Limits...
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.


When previously phosphorylated by CPK21, the SLAC1-NT is de-phosphorylated by the PP2Cs ABI1 and PP2CA.Recombinant SLAC1-NT phosphorylation by CPK21 (lane 1) is inhibited if the protein phosphatases ABI1 and PP2CA are added before starting the reaction (lanes 2–3). The phosphorylated SLAC1-NT derived signal is rapidly and strongly decreased if the PP2Cs ABI1 and PP2CA (lanes 4–7) are added after the addition of staurosporine.DOI:http://dx.doi.org/10.7554/eLife.03599.014
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4507714&req=5

fig4s1: When previously phosphorylated by CPK21, the SLAC1-NT is de-phosphorylated by the PP2Cs ABI1 and PP2CA.Recombinant SLAC1-NT phosphorylation by CPK21 (lane 1) is inhibited if the protein phosphatases ABI1 and PP2CA are added before starting the reaction (lanes 2–3). The phosphorylated SLAC1-NT derived signal is rapidly and strongly decreased if the PP2Cs ABI1 and PP2CA (lanes 4–7) are added after the addition of staurosporine.DOI:http://dx.doi.org/10.7554/eLife.03599.014

Mentions: Our results suggest that PP2Cs neither down-regulate CPK6 activity directly in vitro (Figure 3C,D and Figure 3—figure supplement 5) nor that CPK activities are strongly ABA-regulated independent of [Ca2+] changes in native plant protein extracts (Figure 3A,B). We next investigated the kinetics and specificity of PP2C down-regulation of SLAC1 activation by CPKs through dephosphorylation of the SLAC1 channel, a mechanism reported for CPK-dependent transcription factor regulation (Lynch et al., 2012) and consistent with previous findings (Brandt et al., 2012). First, we determined whether SLAC1 interacts with the PP2C ABI1 in planta using bimolecular fluorescence complementation (BiFC). We observed clear BiFC signals for full length SLAC1 co-expressed with CPK6 and ABI1 (Figure 4A,B) while signal intensities of SLAC1 co-expressed with a control protein phosphatase 2A catalytic subunit 5 (PP2AC5) were very low (Figure 4B). Protein–protein interaction of SLAC1 with PP2CA in BiFC experiments was reported earlier (Lee et al., 2009). As shown in Figure 4C,D, the ABI1-mediated dephosphorylation of the N-terminus of SLAC1 (SLAC1-NT) previously phosphorylated by CPK6 (Brandt et al., 2012) occurs very rapidly. Already 1 min after the addition of ABI1 a strong decrease of the phosphorylation signal was observed (Figure 4D, lane 4). This de-phosphorylation was also found when the PP2C phosphatase PP2CA was added instead of ABI1 (Figure 4C,E, lane 4). To test whether this is a general phenomenon, we phosphorylated the SLAC1-NT with the SLAC1-activating and -phosphorylating kinases CPK21, CPK23, and OST1 (Geiger et al., 2009, 2010; Lee et al., 2009) and analyzed whether ABI1 and PP2CA are able to remove phospho-groups added by these kinases (Figure 4F–H and Figure 4—figure supplement 1). After inhibiting the kinase with staurosporine, band intensities decreased only after addition of the PP2C protein phosphatases for all combinations, showing that this rapid SLAC1 de-phosphorylation is mediated by PP2Cs (Figure 4F–H and Figure 4—figure supplement 1, lanes 5–6).10.7554/eLife.03599.013Figure 4.PP2Cs interact with and directly and rapidly dephosphorylate the N-terminus of SLAC1 (SLAC1-NT) when previously phosphorylated by several SLAC1-activating CPK and OST1 protein kinases.


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)

When previously phosphorylated by CPK21, the SLAC1-NT is de-phosphorylated by the PP2Cs ABI1 and PP2CA.Recombinant SLAC1-NT phosphorylation by CPK21 (lane 1) is inhibited if the protein phosphatases ABI1 and PP2CA are added before starting the reaction (lanes 2–3). The phosphorylated SLAC1-NT derived signal is rapidly and strongly decreased if the PP2Cs ABI1 and PP2CA (lanes 4–7) are added after the addition of staurosporine.DOI:http://dx.doi.org/10.7554/eLife.03599.014
© Copyright Policy
Related In: Results  -  Collection

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

fig4s1: When previously phosphorylated by CPK21, the SLAC1-NT is de-phosphorylated by the PP2Cs ABI1 and PP2CA.Recombinant SLAC1-NT phosphorylation by CPK21 (lane 1) is inhibited if the protein phosphatases ABI1 and PP2CA are added before starting the reaction (lanes 2–3). The phosphorylated SLAC1-NT derived signal is rapidly and strongly decreased if the PP2Cs ABI1 and PP2CA (lanes 4–7) are added after the addition of staurosporine.DOI:http://dx.doi.org/10.7554/eLife.03599.014
Mentions: Our results suggest that PP2Cs neither down-regulate CPK6 activity directly in vitro (Figure 3C,D and Figure 3—figure supplement 5) nor that CPK activities are strongly ABA-regulated independent of [Ca2+] changes in native plant protein extracts (Figure 3A,B). We next investigated the kinetics and specificity of PP2C down-regulation of SLAC1 activation by CPKs through dephosphorylation of the SLAC1 channel, a mechanism reported for CPK-dependent transcription factor regulation (Lynch et al., 2012) and consistent with previous findings (Brandt et al., 2012). First, we determined whether SLAC1 interacts with the PP2C ABI1 in planta using bimolecular fluorescence complementation (BiFC). We observed clear BiFC signals for full length SLAC1 co-expressed with CPK6 and ABI1 (Figure 4A,B) while signal intensities of SLAC1 co-expressed with a control protein phosphatase 2A catalytic subunit 5 (PP2AC5) were very low (Figure 4B). Protein–protein interaction of SLAC1 with PP2CA in BiFC experiments was reported earlier (Lee et al., 2009). As shown in Figure 4C,D, the ABI1-mediated dephosphorylation of the N-terminus of SLAC1 (SLAC1-NT) previously phosphorylated by CPK6 (Brandt et al., 2012) occurs very rapidly. Already 1 min after the addition of ABI1 a strong decrease of the phosphorylation signal was observed (Figure 4D, lane 4). This de-phosphorylation was also found when the PP2C phosphatase PP2CA was added instead of ABI1 (Figure 4C,E, lane 4). To test whether this is a general phenomenon, we phosphorylated the SLAC1-NT with the SLAC1-activating and -phosphorylating kinases CPK21, CPK23, and OST1 (Geiger et al., 2009, 2010; Lee et al., 2009) and analyzed whether ABI1 and PP2CA are able to remove phospho-groups added by these kinases (Figure 4F–H and Figure 4—figure supplement 1). After inhibiting the kinase with staurosporine, band intensities decreased only after addition of the PP2C protein phosphatases for all combinations, showing that this rapid SLAC1 de-phosphorylation is mediated by PP2Cs (Figure 4F–H and Figure 4—figure supplement 1, lanes 5–6).10.7554/eLife.03599.013Figure 4.PP2Cs interact with and directly and rapidly dephosphorylate the N-terminus of SLAC1 (SLAC1-NT) when previously phosphorylated by several SLAC1-activating CPK and OST1 protein kinases.

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.