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Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells.

Brandt B, Munemasa S, Wang C, Nguyen D, Yong T, Yang PG, Poretsky E, Belknap TF, Waadt R, Alemán F, Schroeder JI - Elife (2015)

Bottom Line: Interestingly, protein phosphatase 2Cs prevent non-specific Ca(2+)-signaling.Moreover, we demonstrate an unexpected interdependence of the Ca(2+)-dependent and Ca(2+)-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1.We identify novel mechanisms ensuring specificity and robustness within stomatal Ca(2+)-signaling on a cellular, genetic, and biochemical level.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, San Diego, United States.

ABSTRACT
A central question is how specificity in cellular responses to the eukaryotic second messenger Ca(2+) is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca(2+)-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca(2+)-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca(2+)-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca(2+)-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca(2+)-dependent and Ca(2+)-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca(2+)-signaling on a cellular, genetic, and biochemical level.

No MeSH data available.


Related in: MedlinePlus

ABA-induced S-type anion currents and stomatal closure responses are impaired when both SLAC1 S59 and S120 are substituted with alanine in independent double amino acid mutant line.(A) In whole-cell patch-clamp experiments, slac1-1 guard cells show impaired ABA-activation of S-type anion currents. Expression of SLAC1 WT, S59A, and S120A in slac1-1 plants restores ABA activation of S-type anion currents, but expression of SLAC1 S59A/S120A does not. (B) ABA-insensitive stomatal closing phenotype of slac1-1 was recovered by expression of SLAC1 WT, S59A, and S120A, but not by expression of S59A/S120A. Note that SLAC1 WT, S59A, S120A, and S59A/S120A are expressed as C-terminal mVenus fusion proteins under the native 1.63 kbp of the SLAC1 5′ UTR promoter region (see ‘Materials and methods’). The results shown here were recorded from independent Arabidopsis slac1-1 transformation lines that differ from the transformation lines shown in Figure 6G,H. Note that Col0 and slac1-1 measurements are the same control data as those shown in Figure 6G,H as all lines were investigated under the same conditions. Average steady-state current responses ±SEM at −145 mV are plotted in (A). In (B) average stomatal apertures ±SEM. * indicates p < 0.05; t-test. Exact p-values and number of individual experiments for (A and B) can be found in Figure 6—source data 1.DOI:http://dx.doi.org/10.7554/eLife.03599.021
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fig6s3: ABA-induced S-type anion currents and stomatal closure responses are impaired when both SLAC1 S59 and S120 are substituted with alanine in independent double amino acid mutant line.(A) In whole-cell patch-clamp experiments, slac1-1 guard cells show impaired ABA-activation of S-type anion currents. Expression of SLAC1 WT, S59A, and S120A in slac1-1 plants restores ABA activation of S-type anion currents, but expression of SLAC1 S59A/S120A does not. (B) ABA-insensitive stomatal closing phenotype of slac1-1 was recovered by expression of SLAC1 WT, S59A, and S120A, but not by expression of S59A/S120A. Note that SLAC1 WT, S59A, S120A, and S59A/S120A are expressed as C-terminal mVenus fusion proteins under the native 1.63 kbp of the SLAC1 5′ UTR promoter region (see ‘Materials and methods’). The results shown here were recorded from independent Arabidopsis slac1-1 transformation lines that differ from the transformation lines shown in Figure 6G,H. Note that Col0 and slac1-1 measurements are the same control data as those shown in Figure 6G,H as all lines were investigated under the same conditions. Average steady-state current responses ±SEM at −145 mV are plotted in (A). In (B) average stomatal apertures ±SEM. * indicates p < 0.05; t-test. Exact p-values and number of individual experiments for (A and B) can be found in Figure 6—source data 1.DOI:http://dx.doi.org/10.7554/eLife.03599.021

