CIPK3, a calcium sensor-associated protein kinase that regulates abscisic acid and cold signal transduction in Arabidopsis

Plants respond to environmental stress by activating "stress genes." The plant hormone abscisic acid (ABA) plays an important role in stress-responsive gene expression. Although Ca(2+) serves as a common second messenger in signaling stress and ABA, little is known about the molecular basis of Ca(2+) action in these pathways. Here, we show that CIPK3, a Ser/Thr protein kinase that associates with a calcineurin B-like calcium sensor, regulates ABA response during seed germination and ABA- and stress-induced gene expression in Arabidopsis. The expression of the CIPK3 gene itself is responsive to ABA and stress conditions, including cold, high salt, wounding, and drought. Disruption of CIPK3 altered the expression pattern of a number of stress gene markers in response to ABA, cold, and high salt. However, drought-induced gene expression was not altered in the cipk3 mutant plants, suggesting that CIPK3 regulates select pathways in response to abiotic stress and ABA. These results identify CIPK3 as a molecular link between stress- and ABA-induced calcium signal and gene expression in plant cells. Because the cold signaling pathway is largely independent of endogenous ABA production, CIPK3 represents a cross-talk "node" between the ABA-dependent and ABA-independent pathways in stress responses.     

ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis

Protein phosphorylation has pivotal roles in ABA and osmotic stress signaling in higher plants. Two protein phosphatase genes, ABI1 and ABI2, are known to regulate these signaling pathways in Arabidopsis: The identity of ABA-activated protein kinases required for the ABA signaling, however, remains to be elucidated. Here we demonstrate that two protein kinases, p44 and p42, were activated by ABA in Arabidopsis T87 cultured cells, and at least one protein kinase, p44, was activated not only by ABA but also by low humidity in Arabidopsis plants. Analysis of T-DNA knockout mutants and biochemical analysis using a specific antibody revealed that the p44 is encoded by a SnRK2-type protein kinase gene, SRK2E. The srk2e mutation resulted in a wilty phenotype mainly due to loss of stomatal closure in response to a rapid humidity decrease. ABA-inducible gene expression of rd22 and rd29B was suppressed in srk2e. These results show that SRK2E plays an important role in ABA signaling in response to water stress.     

PYR/PYL/RCAR family members are major in‐vivo ABI1 protein phosphatase 2C‐interacting proteins in Arabidopsis

Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group‐A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP‐tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1‐interacting proteins by mass‐spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA‐signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1‐interacting proteins in all LC‐MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA‐binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1–PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 co‐immunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA‐induced stomatal closure and ABA‐inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1–ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinase‐PYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCAR‐mediated ABA signalling.     

Disruption of the Arabidopsis thaliana inward-rectifier K+ channel AKT1 improves plant responses to water stress

The Arabidopsis thaliana inward-rectifier K(+) channel AKT1 plays an important role in root K(+) uptake. Recent results show that the calcineurin B-like (CBL)-interacting protein kinase (CIPK) 23-CBL1/9 complex activates AKT1 in the root to enhance K(+) uptake. In addition, this CIPK-CBL complex has been demonstrated to regulate stomatal movements and plant transpiration. However, a role for AKT1 in plant transpiration has not yet been demonstrated. Here we show that disruption of AKT1 conferred an enhanced response to water stress in plants. Experiments performed in hydroponics showed that, when water potential was diminished by adding polyethylene glycol, akt1 adult plants lost less water than wild-type (WT) plants. Under long-term water stress in soil, adult akt1 plants displayed lower transpiration and less water consumption than WT plants. Finally, akt1 stomata closed more efficiently in response to ABA. Such results were also observed in cipk23 plants. The similar responses shown by cipk23 and akt1 plants to water stress denote that the regulation of AKT1 by CIPK23 may also take place in stomata and has a negative impact on plant performance under water stress conditions.     

