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Plant Signal Behav. Nov 1, 2011; 6(11): 1687–1690.
PMCID: PMC3329337

CPK12

A Ca2+-dependent protein kinase balancer in abscisic acid signaling

Abstract

Ca2+ is believed to be a critical second messenger in ABA signal transduction. Ca2+-dependent protein kinases (CDPKs) are the best characterized Ca2+ sensors in plants. Recently, we identified an Arabidopsis CDPK member CPK12 as a negative regulator of ABA signaling in seed germination and post-germination growth, which reveals that different members of the CDPK family may constitute a regulation loop by functioning positively and negatively in ABA signal transduction. We observed that both RNA interference and overexpression of CPK12 gene resulted in ABA-hypersensitive phenotypes in seed germination and post-germination growth, suggesting a high complexity of the CPK12-mediated ABA signaling pathway. CPK12 stimulates a negative ABA-signaling regulator (ABI2) and phosphorylates two positive ABA-signaling regulators (ABF1 and ABF4), which may partly explain the ABA hypersensitivity induced by both downregulation and upregulation of CPK12 expression. Our data indicate that CPK12 appears to function as a balancer in ABA signal transduction in Arabidopsis.

Keywords: ABA signal transduction, abscisic acid (ABA), Ca2+-dependent protein kinase (CDPK), CPK12

Introduction

Abscisic acid (ABA) regulates many processes of plant development, including seed maturation and germination, seedling growth and flowering, and plays a vital role in plant adaptation to environment challenges, such as drought, salt and cold stress.1-5 Ca2+ is an important second message involved in ABA signal transduction.6 The Ca2+ sensors identified in plants include calmodulin (CaM) and CaM-related proteins, calcineurin B-like (CBL) proteins, and Ca2+-dependent protein kinases (CDPKs).3,7,8 CDPKs have both kinase and CaM-like domain and are among the best characterized calcium sensors in plants.9 There are 34 genes predicted to encode CDPKs in Arabidopsis.9 In recent years, researchers used genetic approaches to identify a set of CDPK members as ABA signaling components, which regulate plant response to abiotic stresses. CPK32 interacts with and phosphorylates an ABA-responsive transcription factor ABF4, and the CPK32-overexpression plants show hypersensitive phenotype to ABA and NaCl, indicating that CPK32 is positively involved in ABA signaling.10 The loss-of-function mutant of the CPK23 gene shows more tolerant to drought and salt stresses, while the CPK23-overexpression lines are hypersensitive to these stresses.11 CPK23, together with CPK21, is involved in the process of ABA-regulated stomata movement by regulating anion channel SLAC1.12 When plants face drought stress, CPK23 and CPK21 phosphorylate and activate SLAC1, which leads to depolarization of the membrane and activation of the K+ release channel GORK, resulting in anion and K+ release from the guard cells.13 This continuous process finally induces stomata closure.12 Recent report showed that ABA receptor RCAR1-ABI1 coupled signaling regulates CPK21 to control anion channel SLAC1 and SLAH3.13 Two other CDPK members, CPK3 and CPK6, were also shown to be involved in guard cell signaling in response to ABA. ABA and Ca2+ induce stomatal closure and stimulate the activity of slow-type anion channels, but the two effects are inhibited in CPK3 and CPK6 single or double mutants, by which mechanism CPK3 and CPK6 are positively involved in ABA-regulated stomatal movement.14 Recently, CPK10 was shown to be a positive regulator of plant response to drought, which was evidenced by the findings that the loss-of-function mutant of the CPK10 gene is hypersensitive to drought stress, while the CPK10-overexpression plants show enhanced tolerance to drought.15 CPK10 interacts with a heat shock protein HSP1. The HSP1 T-DNA insertion mutant hsp1 shows similar phenotype with cpk10 mutant under drought stress, suggesting that HSP1 functions directly downstream of CPK10.15 We previously observed that the kinase activity of two homologous CDPKs in Arabidopsis, CPK4 and CPK11, is stimulated by ABA.16 Loss-of-function mutants of CPK4 and CPK11 show ABA-insensitive phenotypes in seed germination, seedling growth, and stomatal movement, and also exhibit salt insensitivity in seed germination and decreased tolerance of seedlings to salt stress, while CPK4- or CPK11-overexpressing plants show ABA hypersensitive phenotypes.16 Both CPK4 and CPK11 phosphorylate two ABA-responsive transcription factors, ABF1 and ABF4, suggesting that the two kinases may regulate ABA signaling through these transcription factors.16 These data show that CPK4 and CPK11 are two important positive regulators in CDPK/Ca2+-mediated ABA signaling pathways. All these significant progresses deepen our understanding of the mechanisms of Ca2+ signaling in ABA signaling pathways.

