells in leaves to induce stomatal closure and prevent water loss from transpiration. Using a reconstituted system, we demonstrate that H₂O₂ reversibly prevents the protein phosphatase HAB1, a key component of the core ABA-signaling pathway, from inhibiting its main target in guard cells, SnRK2.6/OST1 kinase. We have identified HAB1 C186 and C274 as H₂O₂-sensitive thiols and demonstrate that their oxidation inhibits both HAB1 catalytic activity and its ability to physically associate with SnRK2.6 by formation of intermolecular dimers.     

The effect of H2O2 and abscisic acid (ABA) interaction on β-amylase activity under osmotic stress during grain development in barley

The effects of exogenous abscisic acid (ABA) and polyethylene glycol (PEG 6000) treatments on grain H₂O₂, ABA and β-amylase activity were studied during grain development in the spike culture experiments with variety Triumph and its ABA-insensitive mutant TL43 as the plant materials. The results showed that during grain development the two genotypes were similar in the pattern of ABA concentration change, but differed greatly in the pattern of H₂O₂ concentration and β-amylase activity changes. The β-amylase activity was positively correlated with H₂O₂ concentration, negatively correlated with ABA concentration, and it is mainly closely associated with continued high levels of ABA with respect to H₂O₂. Water stress (PEG treatment) induced β-amylase was associated with H₂O₂ concentration but not with ABA concentration. Exogenous application of H₂O₂ and Ascorbic acid (AsA) increased β-amylase activity in Triumph but reduced that of TL43. However, the endogenous H₂O₂ concentration in grains was always consistent with β-amylase activity. A novel model was hypothesized from the current results to illustrate the relationship between H₂O₂, ABA and β-amylase synthesis for the barley exposed to abiotic stresses.     

GhMPK17, a Cotton Mitogen-Activated Protein Kinase,Is Involved in Plant Response to High Salinity and Osmotic Stresses and ABA Signaling

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in mediating biotic and abiotic stress responses. Cotton (Gossypium hirsutum) is the most important textile crop in the world, and often encounters abiotic stress during its growth seasons. In this study, a gene encoding a mitogen-activated protein kinase (MAPK) was isolated from cotton, and designated as GhMPK17. The open reading frame (ORF) of GhMPK17 gene is 1494 bp in length and encodes a protein with 497 amino acids. Quantitative RT-PCR analysis indicated that GhMPK17 expression was up-regulated in cotton under NaCl, mannitol and ABA treatments. The transgenic Arabidopsis plants expressing GhMPK17 gene showed higher seed germination, root elongation and cotyledon greening/expansion rates than those of the wild type on MS medium containing NaCl, mannitol and exogenous ABA, suggesting that overexpression of GhMPK17 in Arabidopsis increased plant ABA-insensitivity, and enhanced plant tolerance to salt and osmotic stresses. Furthermore, overexpression of GhMPK17 in Arabidopsis reduced H₂O₂ level and altered expression of ABA- and abiotic stress-related genes in the transgenic plants. Collectively, these data suggested that GhMPK17 gene may be involved in plant response to high salinity and osmotic stresses and ABA signaling.     

Cytochrome c peroxidase is a mitochondrial heme-based H2O2 sensor that modulates antioxidant defense

