A hypermorphic mutation in the protein phosphatase 2C HAB1 strongly affects ABA signaling in Arabidopsis

Protein phosphatases of the 2C family (PP2C) function in the regulation of several signaling pathways from prokaryotes to eukaryotes. In Arabidopsis thaliana, the HAB1 PP2C is a negative regulator of the stress hormone abscisic acid (ABA) signaling. Here, we show that plants expressing a mutant form of HAB1 in which Gly246 was mutated to Asp (G246D) display strong ABA insensitive phenotypes. Our results indicate that the G246D mutation has a hypermorphic rather than a dominant negative effect. The data suggest that this mutation localized in a conserved motif in the PP2C catalytic domain could be used in other PP2Cs to reveal their biological functions.     

Antisense inhibition of protein phosphatase 2C accelerates cold acclimation in Arabidopsis thaliana

Two related protein phosphatases 2C, ABI1 and AtPP2CA have been implicated as negative regulators of ABA signalling. In this study we characterized the role of AtPP2CA in cold acclimation. The pattern of expression of AtPP2CA and ABI1 was studied in different tissues and in response to abiotic stresses. The expression of both AtPP2CA and ABI1 was induced by low temperature, drought, high salt and ABA. The cold and drought-induced expression of these genes was ABA-dependent, but divergent in various ABA signalling mutants. In addition, the two PP2C genes exhibited differences in their tissue-specific expression as well as in temporal induction in response to low temperature. To elucidate the function of AtPP2CA in cold acclimation further, the corresponding gene was silenced by antisense inhibition. Transgenic antisense plants exhibited clearly accelerated development of freezing tolerance. Both exposure to low temperature and application of ABA resulted in enhanced freezing tolerance in antisense plants. These plants displayed increased sensitivity to ABA both during development of frost tolerance and during seed germination, but not in their drought responses. Furthermore, the expression of cold-and ABA-induced genes was enhanced in transgenic antisense plants. Our results suggest that AtPP2CA is a negative regulator of ABA responses during cold acclimation.     

Protein degradation and nitrogen remobilization during leaf senescence

Leaf senescence, a type of programmed cell death, is a complex and highly regulated process that involves the degradation of macromolecules, including proteins, nucleic acids, and lipids. Nutrients, especially nitrogen, are re-mobilized from senescing leaves to newly developing tissues or reserve organs. Our review focuses on three pathways for protein breakdown and the resorption of N during this process: the ubiquitin/proteosome system, the chloroplast degradation pathway, and the vacuolar and autophagic pathway. We propose that two relative biochemical cycles exist for amino acid recycling and N-export — the GS/GOGAT cycle and the PPDK-GS/GOGAT cycle.     

Biotechnological implications from abscisic acid (ABA) roles in cold stress and leaf senescence as an important signal for improving plant sustainable survival under abiotic-stressed conditions

In the past few years, the signal transduction of the plant hormone abscisic acid (ABA) has been studied extensively and has revealed an unanticipated complex. ABA, characterized as an intracellular messenger, has been proven to act a critical function at the heart of a signaling network operation. It has been found that ABA plays an important role in improving plant tolerance to cold, as well as triggering leaf senescence for years. In addition, there have been many reports suggesting that the signaling pathways for leaf senescence and plant defense responses may overlap. Therefore, the objective was to review what is known about the involvement of ABA signaling in plant responses to cold stress and regulation of leaf senescence. An overview about how ABA is integrated into sugars and reactive oxygen species signaling pathways, to regulate plant cold tolerance and leaf senescence, is provided. These roles can provide important implications for biotechnologically improving plant cold tolerance.     

异源过表达水稻OsSAPP3基因促进拟南芥叶片衰老

蛋白磷酸酶催化的蛋白质可逆磷酸化反应是叶片衰老的关键环节。该研究筛选并克隆了1个新的参与水稻(Oryza sativa)叶片衰老调控的PP2C基因OsSAPP3。研究表明, OsSAPP3的启动子在Pro_OsSAPP3-GUS转基因拟南芥(Arabidopsis thaliana)的莲座叶中有活性, 并且活性以依赖叶龄方式增加。利用CaMV 35S启动子驱动组成型异源过表达OsSAPP3导致转基因拟南芥无法正常生长。用可诱导型启动子GVG系统驱动OsSAPP3异源过表达导致转基因拟南芥出现莲座叶变小、数量增加、叶片早衰及抽薹开花提前等早衰表型。外源诱导OsSAPP3基因异源过表达后, 利用实时荧光定量PCR检测到SAG12、WRKY6和NAC2等衰老标志基因显著上调表达。研究结果表明, OsSAPP3是参与水稻叶片衰老的正向调控因子。     

Overexpression of AtPTPA in Arabidopsis increases protein phosphatase 2A activity by promoting holoenzyme formation and ABA negatively affects holoenzyme formation

AtPTPA is a critical regulator for the holoenzyme assembling of protein phosphatase 2A (PP2A) in Arabidopsis. Characterization of AtPTPA improves our understanding of the function and regulation of PP2A in eukaryotes. Further analysis of AtPTPA-overexpressing plants indicates that AtPTPA increases PP2A activity by promoting PP2A''s AC dimer formation, thereby holoenzyme assembling. Plant hormone abscisic acid (ABA) reduces PP2A enzyme activity by negatively affects PP2A''s AC dimer formation. Therefore, AtPTPA is a positive factor that promotes PP2A holoenzyme assembly, and ABA is a negative factor that prevents PP2A holoenzyme assembly.     

