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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: 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.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.

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

Close up view of Ca2+-activated kinase activities.The region of predicted molecular weights of CPKs (CPK1, CPK2, CPK5, CPK6, CPK11, and CPK23 are 68.3 kDa, 72.3 kDa, 62.1 kDa, 61.1 kDa, 55.9 kDa, 58.7 kDa, respectively) of the same autoradiograph which is shown in Figure 3B is magnified to increase the visibility of the individual bands. The apparent loss of the prominent bands with high molecular weight in cpk1/2/5/6 plants could correspond to CPK1 and CPK2 protein isoforms which possess the largest predicted molecular weights of all CPKs. Note that bands that run at a higher molecular weight than the predicted mass could be due to post-translational modifications. The faint band indicated with red asterisks might correspond to the closely related CPK5 and CPK6 as it is not clearly resolved in both mutants and also runs at the expected molecular weight.DOI:http://dx.doi.org/10.7554/eLife.03599.008
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fig3s1: Close up view of Ca2+-activated kinase activities.The region of predicted molecular weights of CPKs (CPK1, CPK2, CPK5, CPK6, CPK11, and CPK23 are 68.3 kDa, 72.3 kDa, 62.1 kDa, 61.1 kDa, 55.9 kDa, 58.7 kDa, respectively) of the same autoradiograph which is shown in Figure 3B is magnified to increase the visibility of the individual bands. The apparent loss of the prominent bands with high molecular weight in cpk1/2/5/6 plants could correspond to CPK1 and CPK2 protein isoforms which possess the largest predicted molecular weights of all CPKs. Note that bands that run at a higher molecular weight than the predicted mass could be due to post-translational modifications. The faint band indicated with red asterisks might correspond to the closely related CPK5 and CPK6 as it is not clearly resolved in both mutants and also runs at the expected molecular weight.DOI:http://dx.doi.org/10.7554/eLife.03599.008

Mentions: Based on the above results we sought to determine the biochemical mechanisms by which PP2Cs down-regulate Ca2+ sensitivity in the absence of ABA. The main SLAC1-activating protein kinase in the Ca2+-independent branch, OST1 (Mustilli et al., 2002; Yoshida et al., 2002), is directly inactivated by PP2Cs through de-phosphorylation of the activation loop (Umezawa et al., 2009; Vlad et al., 2009). We tested whether CPKs might be down-regulated by PP2Cs in a similar manner and whether pp2c quadruple mutant plants may also exhibit a constitutive OST1 activity. Our first approach to test whether CPK activity is regulated by ABA through PP2Cs was an in-gel protein kinase assay using protein extracts of Arabidopsis seedlings, which is routinely used to test OST1 activation by ABA (Mustilli et al., 2002) and also CPK activation by flg22 (Boudsocq et al., 2010). Guard cell [Ca2+]cyt ranges from resting levels of ≈0.15 μM to stimulus induced elevated levels of above 1 μM (McAinsh et al., 1990). Similar to studies reporting the ABA-activation of SnRK2.2, SnRK2.3, and SnRK2.6/OST1 (Mustilli et al., 2002; Yoshida et al., 2002; Fujii et al., 2007), we compared the phosphorylation pattern of a reaction carried out at 0.15 μM free Ca2+ with the phosphorylation pattern at 3 μM free Ca2+ (Figure 3A,B; for intermediate free Ca2+ concentration of 0.4 μM Ca2+ see Figure 3—figure supplement 2). Incubating the gels in a reaction buffer with 3 μM free Ca2+ led to strong Ca2+-activated phosphorylation signals compared to resting Ca2+ at 0.15 μM (Figure 3A,B). To determine whether these Ca2+-activated signals are CPK-derived we included two distinct quadruple mutants, cpk5/6/11/23 and cpk1/2/5/6, in the in-gel kinase assays. Several Ca2+-activated bands disappeared or became notably weaker when extracts were tested from cpk5/6/11/23 and cpk1/2/5/6 (Boudsocq et al., 2010) plants (Figure 3B and for improved visibility Figure 3—figure supplement 1).10.7554/eLife.03599.007Figure 3.CPK activity is not changed by ABA or hyper-activated in pp2c quadruple mutants at defined Ca2+ concentrations.