Hydrogen peroxide (H₂O₂) is a key signaling molecule that also induces apoptosis. Thus, cells must rapidly sense and tightly control H₂O₂ levels. Well-characterized cellular responses to exogenous H₂O₂ involve oxidation of specific cytosolic protein-based thiols but sensing of H₂O₂ generated by mitochondrial respiration is less well described. Here we provide substantial biochemical evidence that the heme enzyme Ccp1 (cytochrome c peroxidase), which is targeted to the intermembrane space, functions primarily as a mitochondrial H₂O₂ sensing and signaling protein in Saccharomyces cerevisiae. Key evidence for a sensing role for Ccp1 is the significantly higher H₂O₂ accumulation in ccp1-null cells(ccp1Δ) vs ccp1^W191F cells producing the catalytically inactive Ccp1^W191F variant. In fact, intracellular H₂O₂ levels (ccp1Δ>wildtype >ccp1^W191F) correlate inversely with the activity of the mitochondrial (and peroxisomal) heme catalase, Cta1 (ccp1Δ<wildtype <ccp1^W191F). Mitochondrial Sod2 activity also varies in the three strains (ccp1Δ>wildtype >ccp1^W191F) and ccp1Δ cells exhibit low superoxide levels. Notably, Ccp1^W191F is a more persistent H₂O₂ signaling protein than wild-type Ccp1, and this enhanced mitochondrial H₂O₂ signaling decreases the mitochondrial fitness of ccp1^W191F cells. However, these cells are fully protected from a bolus (0.4 mM) of exogenous H₂O₂ added after 12 h of growth, whereas the viability of ccp1Δ cells drops below 20%, which additionally associates Ccp1 with Yap1-dependent H₂O₂ signaling. Combined, our results strongly implicate Ccp1, independent of its peroxidase activity, in mitochondrial H₂O₂ sensing and signaling to maintain reactive oxygen species homeostasis.     

Phospholipase Dα1 and Phosphatidic Acid Regulate NADPH Oxidase Activity and Production of Reactive Oxygen Species in ABA-Mediated Stomatal Closure in Arabidopsis

We determined the role of Phospholipase Dα1 (PLDα1) and its lipid product phosphatidic acid (PA) in abscisic acid (ABA)-induced production of reactive oxygen species (ROS) in Arabidopsis thaliana guard cells. The pldα1 mutant failed to produce ROS in guard cells in response to ABA. ABA stimulated NADPH oxidase activity in wild-type guard cells but not in pldα1 cells, whereas PA stimulated NADPH oxidase activity in both genotypes. PA bound to recombinant Arabidopsis NADPH oxidase RbohD (respiratory burst oxidase homolog D) and RbohF. The PA binding motifs were identified, and mutation of the Arg residues 149, 150, 156, and 157 in RbohD resulted in the loss of PA binding and the loss of PA activation of RbohD. The rbohD mutant expressing non-PA-binding RbohD was compromised in ABA-mediated ROS production and stomatal closure. Furthermore, ABA-induced production of nitric oxide (NO) was impaired in pldα1 guard cells. Disruption of PA binding to ABI1 protein phosphatase 2C did not affect ABA-induced production of ROS or NO, but the PA–ABI1 interaction was required for stomatal closure induced by ABA, H₂O₂, or NO. Thus, PA is as a central lipid signaling molecule that links different components in the ABA signaling network in guard cells.     

Ectopic expression of wheat TaCIPK14, encoding a calcineurin B‐like protein‐interacting protein kinase,confers salinity and cold tolerance in tobacco

Calcineurin B‐like protein‐interacting protein kinases (CIPKs) are components of Ca²⁺ signaling in responses to abiotic stresses. In this work, the full‐length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT‐PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H₂O₂. Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H₂O₂ and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na⁺ content in the transgenic lines under salt stress. Higher expression of stress‐related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress‐responsive gene in plants.     

New cross talk between ROS,ABA and auxin controlling seed maturation and germination unraveled in APX6 deficient Arabidopsis seeds

Successful execution of germination program greatly depends on the seeds’ oxidative homeostasis. We recently identified new roles for the H₂O₂-reducing enzyme ascorbate peroxidase 6 (APX6) in germination control and seeds’ stress tolerance. APX6 replaces APX1 as the dominant APX in dry seeds, and its loss-of-function results in reduced germination due to over accumulation of ROS and oxidative damage. Metabolic analyses in dry apx6 seeds, revealed altered homeostasis of primary metabolites including accumulation of TCA cycle metabolites, ABA and auxin, supporting a novel role for APX6 in regulating cellular metabolism. Increased sensitivity of apx6 mutants to ABA or IAA in germination assays indicated impaired perception of these signals. Relative suppression of ABI3 and ABI5 expression, and induction of ABI4, suggested the activation of a signaling route inhibiting germination in apx6 seeds that is independent of ABI3. Here we provide additional evidence linking ABI4 with ABA- and auxin-controlled inhibition of germination and suggest a hypothetical model for the role of APX6 in the regulation of the crosstalk between these hormones and ROS.     