Blue light‐induced apoplastic acidification of Arabidopsis thaliana guard cells: Inhibition by ABA is mediated through protein phosphatases

The phytohormone abscisic acid (ABA) inhibits blue light‐induced apoplastic acidification of guard cells. The signal transduction pathway of ABA, mediating this response, was studied using ABA‐insensitive ( abi ) mutants of Arabidopsis thaliana . Apoplastic acidification was monitored with a flat tipped pH‐electrode placed on epidermal strips, in which only guard cells were viable. Blue light‐induced apoplastic acidification was reduced by vanadate and diethylstilbestrol (DES), indicating involvement of plasma membrane‐bound H⁺‐ATPases. In wild type epidermal strips, ABA reduced blue light‐induced acidification to 63%. The inhibition did not result from an increased cytoplasmic free Ca²⁺ concentration in guard cells, since factors that increase the Ca²⁺ concentration stimulated apoplastic acidification. Apoplastic acidification was not inhibited by ABA in abi1 and abi2 mutants. In abi1 epidermal strips ABA had no effect on the acidification rate, while it stimulated apoplastic acidification in abi2 . The ABA response in both mutants could be partially restored with protein kinase and phosphatase inhibitors. The abi1 guard cells became ABA responsive in the presence of okadaic acid, a protein phosphatase inhibitor. In abi2 guard cells the wild type ABA response was partially restored by K‐252a, a protein kinase inhibitor. Apoplastic inhibition is thus mediated through the protein phosphatases encoded by ABI1 and ABI2 . The results with protein kinase and protein phosphatase inhibitors indicate that ABI1 and ABI2 are involved in separate signal transduction pathways.     

A calcium sensor and its interacting protein kinase are global regulators of abscisic acid signaling in Arabidopsis

The phytohormone abscisic acid (ABA) triggers an oscillation in the cytosolic Ca(2+) concentration, which is then perceived by unknown Ca(2+) binding proteins to initiate a series of signaling cascades that control many physiological processes, including adaptation to environmental stress. We report here that a Ca(2+) binding protein, SCaBP5, and its interacting protein kinase, PKS3, function as global regulators of ABA responses. Arabidopsis mutants with silenced SCaBP5 or PKS3 are hypersensitive to ABA in seed germination, seedling growth, stomatal closing, and gene expression. PKS3 physically interacts with the 2C-type protein phosphatase ABI2 (ABA-insensitive 2) and to a lesser extent with the homologous ABI1 (ABA-insensitive 1) protein. Thus, SCaBP5 and PKS3 are part of a calcium-responsive negative regulatory loop controlling ABA sensitivity.     

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     

Identification of the abscisic acid receptor VvPYL1 in Vitis vinifera

The phytohormone abscisic acid (ABA) plays a central role in many developmental processes and in responses to several abiotic stresses. Identification of the ABA receptor is a first step towards understanding ABA signalling. In this study, using homology analysis, we cloned three genes, named VvPYL1, VvPYL2 and VvPYL3, from Vitis vinifera. An isothermal titration calorimetry assay suggested that VvPYL1 could bind to ABA. A phosphatase activity assay demonstrated that VvPYL1 inhibits phosphatase activity of ABI1, a negative regulator of ABA signalling, in the presence of ABA. Subcellular localisation demonstrates that VvPYL1 is distributed in both the nucleus and cytosol, which is similar to the subcellular localisation of ABA receptors in Arabidopsis. We therefore conclude that VvPYL1 is an ABA receptor that modulates ABA signalling by inhibiting type 2C protein phosphatases (PP2Cs).     

The abi1-1 mutation blocks ABA signaling downstream of cADPR action

Arabidopsis thaliana abscisic acid insensitive 1-1 (abi1-1) is a dominant mutant that is insensitive to the inhibition of germination and growth by the plant hormone, abscisic acid (ABA). The mutation severely decreases the catalytic activity of the ABI1 type 2C protein phosphatase (PP2C). However, the site of action of the abi1-1/ABI1 in the ABA signal transduction pathway has not yet been determined. Using single cell assays, we showed that microinjecting mutant abi1-1 protein inhibited the activation of RD29A-GUS and KIN2-GUS in response to ABA, cyclic ADP-ribose (cADPR), and Ca2+. The inhibitory effect of the mutant protein, however, was reversed by co-microinjection of an excess amount of the ABI1 protein. In transgenic Arabidopsis plants, overexpression of abi1-1 rendered the plants insensitive to ABA during germination, whereas overexpression of ABI1 did not have any apparent effect. Moreover, transgenic plants overexpressing abi1-1 were blocked in the induction of ABA-responsive genes; however, overexpression of ABI1 did not affect gene expression. Taken together, our results demonstrate that abi1-1 is likely to be a dominant negative mutation and ABI1 likely acts downstream of cADPR in the ABA-signaling pathway. Our results on ABI1 overexpression in Arabidopsis are not compatible with a negative regulatory role of this phosphatase in ABA responses.     