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)

Close up view of Ca2+-activated kinase activities.The region of predicted molecular weights of CPKs (CPK1, CPK2, CPK5, CPK6, CPK11, and CPK23 are 68.3 kDa, 72.3 kDa, 62.1 kDa, 61.1 kDa, 55.9 kDa, 58.7 kDa, respectively) of the same autoradiograph which is shown in Figure 3B is magnified to increase the visibility of the individual bands. The apparent loss of the prominent bands with high molecular weight in cpk1/2/5/6 plants could correspond to CPK1 and CPK2 protein isoforms which possess the largest predicted molecular weights of all CPKs. Note that bands that run at a higher molecular weight than the predicted mass could be due to post-translational modifications. The faint band indicated with red asterisks might correspond to the closely related CPK5 and CPK6 as it is not clearly resolved in both mutants and also runs at the expected molecular weight.DOI:http://dx.doi.org/10.7554/eLife.03599.008
© Copyright Policy
Related In: Results  -  Collection

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

fig3s1: Close up view of Ca2+-activated kinase activities.The region of predicted molecular weights of CPKs (CPK1, CPK2, CPK5, CPK6, CPK11, and CPK23 are 68.3 kDa, 72.3 kDa, 62.1 kDa, 61.1 kDa, 55.9 kDa, 58.7 kDa, respectively) of the same autoradiograph which is shown in Figure 3B is magnified to increase the visibility of the individual bands. The apparent loss of the prominent bands with high molecular weight in cpk1/2/5/6 plants could correspond to CPK1 and CPK2 protein isoforms which possess the largest predicted molecular weights of all CPKs. Note that bands that run at a higher molecular weight than the predicted mass could be due to post-translational modifications. The faint band indicated with red asterisks might correspond to the closely related CPK5 and CPK6 as it is not clearly resolved in both mutants and also runs at the expected molecular weight.DOI:http://dx.doi.org/10.7554/eLife.03599.008
Mentions: Based on the above results we sought to determine the biochemical mechanisms by which PP2Cs down-regulate Ca2+ sensitivity in the absence of ABA. The main SLAC1-activating protein kinase in the Ca2+-independent branch, OST1 (Mustilli et al., 2002; Yoshida et al., 2002), is directly inactivated by PP2Cs through de-phosphorylation of the activation loop (Umezawa et al., 2009; Vlad et al., 2009). We tested whether CPKs might be down-regulated by PP2Cs in a similar manner and whether pp2c quadruple mutant plants may also exhibit a constitutive OST1 activity. Our first approach to test whether CPK activity is regulated by ABA through PP2Cs was an in-gel protein kinase assay using protein extracts of Arabidopsis seedlings, which is routinely used to test OST1 activation by ABA (Mustilli et al., 2002) and also CPK activation by flg22 (Boudsocq et al., 2010). Guard cell [Ca2+]cyt ranges from resting levels of ≈0.15 μM to stimulus induced elevated levels of above 1 μM (McAinsh et al., 1990). Similar to studies reporting the ABA-activation of SnRK2.2, SnRK2.3, and SnRK2.6/OST1 (Mustilli et al., 2002; Yoshida et al., 2002; Fujii et al., 2007), we compared the phosphorylation pattern of a reaction carried out at 0.15 μM free Ca2+ with the phosphorylation pattern at 3 μM free Ca2+ (Figure 3A,B; for intermediate free Ca2+ concentration of 0.4 μM Ca2+ see Figure 3—figure supplement 2). Incubating the gels in a reaction buffer with 3 μM free Ca2+ led to strong Ca2+-activated phosphorylation signals compared to resting Ca2+ at 0.15 μM (Figure 3A,B). To determine whether these Ca2+-activated signals are CPK-derived we included two distinct quadruple mutants, cpk5/6/11/23 and cpk1/2/5/6, in the in-gel kinase assays. Several Ca2+-activated bands disappeared or became notably weaker when extracts were tested from cpk5/6/11/23 and cpk1/2/5/6 (Boudsocq et al., 2010) plants (Figure 3B and for improved visibility Figure 3—figure supplement 1).10.7554/eLife.03599.007Figure 3.CPK activity is not changed by ABA or hyper-activated in pp2c quadruple mutants at defined Ca2+ concentrations.

Bottom Line: 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.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.

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