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. 2006 Oct;4(10):e327.
doi: 10.1371/journal.pbio.0040327.

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure

Izumi C Mori  1 , Yoshiyuki Murata, Yingzhen Yang, Shintaro Munemasa, Yong-Fei Wang, Shannon Andreoli, Hervé Tiriac, Jose M Alonso, Jeffery F Harper, Joseph R Ecker, June M Kwak, Julian I Schroeder
Affiliations

CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca(2+)-permeable channels and stomatal closure

Izumi C Mori et al. PLoS Biol. 2006 Oct.

Abstract

Abscisic acid (ABA) signal transduction has been proposed to utilize cytosolic Ca(2+) in guard cell ion channel regulation. However, genetic mutants in Ca(2+) sensors that impair guard cell or plant ion channel signaling responses have not been identified, and whether Ca(2+)-independent ABA signaling mechanisms suffice for a full response remains unclear. Calcium-dependent protein kinases (CDPKs) have been proposed to contribute to central signal transduction responses in plants. However, no Arabidopsis CDPK gene disruption mutant phenotype has been reported to date, likely due to overlapping redundancies in CDPKs. Two Arabidopsis guard cell-expressed CDPK genes, CPK3 and CPK6, showed gene disruption phenotypes. ABA and Ca(2+) activation of slow-type anion channels and, interestingly, ABA activation of plasma membrane Ca(2+)-permeable channels were impaired in independent alleles of single and double cpk3cpk6 mutant guard cells. Furthermore, ABA- and Ca(2+)-induced stomatal closing were partially impaired in these cpk3cpk6 mutant alleles. However, rapid-type anion channel current activity was not affected, consistent with the partial stomatal closing response in double mutants via a proposed branched signaling network. Imposed Ca(2+) oscillation experiments revealed that Ca(2+)-reactive stomatal closure was reduced in CDPK double mutant plants. However, long-lasting Ca(2+)-programmed stomatal closure was not impaired, providing genetic evidence for a functional separation of these two modes of Ca(2+)-induced stomatal closing. Our findings show important functions of the CPK6 and CPK3 CDPKs in guard cell ion channel regulation and provide genetic evidence for calcium sensors that transduce stomatal ABA signaling.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Guard Cell Expression of CPK3 and CPK6 CDPKs
(A) Expression of CPK3 and CPK6 in guard cell (GC) and mesophyll cell (MC) protoplasts was examined by RT-PCR. Control amplifications of the guard cell-expressed KAT1 gene and the mesophyll-expressed CBP marker genes [54] (Leonhardt et al., 2004) were used to test the purity of cell preparations (see Results). ACTIN2 was used for an internal loading control. To amplify each CDPK-specific band, RT-PCR was performed with primer sets as indicated by arrowheads in (B) for 36 cycles. Plants were sprayed with water (−ABA) or 100 μM ABA (+ABA) 4 h before isolation of protoplasts and RNA extraction. (B) Cartoon showing the T-DNA insertion positions in cpk3 and cpk6 T-DNA insertion alleles. PCR was performed with a left boarder primer of the T-DNA and a gene-specific primer, and the PCR products were sequenced to determine the T-DNA insertion positions. Arrowheads indicate primer locations for RT-PCR in (A) and (C). ATG and TGA indicate start and stop codons. White boxes indicate exons. (C) RT-PCR confirmed that cpk3–1 and cpk6–1 alleles were disruption mutants. PCRs (32 cycles) were performed with primer sets as indicated in (B) (black arrowheads) in the left three panels. Transcripts of wild-type (WT) and cpk6–2 were examined with two sets of primers [white and black arrowheads in (B)] showing that cpk6–2 lacks exon 1 and that the cpk6–2 has 8% or less the mRNA level of wild-type based on densitometry analyses (n = 2). RNA was extracted from leaves of WT, homozygous cpk3–1, cpk6–1, and cpk6–2 single mutants, and the cpk3-1cpk6–1 double mutant.
Figure 2
Figure 2. Impairment in [Ca2+]cyt-Dependent Activation of S-Type Anion Channel Currents in Guard Cells of cpk3 and cpk6 Single and cpk3cpk6 Double Mutants
(A and B) Typical S-type anion channel current traces in wild-type (WT) (A) and cpk3-1cpk6–1 double mutant (B) guard cells are shown in response to 2 μM free Ca2+ in the patch-clamp pipette solution that dialyzes the cytoplasm. (C) Average current-voltage curves of wild-type (n = 17 cells), cpk3–1 (n = 11), cpk3–2 (n = 7), cpk6–1 (n = 11), cpk6–2 (n = 4), cpk3-1cpk6–1 (n = 17), and cpk3-2cpk6–2 (n = 11). Error bars show SEM.
Figure 3
Figure 3. Impairment in ABA Activation of S-Type Anion Channel Currents in cpk3cpk6 Mutant Guard Cells
(A and B) Whole-cell S-type anion channel current traces in wild-type (WT) guard cells in the absence of ABA (A) and in the presence of 50 μM ABA (B). (C and D) Whole-cell S-type anion channel current traces in cpk3-1cpk6–1 double mutant guard cells in the absence of ABA (C) and in the presence of ABA (D). (E) Average current-voltage curves of wild-type and cpk3-1cpk6–1 and cpk3-2cpk6–2 double mutant guard cells in the absence and presence of ABA (n = 8 WT; n = 7 cpk3-1cpk6–1; n = 3 cpk3-2cpk6–2 guard cells). GCPs were treated with 50 μM ABA or solvent control (0.1% ethanol) for 2 h prior to establishing gΩ seals. Open and closed symbols indicate −ABA and +ABA, respectively.