The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway

The Arabidopsis ABI1 and ABI2 genes encode two protein serine/threonine phosphatases 2C (PP2C). These genes have been originally identified by the dominant mutations abi1--1 and abi2--1, which reduce the plant's responsiveness to the hormone abscisic acid (ABA). However, recessive mutants of ABI1 were recently shown to be supersensitive to ABA, which demonstrated that the ABI1 phosphatase is a negative regulator of ABA signalling. We report here the isolation and characterisation of the first reduction-of-function allele of ABI2, abi2--1R1. The in vitro phosphatase activity of the abi2--1R1 protein is approximately 100-fold lower than that of the wild-type ABI2 protein. Abi2--1R1 plants displayed a wild-type ABA sensitivity. However, doubly mutant plants combining the abi2--1R1 allele and a loss-of-function allele at the ABI1 locus were more responsive to ABA than each of the parental single mutants. These data indicate that the wild-type ABI2 phosphatase is a negative regulator of ABA signalling, and that the ABI1 and ABI2 phosphatases have overlapping roles in controlling ABA action. Measurements of PP2C activity in plant extracts showed that the phosphatase activity of ABI1 and ABI2 increases in response to ABA. These results suggest that ABI1 and ABI2 act in a negative feedback regulatory loop of the ABA signalling pathway.     

The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction.

Abscisic acid (ABA) mediates seed maturation and adaptive responses to environmental stress. In Arabidopsis, the ABA-INSENSITIVE1 (ABI1) protein phosphatase 2C is required for proper ABA responsiveness both in seeds and in vegetative tissues. To determine whether the lack of recessive alleles at the corresponding locus could be explained by the existence of redundant genes, we initiated a search for ABI1 homologs. One such homolog turned out to be the ABI2 locus, whose abi2-1 mutation was previously known to decrease ABA sensitivity. Whereas abi1-1 is (semi)dominant, abi2-1 has been described as recessive and maternally controlled at the germination stage. Unexpectedly, the sequence of the abi2-1 mutation showed that it converts Gly-168 to Asp, which is precisely the same amino acid substitution found in abi1-1 and at the coincidental position within the ABI1 phosphatase domain (Gly-180 to Asp). In vitro assays and functional complementation studies in yeast confirmed that the ABI2 protein is an active protein phosphatase 2C and that the abi2-1 mutation reduced phosphatase activity as well as affinity to Mg2+. Although a number of differences between the two mutants in adaptive responses to stress have been reported, quantitative comparisons of other major phenotypes showed that the effects of both abi1-1 and abi2-1 on these processes are nearly indistinguishable. Thus, the homologous ABI1 and ABI2 phosphatases appear to assume partially redundant functions in ABA signaling, which may provide a mechanism to maintain informational homeostasis.     

拟南芥CIPK1基因的功能初步分析

以模式植物拟南芥为材料,采用RT-PCR分析、基因克隆、转化等方法对CIPK1基因功能进行了研究.结果发现,CIPK1(CBL-interacting protein kinase 1)基因在拟南芥茎和花中表达量最高,在叶中表达量最低;ABA能迅速诱导CIPK1基因的表达,GA则抑制该基因的表达,但2,4-D和6-BA对该基因的表达无明显影响;通过对CIPK1基因转基因突变体进行ABA处理,发现转基因拟南芥种子的萌发率明显高于野生型.以上结果表明CIPK1基因的表达具有组织特异性,并且该基因参与ABA和GA信号传导,尤其在ABA促进种子休眠的信号传导途径中具有非常重要的作用.