CPK12 may function as an antagonist to positive roles of other CDPK members in ABA signaling

In a cell signaling pathway involving reversible protein phosphorylation, protein kinase-mediated signaling process by phosphorylation is believed to be terminated or balanced by a de-phosphorylation event that is mediated by a protein phosphatase. Whereas many members of CDPK family have been identified as positive players in ABA signaling, our knowledge about protein phosphatases that antagonize CDPKs in ABA signaling has been limited. Interestingly, we observed that CPK12, a homolog of CPK4 and CPK11, functions as a negative regulator of ABA signaling,17 suggesting that one CDPK member-triggered ABA signaling event may be balanced by another CDPK member, and that different members of CDPK family may constitute thus a regulation loop by functioning positively and negatively in ABA signal transduction. This mechanism may provide cells with diversity of tactics to balance or terminate ABA signaling when stressful conditions are well passed. Also, a CDPK-signaling balancer with another CDPK member may be useful in cell signaling in response to Ca2+, as a CDPK-mediated process may be terminated by a Ca2+ sensor in the presence of Ca2+ without Ca2+ signal receding.

Both up- and downregulation of CPK12 expression result in ABA hypersensitivity

We previously observed that the CPK12-RNAi mutants were hypersensitive to ABA in seed germination and post germination seedling growth.17 To investigate whether upregulation of CPK12 gene alters ABA signaling, we generated CPK12-overexpression lines under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Fig. 1A). The seeds of the CPK12-overexpressors germinated normally as the wild-type seeds did in the ABA-free medium (0 μM ABA), but in the media supplemented with different concentrations of (±)-ABA, their germination rate was significantly more inhibited by ABA than that of the wild-type seeds (Fig. 1B). In seedling growth, there was no difference between CPK12-overexpression plants and wild-type seedlings on ABA-free medium (Fig. 2; 0 μM ABA). However, on the medium containing 0.3 μM and 0.5 μM (±)-ABA, the growth of the CPK12-overexpression seedlings was more inhibited than that of the wild-type seedlings (Fig. 2; 0.3 μM and 0.5 μM ABA). We screened more than ten CPK12-overexpression lines, and all the lines showed ABA hypersensitivity in seed germination and post-germination growth. Thus, the CPK12-overexpressors showed unexpectedly the same phenotypes as those of CPK12-RNAi lines.17 These findings indicate a high complexity of the CPK12-mediated ABA signal transduction.

Figure 1.
Overexpression of CPK12 gene enhances the sensitivity of seed germination to ABA. (A) The mRNA amounts estimated by both real-time PCR (top columns; relative units, normalized relative to the mRNA level of the wild-type Col-5) and RNA gel blot (bottom, ...
Figure 2.
Overexpression of CPK12 gene enhances the sensitivity of post-germination growth to ABA. Seeds of the two CPK12-overexpression lines (12OE1 and 12OE2) and wild-type Col-5 plants were directly planted in the ABA-free (0 μM) medium and the media ...

How does CPK12 work as a balancer in ABA signaling?

CPK12 phosphorylates two ABA responsive transcription factor ABF1 and ABF4, and interacts with, phosphorylates, and stimulates a type 2C protein phosphatase ABI2.17 ABF1 and ABF4 are members of ABA-responsive, basic leucine zipper transcription factors positively involved in ABA signaling transduction,18,19 but ABI2 is an important member of the clade A type 2C protein phosphatases negatively involved in ABA signaling.20,21 CPK12 may use these important downstream targets that function distinctly in ABA signaling. The CPK12-induced stimulation of phosphatase activity of ABI2, which inhibits downstream positive ABA-signaling regulators SnRK2s,21 may be abolished when CPK12 is downregulated, while the CPK12-induced inhibition of ABF1 and ABF4 transcription factors may be relieved when CPK12 is upregulated. Consequently, ABA signaling may be improved in both cases, which may partly explain the ABA hypersensitive phenotypes with both the CPK12 overexpressors (Figs. 1 and and2)2) and RNAi lines.17 However, whether and how CPK12 can function to selectively act on its targets needs further studies in the future. In addition, signaling components upstream of CPK12 and other identified ABA signaling CDPK members remain to be identified in the future, though several functional substrates have been identified. Identification of additional CDPK members and their downstream substrates and elucidation of the mechanisms underlying these CDPKs in ABA signaling pathways will greatly improve our understanding of plant signaling in response to environmental challenges.

Acknowledgments

This work was supported by National Natural Science Foundation of China (grant nos. 30671444 and 90817104 to D.-P.Z. and 30700053 to X.-F.W.) and by a grant from Agricultural Ministry of China (grant no. 2008ZX08009–003 to D.P.Z.).

Glossary

Abbreviations:

ABA
Abscisic acid
Arabidopsis
ABF1/ABF4, ABA-responsive transcription factor1/4
ABI2
ABA insensitive 2
Arabidopsis thaliana
CDPK
Ca2+ dependent protein kinase
SnRK2
sucrose non-fermenting-1-related protein kinase 2

Footnotes

References

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