CDPK-mediated abiotic stress signaling

Abstract

Calcium-dependent protein kinases (CDPKs) constitute a large multigene family in various plant species. CDPKs have been shown to have important roles in various physiological processes, including plant growth and development and abiotic and biotic stress responses in plants. Functional analysis using gain-of-function and loss-of-function mutants has revealed the biological function of CDPKs in planta. Several CDPKs have been shown to be essential factors in abiotic stress tolerance, positively or negatively regulating stress tolerance by modulating ABA signaling and reducing the accumulation of reactive oxygen species (ROS). This review summarizes recent results describing the biological function of CDPKs that are involved in abiotic stress tolerance.

Plants have developed a network of signal transduction pathways to control their metabolism and to adapt to their environment. Among these pathways, calcium plays an important role as a messenger in various signal transduction pathways.^1-3 In plants, several Ca²⁺ sensors or Ca²⁺-binding proteins recognize transient Ca²⁺ elevation in response to various stimuli, including hormones, pathogens, light and abiotic stresses, and induce downstream effects, such as altered protein phosphorylation and gene expression patterns.^1,4,5 Three major classes of Ca²⁺-binding proteins have been characterized in higher plants: calcium-dependent protein kinases (CDPKs), calmodulins (CaMs) and CaM-like proteins,⁶ and calcineurin B-like proteins.^7-9

Each calcium-dependent protein kinase (CDPK) consists of a variable N-terminal domain, a protein kinase domain, an autoinhibitory region and a calmodulin-like domain with EF-hand Ca²⁺-binding sites.^10-13 CDPKs are directly activated by the binding of Ca²⁺ to the calmodulin-like domain, and the activated CDPKs regulate downstream targets.^14,15 CDPKs have been identified throughout the plant kingdom^15,16 and in some protozoans,¹⁷ but not in animals.^11,12,15 CDPKs constitute a large multigene family in various plant species;^11,12,18,19 34 CDPK genes have been identified in Arabidopsis thaliana,^11,12 and 29 CDPK genes have been found in Oryza sativa (rice)¹⁸ (Fig. 1). The expression and activities of CDPKs are upregulated by a variety of stimuli, such as hormones, abiotic stresses and biotic stresses.¹⁶ Furthermore, CDPKs show widespread subcellular distribution, including the plasma membrane, cytosol, nucleus, endoplasmic reticulum, peroxisomes, mitochondrial outer membrane, and oil bodies.^14,20 These results suggest that CDPKs are involved in many physiological processes in various cellular compartments. In this review, we focus on recent finding describing the molecular mechanisms by which CDPKs modulate tolerance to abiotic stress in plants.

Figure 1. (A) Structure of CDPK. The junction contains an autoinhibitory sequence that blocks the active site of the CDPK under basal state. The four bars within the calmodulin-like domain indicate the EF-hand Ca²⁺ binding motif. (B) Phylogenetic relationships among CDPKs from rice (OsCPK1-OsCPK29) and Arabidopsis (AtCPK1-AtCPK34). Red letters indicate CDPKs involved in abiotic stress signaling.

The Molecular Functions of CDPKs in ABA and Abiotic Stress Responses

The phytohormone abscisic acid (ABA) plays important roles in tolerance to abiotic stress in plants.^21-23 Some Arabidopsis CDPKs have been reported to be involved in abiotic and ABA signaling pathways. CPK3 and CPK6 were shown to be involved in the regulation of guard cell ion channels and in ABA-regulated stomatal signaling in experiments using Arabidopsis loss-of-function mutants. These mutants maintained normal ABA response during seed germination or early seedling growth.²⁴ Later, CPK3 and CPK6 were reported to be positive regulators of the abiotic stress response. cpk3 mutants have a salt-sensitive phenotype. CPK3 is found to be associated with the plasma membrane and the vacuole. CPK3 is likely to regulate abiotic stress signaling independently of the MAPK-mediated signaling pathway.²⁵ Meanwhile, CPK6-overexpressing plants have enhanced tolerance to salt and drought stresses, whereas cpk6 mutants have no obvious phenotypes. CPK6 is likely to act as a positive regulator of the tolerance to salt and drought stresses, but CPK6 is functionally redundant in abiotic stress signaling in Arabidopsis.²⁶ Arabidopsis CPK10 is involved in tolerance to drought stress. Furthermore, cpk10 mutants exhibit impaired induction of stomatal closure and inhibition of stomatal opening in response to ABA and Ca²⁺.²⁷ Therefore, CPK10 may play important roles in the ABA- and Ca²⁺-mediated regulation of stomatal movements.

Arabidopsis CDPKs are reported to be involved in the ABA signaling pathway by phosphorylating ABA-responsive element binding factors (ABFs), a subfamily of basic leucine zipper class transcription factor proteins. The closely related Arabidopsis CPK4 and CPK11 are involved in ABA-regulated physiological processes, including seed germination, seedling growth, stomatal movement, and tolerance to salt and drought stresses. CPK4 and CPK11 phosphorylate two ABA-responsive transcription factors, ABF1 and ABF4,²⁸ indicating that they are positive regulators in the CDPK-mediated ABA signaling pathway. Arabidopsis CPK32 interacts with ABF4 and phosphorylates ABF4 in vitro. The overexpression of CPK32 in plants affects the expression of ABF4 regulated genes, the ABA response, and salt sensitivity. Thus, CPK32 participates in ABA and stress responses by regulating the ABA-responsive gene expression via ABF4.²⁹ The Arabidopsis cpk23 mutant shows markedly enhanced tolerance to drought and salt stresses and reduced stomatal apertures, while CPK23 overexpressing lines are more sensitive to drought and salt stresses and have increased stomatal apertures.³⁰ Arabidopsis seedlings with a loss-of-function of CPK21, which is the closest homolog of CPK23, show increased tolerance to hyperosmotic stress. In contrast, the salt tolerance of the cpk21 mutants is similar to that of wild-type plants.³¹ The closely related CPK21 and CPK23 proteins have partially overlapping functions in ABA and abiotic stress signaling and appear to act as negative regulators of the plant abiotic stress response. Independently, using Xenopus laevis oocytes and Arabidopsis protoplast systems, CPK21 and CPK23 were reported to control the activation state of the slow guard cell anion channel SLAC1 in response to different Ca²⁺ concentrations as well as in a Ca²⁺-independent manner. Open stomata 1 protein kinase (OST1)/CPK23 and ABA-INSENSITIVE1 (ABI1) seem to be associated with the calcium-independent steps of ABA signaling, whereas CPK21/ABI1 regulates SLAC1 in response to cytosolic calcium signaling.³² Thus, these contradictory results may be attributed to differences in the experimental systems used. This observation suggests that a complex CDPK-mediated signaling pathway functions in the ABA and abiotic stress responses in plants, and it is clear that several additional factors function in concert with CPK21/CPK23 in this signaling pathway.