小麦TaMAPK2基因的克隆及表达分析

该研究从小麦中克隆了1个MAPK基因TaMAPK2。序列分析表明,TaMAPK2基因的ORF为1 110bp,编码369个氨基酸。序列比对分析表明,该基因所编码的蛋白与粗山羊草、水稻、谷子等MAPK蛋白具有较高的一致性,分别为99%、94%、94%。进化树分析表明,TaMAPK2与水稻OsMAPK2的亲缘关系最近。实时荧光定量PCR分析表明,该基因的表达显著受渗透胁迫、低温胁迫、高盐胁迫、乙烯和双氧水诱导,受ABA抑制。研究表明,TaMAPK2可能参与非生物逆境胁迫及相关信号分子应答。     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

The High Light Response in Arabidopsis Involves ABA Signaling between Vascular and Bundle Sheath Cells

Previously, it has been shown that Arabidopsis thaliana leaves exposed to high light accumulate hydrogen peroxide (H₂O₂) in bundle sheath cell (BSC) chloroplasts as part of a retrograde signaling network that induces ASCORBATE PEROXIDASE2 (APX2). Abscisic acid (ABA) signaling has been postulated to be involved in this network. To investigate the proposed role of ABA, a combination of physiological, pharmacological, bioinformatic, and molecular genetic approaches was used. ABA biosynthesis is initiated in vascular parenchyma and activates a signaling network in neighboring BSCs. This signaling network includes the Gα subunit of the heterotrimeric G protein complex, the OPEN STOMATA1 protein kinase, and extracellular H₂O₂, which together coordinate with a redox-retrograde signal from BSC chloroplasts to activate APX2 expression. High light–responsive genes expressed in other leaf tissues are subject to a coordination of chloroplast retrograde signaling and transcellular signaling activated by ABA synthesized in vascular cells. ABA is necessary for the successful adjustment of the leaf to repeated episodes of high light. This process involves maintenance of photochemical quenching, which is required for dissipation of excess excitation energy.     

ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in Arabidopsis

Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.     

Drought-induced H2O2 accumulation in subsidiary cells is involved in regulatory signaling of stomatal closure in maize leaves

Increasing H₂O₂ levels in guard cells in response to environmental stimuli are recently considered a general messenger involved in the signaling cascade for the induction of stomatal closure. But little is known as to whether subsidiary cells participate in the H₂O₂-mediated stomatal closure of grass plants. In the present study, 2-week-old seedlings of maize (Zea mays) were exposed to different degrees of soil water deficit for 3 weeks. The effects of soil water contents on leaf ABA and H₂O₂ levels and stomatal aperture were investigated using physiological, biochemical, and histochemical approaches. The results showed that even under well-watered conditions, significant amounts of H₂O₂ were observed in guard cells, whereas H₂O₂ concentrations in the subsidiary cells were negligible. Decreasing soil water contents led to a significant increase in leaf ABA levels associated with significantly enhanced O₂ ^? and H₂O₂ contents, consistent with reduced degrees of stomatal conductance and aperture. The significant increase in H₂O₂ appeared in both guard cells and subsidiary cells of the stomatal complex, and H₂O₂ levels increased with decreasing soil water contents. Drought-induced increase in the activity of antioxidative enzymes could not counteract the significant increase in H₂O₂ levels in guard cells and subsidiary cells. These results indicate that subsidiary cells participate in H₂O₂-mediated stomatal closure, and drought-induced H₂O₂ accumulation in subsidiary cells is involved in the signaling cascade regulating stomatal aperture of grass plants such as maize.