Differential changes in the synthesis and steady-state levels of thylakoid proteins during bean leaf senescence

During senescence of primary bean leaves (Phaseolus vulgaris), there are differential changes in the rates at which thylakoid proteins are synthesized. In particular, synthesis of the 32 kD herbicide-binding protein continues throughout senescence, whereas formation of the and subunits of ATPase, the 68 kD photosystem I reaction center polypeptide, cytochrome f, cytochrome b₆ and the structural apoprotein of the lightharvesting chlorophyll protein complex (LHCP) declines. Pulse-chase experiments with intact leaves indicated rapid degradation of the 32 kD protein, which is consistent with its known rapid rate of turnover. This degradation was light-dependent and inhibited by DCMU, and the kinetics of degradation were similar for young and senescent membranes. In Coomassie-stained gels, the 68 kD reaction center polypeptide of photosystem I, the and subunits of ATPase and the LHCP were the dominant proteins for all ages of membranes. Western blot analysis indicated that cytochrome f and cytochrome b₆ are selectively depleted during senescence. The data have been interpreted as indicating that translational disruptions in both the cytoplasmic and chloroplastic compartments may contribute to the decline in photosynthetic electron transport in the senescing leaf.     

Pepper protein phosphatase type 2C,CaADIP1 and its interacting partner CaRLP1 antagonistically regulate ABA signalling and drought response

Abscisic acid (ABA) is a key phytohormone that regulates plant growth and developmental processes, including seed germination and stomatal closing. Here, we report the identification and functional characterization of a novel type 2C protein phosphatase, CaADIP1 (Capsicum annuum A BA and D rought‐I nduced P rotein phosphatase 1). The expression of CaADIP1 was induced in pepper leaves by ABA, drought and NaCl treatments. Arabidopsis plants overexpressing CaADIP1 (CaADIP1‐OX) exhibited an ABA‐hyposensitive and drought‐susceptible phenotype. We used a yeast two‐hybrid screening assay to identify CaRLP1 (Capsicum annuum R CAR‐L ike P rotein 1), which interacts with CaADIP1 in the cytoplasm and nucleus. In contrast to CaADIP1‐OX plants, CaRLP1‐OX plants displayed an ABA‐hypersensitive and drought‐tolerant phenotype, which was characterized by low levels of transpirational water loss and increased expression of stress‐responsive genes relative to those of wild‐type plants. In CaADIP1‐OX/CaRLP1‐OX double transgenic plants, ectopic expression of the CaRLP1 gene led to strong suppression of CaADIP1‐induced ABA hyposensitivity during the germinative and post‐germinative stages, indicating that CaADIP1 and CaRLP1 act in the same signalling pathway and CaADIP1 functions downstream of CaRLP1. Our results indicate that CaADIP1 and its interacting partner CaRLP1 antagonistically regulate the ABA‐dependent defense signalling response to drought stress.     

植物PP2C蛋白磷酸酶ABA信号转导及逆境胁迫调控机制研究进展

蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,而PP2C磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,是高等植物中最大的蛋白磷酸酶家族,包含76个家族成员,广泛存在于生物体中。迄今为止,在植物体内已经发现了4种PP2C蛋白磷酸酶。蛋白激酶和蛋白磷酸酶协同催化蛋白质可逆磷酸化,在植物体内信号转导和生理代谢中起着重要的调节作用,蛋白质的磷酸化几乎存在于所有的信号转导途径中。大量研究表明,PP2Cs参与多条信号转导途径,包括PP2C参与ABA调控,对干旱、低温、高盐等逆境胁迫的响应,参与植物创伤和种子休眠或萌发等信号途径,其调控机制不同,但酶催化活性都依赖于Mg2+或Mn2+的浓度。植物PP2C蛋白的C端催化结构域高度保守,而N端功能各异。文中还综述了高等植物PP2C的分类、结构、ABA受体与PP2Cs蛋白互作、PP2C基因参与ABA信号途径以及其他逆境信号转导途径的研究进展。     

Specific association of a small protein with the telomeric DNA-protein complex during the onset of leaf senescence in Arabidopsis thaliana