In contrast to Arabidopsis CDPKs, the activities of rice CDPKs in abiotic stress and ABA signaling are largely unknown. The overexpression of OsCDPK7 in rice has been shown to enhance tolerance to cold, salt and drought stresses³³; meanwhile, the overexpression of OsCDPK13 in rice has been shown to increase cold stress tolerance.³⁴ The overexpression of OsCPK21 in rice has been shown to increase tolerance to salt stress and sensitivity to ABA. Therefore, OsCPK21 is likely to be involved in the positive regulation of ABA and salt signaling pathways.³⁵

Abiotic Stress Tolerance is Mediated through the Decreased Accumulation of Reactive Oxygen Species

Abiotic stress induces the accumulation of reactive oxygen species (ROS).^36-38 ROS are toxic molecules that cause oxidative damage, although some ROS function as signal transduction molecules in many biological processes.^36,37,39,40 Plants have evolved defense systems to minimize and/or prevent oxidative damage to cells by ROS and to maintain cellular redox homeostasis under abiotic stress conditions. Various studies have shown that ROS-scavenging enzymes such as ascorbate peroxidase (APx), superoxide dismutase, catalase, and glutathione peroxidase are involved in tolerance to abiotic stress in plants.^38,41-45

NADPH oxidase is reported to be involved in ABA-induced ROS production and ABA-induced stomatal closure.⁴⁶ In addition, ROS produced by NADPH oxidase plays a central role in the oxidative burst and is responsible for triggering defense responses in plants. Solanum tuberosum (potato) CDPK4 and CDPK5 were reported to regulate ROS production by phosphorylating NADPH oxidase.⁴⁷ Arabidopsis CPK5/CPK6 and CPK4/CPK11 also appear to regulate ROS production.⁴⁸ Thus, CDPKs seem to function as positive regulators of ROS production in biotic stress signaling. The rice CDPK OsCPK12 is reported to be an essential positive regulator of tolerance to salt stress.⁴⁹ In contrast to the abovementioned potato and Arabidopsis CDPKs, OsCPK12 is thought to promote tolerance to salt stress by reducing the accumulation of ROS. The overexpression of OsCPK12 confers increased tolerance to salt stress and decreased the accumulation of hydrogen peroxide (H₂O₂) in planta under high salinity conditions. Genes encoding ROS-scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-overexpressing plants than in wild-type plants, whereas the expression of the NADPH oxidase gene OsrbohI was decreased in OsCPK12-overexpressing plants under high salinity conditions. These results suggest that OsCPK12 positively regulates ROS detoxification by controlling the expression of OsAPx2, OsAPx8 and OsrbohI. In addition, OsCPK12 overexpression confers increased sensitivity to exogenous ABA and enhanced susceptibility to blast fungus, most likely due to the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, these data indicate that OsCPK12 functions in multiple signaling pathways and inversely modulates salt stress tolerance and blast disease resistance.

Conclusions and Perspectives

Several CDPKs have been shown to be involved in abiotic stress tolerance in various plants, and these proteins have been especially well characterized in Arabidopsis. CDPKs appear to have important roles in mediating tolerance to abiotic stress through their involvement in ABA signaling and ROS detoxification. In addition, the CDPKs are localized to various compartments in plant cells. These data suggest that CDPKs may have multiple functions that enhance tolerance to abiotic stress. Although several target proteins, that interact with CDPKs and/or are phosphorylated by CDPKs, have been identified, the CDPK-mediated signaling pathways underlying the response to ABA and abiotic stress remain largely unknown. Furthermore, opposing functions for CPK21 and CPK23 in ABA signaling and abiotic stress signaling have been reported. This suggests that the system regulating CDPK-mediated ABA and abiotic stress responses is likely complex. Further physiological and biological analyses are necessary to elucidate the functions of CDPKs in abiotic stress responses in more detail. Understanding the mechanism by which increased cytosolic Ca²⁺ triggers the abiotic stress response and identifying additional components that act downstream of CDPKs or cooperate with CDPKs in abiotic stress signaling will facilitate the resolution of the complex regulatory networks involved in CDPK-mediated ABA and abiotic stress signaling.

Figure 2. Summary of the function of CDPKs in ABA and abiotic stress responses, as reported by several authors.^{20,24-35,49,50} The subcellular localization of each CDPK and the phenotypes of overexpression (OX) or knockout or knockdown (KO) lines are described. Red letters, Arabidopsis CDPKs; green letters, rice CDPKs; P, phosphorylation; ABF, ABA-responsive element binding factor; HSP1, a heat shock protein; OST1, open stomata 1 protein kinase.