Figure 4
Figure 4. Impairment in ABA Activation of ICa Channel Currents in cpk3 and cpk6 Single Mutants and cpk3cpk6 Double Mutants
(A and B) ABA (50 μM) activated ICa channel current in wild-type (WT) guard cells. (A) Current traces before (−ABA) and after ABA (+ABA) activation of ICa channels are shown in a representative cell. (B) Average current-voltage curves (n = 13) are shown. (C and D) ABA failed to activate ICa channel currents in cpk3-1cpk6–1 double mutant guard cells. (C) A response in a representative cell is shown. (D) Average current-voltage curves (n = 14) are shown. Traces before and after ABA overlap in (C). (E–I) Averages of current-voltage curves in guard cells isolated from (E) cpk3-2cpk6–2 double mutant (n = 8), (F) cpk3–1 (n = 11), (G) cpk6–1 (n = 9), (H) cpk3–2 (n = 3), and (I) cpk6–2 (n = 5) are shown. (J and K) ABA activation of ICa channel currents in wild-type (WT) guard cells pretreated with the protein kinase inhibitor K252a (2 μM) for 15 min prior to and during patch clamping and with no ATP added in the patch pipette solution. Response in a guard cell is shown in (J), and average current-voltage curves (n = 6) are shown in (K). Open symbols indicate −ABA and closed symbols indicate +ABA. Error bars represent SEM.
Figure 5
Figure 5. Hydrogen Peroxide Activates ICa Channels Both in Wild-type and cpk3cpk6 Double Mutant Guard Cells
(A and B) Exogenous H2O2 (5 mM) activated ICa channels in (A) wild-type (WT) (n = 9) and (B) cpk3-1cpk6–1 double mutant guard cells (n = 12). Error bars represent SEM.
Figure 6
Figure 6. ABA- and Ca2+-Induced Stomatal Closure Is Partially Impaired in cpk3cpk6 Mutants
(A) ABA-induced stomatal closing (white bars: wild-type; shaded bars: cpk3-1cpk6–1 double mutant; black bars: cpk3-2cpk6–2 double mutant). Average data from representative experiments are shown (wild-type: WT, n = 13 experiments, 260 total stomata; cpk3-1cpk6–1 double mutant: n = 7 experiments, 140 stomata; cpk3-2cpk6–2 n = 4 experiments, n = 120 stomata). (B) External Ca2+-induced stomatal closing (white bars: wild-type, shaded bars: cpk3-1cpk6–1 double mutant, black bars: cpk3-2cpk6–2 double mutant). Average data from representative experiments are shown (wild-type: WT, n = 9 experiments including 4 blind experiments, 180 total stomata; cpk3-1cpk6–1 double mutant, n = 9 experiments including 4 blind experiments, 180 total stomata; cpk3-2cpk6–2, n = 4 experiments, n = 80 stomata). Stomatal aperture widths are illustrated. Error bars represent SEM.
Figure 7
Figure 7. Ca2+-Reactive Closure Is Impaired in cpk3cpk6 Double Mutant Stomata
Four [Ca2+]cyt transients (inset) were imposed in wild-type (WT) (n = 6 experiments) and cpk3-1cpk6–1 double mutant (n = 6 experiments) stomata. Individually mapped stomatal apertures were measured for the last 30 min before imposed [Ca2+]cyt transients (before Time = 0) and for the ensuing 180 min at the indicated time points. Error bars represent SEM. Inset (top) shows imposed [Ca2+]cyt transients in wild-type (blue trace) and in cpk3-1cpk6–1 (red trace) guard cells expressing Yellow Cameleon 3.6 (R.U.: ratio units).
Figure 8
Figure 8. R-Type Anion Channel Currents in cpk3cpk6 Double Mutant and Wild-Type Guard Cells
(A) Representative traces of R-type anion channel current in wild-type (black trace) and cpk3-1cpk6–1 double mutant (gray trace). (B) Averages of peak currents of R-type anion channel currents in wild-type (white bar, n = 7) and cpk3-1cpk6–1 double mutant (black bar, n = 7 ± SEM) guard cells.
Figure 9
Figure 9. Simplified Models for Early ABA Signal Transduction Network and CPK3 and CPK6 Functions in Guard Cells
Plasma membrane ICa channel and intracellular Ca2+-release mechanisms cause cytosolic Ca2+ elevations. CPK3 and CPK6 mediate [Ca2+]cyt activation of S-type anion channels. CPK3 and CPK6 regulate ICa Ca2+ channels by a proposed feedback (A) or parallel pathway (B) or by transcriptional/translational regulation mechanism (see Discussion). R-type anion channels are regulated in a parallel CPK3-CPK6-independent signal transduction pathway. The arrow connecting R-type and S-type anion channels indicates that these channels may share molecular components [89]. See Discussion for details.
Figure 10
Figure 10. Alterations in the Extracellular Calcium Concentration in the Absence of Changes in the Extracellular Cl− Concentration Cause Large Ratio Changes in YC3.6 in Arabidopsis Guard Cells Illustrating that YC3.6, Like YC2.1, Reports Cytosolic Ca2+ Changes and Not Cl− Changes during Imposed Transients
See Materials and Methods. The YFP/CFP emission ratio is monitored in a guard cell (wild-type) expressing YC3.6. Ratio increases accompany exposure to high extracellular calcium using the membrane-impermeable counteranion, imminodiacetate.
See this image and copyright information in PMC

Comment in

  • Protein kinases and plant pores.
    Gross L. Gross L. PLoS Biol. 2006 Oct;4(10):e358. doi: 10.1371/journal.pbio.0040358. Epub 2006 Oct 10. PLoS Biol. 2006. PMID: 20076480 Free PMC article. No abstract available.

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