Mentions: (A–C) SLAC1 activation by CPK6 in Xenopus oocytes was abolished when serine 59 is mutated to alanine (S59A) (A and C) (Brandt et al., 2012) but was comparable to wild type SLAC1 activation for the SLAC1 S120A mutated version (B and C). (D–F) OST1 activation of SLAC1 was abolished in the SLAC1 S120A mutant (E and F) (Geiger et al., 2009), while OST1 robustly activated SLAC1 S59A (D and F). (G) In whole-cell patch-clamp experiments, slac1-1 guard cells show impaired ABA-activation of S-type anion currents. Expression of SLAC1 WT, S59A, and S120A in slac1-1 plants restores ABA activation of S-type anion currents in guard cells, but expression of SLAC1 S59A/S120A does not. (H) The ABA-insensitive phenotype of slac1-1 stomata was recovered by expression of SLAC1 WT, S59A, and S120A, but not by expression of S59A/S120A. Note that SLAC1 WT, S59A, S120A, and S59A/S120A are expressed as C-terminal mVenus fusion proteins under native SLAC1 promoter (see ‘Materials and methods’). Representative current traces are depicted in (A, B, D and E) and average current voltage relationships are shown (C and F; ±SEM). Average steady-state current responses ±SEM at −145 mV are plotted in (G) and average stomatal apertures ±SEM in (H). * indicates p < 0.05; unpaired Student's t-test. Exact p-values and number of individual experiments for (G and H) can be found in Figure 6—figure supplement 4. Note that WT (Col0) and slac1-1 control measurements shown in (G and H) are the same control data as those shown in Figure 6—figure supplement 3A,B as all lines were investigated under the same conditions. Several error bars are not visible, as these were smaller than the illustrated symbols.


Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells.

Brandt B, Munemasa S, Wang C, Nguyen D, Yong T, Yang PG, Poretsky E, Belknap TF, Waadt R, Alemán F, Schroeder JI - Elife (2015)