Telomeres and their changes in length throughout the life span of cells have been intensively investigated in different organisms. Telomere length is assumed to control replicative senescence in mammalian cells. However, only very few data are available on the developmental dynamics of plant telomeres. Here, changes of telomere length and DNA-protein structure of Arabidopsis thaliana telomeres were analysed in different stages of development, with the main focus resting on the transition from pre-senescent to senescent leaves. The lengths of the telomeres, ranging from ca. 2.0 to 6.5 kb, do not significantly change during plant development indicating that telomere length is not involved in differentiation and replicative senescence nor in post-mitotic senescence of A. thaliana. In dedifferentiated cultured cells a slight increase in length can be determined. The nucleoprotein structure of the telomeric DNA was investigated by gel mobility shift assays, with synthetic oligonucleotides and nuclear protein extracts derived from four defined stages of post-mitotic leaf senescence. In all four stages, a highly salt-resistant DNA-protein complex was formed with the double-stranded as well as with the single-stranded G-rich telomeric DNA. An additional DNA-protein complex was identified in nuclear protein extracts isolated from plants in the transition stage from pre-senescence to senescence. The protein components of the DNA-protein complexes were analysed on native PAGE and SDS-PAGE gels. A protein of 67 kDa (ATBP1) bound to the telomeric DNA in all developmental stages. An additional protein of merely 22 kDa (ATBP2) was associated via protein-protein interaction with ATBP1 to form a higher-order complex exclusively during the onset of senescence. DNA interaction of this higher-order protein complex seems to be restricted to double-stranded telomeric DNA. The defined period of ATBP1/ATBP2 complex formation with the telomeric DNA probably indicates that ATBP2 is involved in the onset of post-mitotic leaf senescence by either disturbing an established or establishing an additional function exhibited by the telomeres in the interphase nuclei.     

The reorganization of actin filaments is required for vacuolar fusion of guard cells during stomatal opening in Arabidopsis

The reorganization of actin filaments (AFs) and vacuoles in guard cells is involved in the regulation of stomatal movement. However, it remains unclear whether there is any interaction between the reorganization of AFs and vacuolar changes during stomatal movement. Here, we report the relationship between the reorganization of AFs and vacuolar fusion revealed in pharmacological experiments, and characterizing stomatal opening in actin‐related protein 2 (arp2) and arp3 mutants. Our results show that cytochalasin‐D‐induced depolymerization or phalloidin‐induced stabilization of AFs leads to an increase in small unfused vacuoles during stomatal opening in wild‐type (WT) Arabidopsis plants. Light‐induced stomatal opening is retarded and vacuolar fusion in guard cells is impaired in the mutants, in which the reorganization and the dynamic parameters of AFs are aberrant compared with those of the WT. In WT, AFs tightly surround the small separated vacuoles, forming a ring that encircles the boundary membranes of vacuoles partly fused during stomatal opening. In contrast, in the mutants, most AFs and actin patches accumulate abnormally around the nuclei of the guard cells, which probably further impair vacuolar fusion and retard stomatal opening. Our results suggest that the reorganization of AFs regulates vacuolar fusion in guard cells during stomatal opening.     

The YCR079w gene confers a rapamycin-resistant function and encodes the sixth type 2C protein phosphatase in Saccharomyces cerevisiae

Type 2C protein phosphatase (PP2C) is a monomeric enzyme and requires Mg(2+) or Mn(2+) for its activity. Up to now, seven PP2C-like genes have been identified in the genome of Saccharomyces cerevisiae. However, the protein encoded by the sixth PP2C-like gene, YCR079w, has not been demonstrated to have PP2C activity. In this study, we show that YCR079w confers a rapamycin-resistant function in yeast cells, and we also demonstrate that the YCR079w-encoded protein exhibits characteristics of a typical PP2C. Therefore, YCR079w encodes the sixth PP2C, PTC6, in budding yeast.     

Heavy metal stress and leaf senescence induce the barley gene HvC2d1 encoding a calcium-dependent novel C2 domain-like protein

By comparing cDNA populations derived from chromium-stressed primary leaves of barley (Hordeum vulgare L.) with controls, differentially expressed cDNA fragments could be identified. The deduced amino acid sequence of one of these cDNAs [named 'C2 domain 1' (HvC2d1)] exhibits a motif that is similar to the known C2 domain and a nuclear localization signal (NLS). Expression of this member of a novel class of plant C2 domain-like proteins was studied using real-time PCR, and subcellular localization was investigated using green fluorescent protein (GFP) fusion constructs. Calcium binding was analysed using a (45)Ca(2+) overlay assay. HvC2d1 was transiently induced after exposure to different heavy metals and its mRNA accumulated during the phase of leaf senescence. HvC2d1 expression responded to changes in calcium levels caused by the calcium ionophore A23187 and to treatment with methylviologen resulting in the production of reactive oxygen species (ROS). Using overexpressed and purified HvC2d1, the binding of calcium could be confirmed. Chimeric HvC2d1-GFP protein was localized in onion epidermal cells at the plasma membrane, cytoplasm and the nucleus. After addition of calcium ionophore A23187 green fluorescence was only visible in the nucleus. The data suggest a calcium-dependent translocation of HvC2d1 to the nucleus. A possible role of HvC2d1 in stress- and development-dependent signalling in the nucleus is discussed.