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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.


CPK6 kinase activity is not inhibited in the presence of ABI1 or PP2CA.In vitro protein kinase assays measuring the kinase activity via ATP consumption show that staurosporine (Stau.) but not ABI1 or PP2CA inhibited CPK6 kinase activity. The increased ATP-consumption signal in the presence of ABI1 and PP2CA can be explained by higher ATP consumption triggered by kinase auto-phosphorylation of residues removed by the PP2C protein phosphatases. Data shown represent the mean of three experiments ± SD.DOI:http://dx.doi.org/10.7554/eLife.03599.012
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fig3s5: CPK6 kinase activity is not inhibited in the presence of ABI1 or PP2CA.In vitro protein kinase assays measuring the kinase activity via ATP consumption show that staurosporine (Stau.) but not ABI1 or PP2CA inhibited CPK6 kinase activity. The increased ATP-consumption signal in the presence of ABI1 and PP2CA can be explained by higher ATP consumption triggered by kinase auto-phosphorylation of residues removed by the PP2C protein phosphatases. Data shown represent the mean of three experiments ± SD.DOI:http://dx.doi.org/10.7554/eLife.03599.012

Mentions: To determine whether PP2Cs can directly down-regulate CPKs we next investigated whether the SLAC1-activating CPK6 (Mori et al., 2006; Brandt et al., 2012), is negatively regulated by the PP2Cs ABI1 and PP2CA. In-gel protein kinase assays using recombinant proteins were pursued in which kinases and phosphatases are separated by size prior to substrate phosphorylation. CPK6, and as positive control OST1, were pre-incubated either alone or with ABI1 or PP2CA with and without ATP before being subjected to in-gel protein kinase assays. Pre-incubation with either ABI1 or PP2CA did not inhibit CPK6 trans-phosphorylation activity (Figure 3C, lanes 2–3 and 5–6). In contrast, control OST1-derived substrate phosphorylation band intensities strongly decreased when ABI1 or PP2CA proteins were present during the pre-incubation period (Figure 3D, lanes 2–3 and 5–6). These results indicate that OST1, but not CPK6 activity, is directly down-regulated by ABI1 and PP2CA. CPKs have been previously reported to interact with ABI1 (Geiger et al., 2010). An electro-mobility shift can be observed for OST1 as well as for CPK6 (Figure 3C,D). These shifts could be due to dephosphorylation of CPK6 (Figure 3—figure supplement 4) and OST1 (Umezawa et al., 2009; Vlad et al., 2009) by PP2Cs. However, dephosphorylation by PP2Cs did not inhibit CPK6 activity (Figure 3C). An additional independent biochemical assay measuring ATP consumption also did not show down-regulation of CPK6 activity in the presence of ABI1 and PP2CA (Figure 3—figure supplement 5), further underlining no direct down-regulation of CPK6 activity by these three PP2Cs, in contrast to OST1 controls.


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)

CPK6 kinase activity is not inhibited in the presence of ABI1 or PP2CA.In vitro protein kinase assays measuring the kinase activity via ATP consumption show that staurosporine (Stau.) but not ABI1 or PP2CA inhibited CPK6 kinase activity. The increased ATP-consumption signal in the presence of ABI1 and PP2CA can be explained by higher ATP consumption triggered by kinase auto-phosphorylation of residues removed by the PP2C protein phosphatases. Data shown represent the mean of three experiments ± SD.DOI:http://dx.doi.org/10.7554/eLife.03599.012
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4507714&req=5

fig3s5: CPK6 kinase activity is not inhibited in the presence of ABI1 or PP2CA.In vitro protein kinase assays measuring the kinase activity via ATP consumption show that staurosporine (Stau.) but not ABI1 or PP2CA inhibited CPK6 kinase activity. The increased ATP-consumption signal in the presence of ABI1 and PP2CA can be explained by higher ATP consumption triggered by kinase auto-phosphorylation of residues removed by the PP2C protein phosphatases. Data shown represent the mean of three experiments ± SD.DOI:http://dx.doi.org/10.7554/eLife.03599.012
Mentions: To determine whether PP2Cs can directly down-regulate CPKs we next investigated whether the SLAC1-activating CPK6 (Mori et al., 2006; Brandt et al., 2012), is negatively regulated by the PP2Cs ABI1 and PP2CA. In-gel protein kinase assays using recombinant proteins were pursued in which kinases and phosphatases are separated by size prior to substrate phosphorylation. CPK6, and as positive control OST1, were pre-incubated either alone or with ABI1 or PP2CA with and without ATP before being subjected to in-gel protein kinase assays. Pre-incubation with either ABI1 or PP2CA did not inhibit CPK6 trans-phosphorylation activity (Figure 3C, lanes 2–3 and 5–6). In contrast, control OST1-derived substrate phosphorylation band intensities strongly decreased when ABI1 or PP2CA proteins were present during the pre-incubation period (Figure 3D, lanes 2–3 and 5–6). These results indicate that OST1, but not CPK6 activity, is directly down-regulated by ABI1 and PP2CA. CPKs have been previously reported to interact with ABI1 (Geiger et al., 2010). An electro-mobility shift can be observed for OST1 as well as for CPK6 (Figure 3C,D). These shifts could be due to dephosphorylation of CPK6 (Figure 3—figure supplement 4) and OST1 (Umezawa et al., 2009; Vlad et al., 2009) by PP2Cs. However, dephosphorylation by PP2Cs did not inhibit CPK6 activity (Figure 3C). An additional independent biochemical assay measuring ATP consumption also did not show down-regulation of CPK6 activity in the presence of ABI1 and PP2CA (Figure 3—figure supplement 5), further underlining no direct down-regulation of CPK6 activity by these three PP2Cs, in contrast to OST1 controls.

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.