ABA-induced S-type anion currents and stomatal closure responses are impaired when both SLAC1 S59 and S120 are substituted with alanine in independent double amino acid mutant line.(A) In whole-cell patch-clamp experiments, slac1-1 guard cells show impaired ABA-activation of S-type anion currents. Expression of SLAC1 WT, S59A, and S120A in slac1-1 plants restores ABA activation of S-type anion currents, but expression of SLAC1 S59A/S120A does not. (B) ABA-insensitive stomatal closing phenotype of slac1-1 was recovered by expression of SLAC1 WT, S59A, and S120A, but not by expression of S59A/S120A. Note that SLAC1 WT, S59A, S120A, and S59A/S120A are expressed as C-terminal mVenus fusion proteins under the native 1.63 kbp of the SLAC1 5′ UTR promoter region (see ‘Materials and methods’). The results shown here were recorded from independent Arabidopsis slac1-1 transformation lines that differ from the transformation lines shown in Figure 6G,H. Note that Col0 and slac1-1 measurements are the same control data as those shown in Figure 6G,H as all lines were investigated under the same conditions. Average steady-state current responses ±SEM at −145 mV are plotted in (A). In (B) average stomatal apertures ±SEM. * indicates p < 0.05; t-test. Exact p-values and number of individual experiments for (A and B) can be found in Figure 6—source data 1.DOI:http://dx.doi.org/10.7554/eLife.03599.021
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fig6s3: ABA-induced S-type anion currents and stomatal closure responses are impaired when both SLAC1 S59 and S120 are substituted with alanine in independent double amino acid mutant line.(A) In whole-cell patch-clamp experiments, slac1-1 guard cells show impaired ABA-activation of S-type anion currents. Expression of SLAC1 WT, S59A, and S120A in slac1-1 plants restores ABA activation of S-type anion currents, but expression of SLAC1 S59A/S120A does not. (B) ABA-insensitive stomatal closing phenotype of slac1-1 was recovered by expression of SLAC1 WT, S59A, and S120A, but not by expression of S59A/S120A. Note that SLAC1 WT, S59A, S120A, and S59A/S120A are expressed as C-terminal mVenus fusion proteins under the native 1.63 kbp of the SLAC1 5′ UTR promoter region (see ‘Materials and methods’). The results shown here were recorded from independent Arabidopsis slac1-1 transformation lines that differ from the transformation lines shown in Figure 6G,H. Note that Col0 and slac1-1 measurements are the same control data as those shown in Figure 6G,H as all lines were investigated under the same conditions. Average steady-state current responses ±SEM at −145 mV are plotted in (A). In (B) average stomatal apertures ±SEM. * indicates p < 0.05; t-test. Exact p-values and number of individual experiments for (A and B) can be found in Figure 6—source data 1.DOI:http://dx.doi.org/10.7554/eLife.03599.021
Mentions: (A–C) SLAC1 activation by CPK6 in Xenopus oocytes was abolished when serine 59 is mutated to alanine (S59A) (A and C) (Brandt et al., 2012) but was comparable to wild type SLAC1 activation for the SLAC1 S120A mutated version (B and C). (D–F) OST1 activation of SLAC1 was abolished in the SLAC1 S120A mutant (E and F) (Geiger et al., 2009), while OST1 robustly activated SLAC1 S59A (D and F). (G) In whole-cell patch-clamp experiments, slac1-1 guard cells show impaired ABA-activation of S-type anion currents. Expression of SLAC1 WT, S59A, and S120A in slac1-1 plants restores ABA activation of S-type anion currents in guard cells, but expression of SLAC1 S59A/S120A does not. (H) The ABA-insensitive phenotype of slac1-1 stomata was recovered by expression of SLAC1 WT, S59A, and S120A, but not by expression of S59A/S120A. Note that SLAC1 WT, S59A, S120A, and S59A/S120A are expressed as C-terminal mVenus fusion proteins under native SLAC1 promoter (see ‘Materials and methods’). Representative current traces are depicted in (A, B, D and E) and average current voltage relationships are shown (C and F; ±SEM). Average steady-state current responses ±SEM at −145 mV are plotted in (G) and average stomatal apertures ±SEM in (H). * indicates p < 0.05; unpaired Student's t-test. Exact p-values and number of individual experiments for (G and H) can be found in Figure 6—figure supplement 4. Note that WT (Col0) and slac1-1 control measurements shown in (G and H) are the same control data as those shown in Figure 6—figure supplement 3A,B as all lines were investigated under the same conditions. Several error bars are not visible, as these were smaller than the illustrated symbols.

Bottom Line: Interestingly, protein phosphatase 2Cs prevent non-specific Ca(2+)-signaling.Moreover, we demonstrate an unexpected interdependence of the Ca(2+)-dependent and Ca(2+)-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1.We identify novel mechanisms ensuring specificity and robustness within stomatal Ca(2+)-signaling on a cellular, genetic, and biochemical level.

View Article: PubMed Central - PubMed

Affiliation: Division of Biological Sciences, Cell and Developmental Biology Section, University of California, San Diego, San Diego, United States.

ABSTRACT
A central question is how specificity in cellular responses to the eukaryotic second messenger Ca(2+) is achieved. Plant guard cells, that form stomatal pores for gas exchange, provide a powerful system for in depth investigation of Ca(2+)-signaling specificity in plants. In intact guard cells, abscisic acid (ABA) enhances (primes) the Ca(2+)-sensitivity of downstream signaling events that result in activation of S-type anion channels during stomatal closure, providing a specificity mechanism in Ca(2+)-signaling. However, the underlying genetic and biochemical mechanisms remain unknown. Here we show impairment of ABA signal transduction in stomata of calcium-dependent protein kinase quadruple mutant plants. Interestingly, protein phosphatase 2Cs prevent non-specific Ca(2+)-signaling. Moreover, we demonstrate an unexpected interdependence of the Ca(2+)-dependent and Ca(2+)-independent ABA-signaling branches and the in planta requirement of simultaneous phosphorylation at two key phosphorylation sites in SLAC1. We identify novel mechanisms ensuring specificity and robustness within stomatal Ca(2+)-signaling on a cellular, genetic, and biochemical level.

No MeSH data available.


Related in: MedlinePlus