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.


Related in: MedlinePlus

SLAC1 exhibits enhanced activity by co-expression of CPK6 and OST1 in Xenopus oocytes.(A–D) If SLAC1 (5 ng cRNA) is expressed alone or with non-BIFC OST1 (7.5 ng), no anion currents can be detected (C and D). If CPK6 (0.5 ng) is co-expressed, SLAC1-mediated currents can be seen (A, C, and D) which are enhanced when OST1 (7.5 ng) is added (B–D). Due to overlapping data of ‘SLAC1’ and ‘SLAC1 + OST1’ alternating data points are shown in (C). (E) This enhancement is almost completely impaired when the kinase inactive mutant OST1 D140A is co-injected with SLAC1 and CPK6. In (A and B) typical current responses are shown while in (C and E) average current–voltage relationships ±SEM and the number of the measured cells are presented. (D) Shows average currents at −140 mV ±SEM (*** indicates p = 0.005). Several error bars are not visible, as these were smaller than the illustrated symbols.DOI:http://dx.doi.org/10.7554/eLife.03599.020
© Copyright Policy
Related In: Results  -  Collection

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

fig6s2: SLAC1 exhibits enhanced activity by co-expression of CPK6 and OST1 in Xenopus oocytes.(A–D) If SLAC1 (5 ng cRNA) is expressed alone or with non-BIFC OST1 (7.5 ng), no anion currents can be detected (C and D). If CPK6 (0.5 ng) is co-expressed, SLAC1-mediated currents can be seen (A, C, and D) which are enhanced when OST1 (7.5 ng) is added (B–D). Due to overlapping data of ‘SLAC1’ and ‘SLAC1 + OST1’ alternating data points are shown in (C). (E) This enhancement is almost completely impaired when the kinase inactive mutant OST1 D140A is co-injected with SLAC1 and CPK6. In (A and B) typical current responses are shown while in (C and E) average current–voltage relationships ±SEM and the number of the measured cells are presented. (D) Shows average currents at −140 mV ±SEM (*** indicates p = 0.005). Several error bars are not visible, as these were smaller than the illustrated symbols.DOI:http://dx.doi.org/10.7554/eLife.03599.020

Mentions: In addition to possible direct cross-regulation of CPKs and SnRK2s, another non-mutually exclusive potential mechanism for the requirement of both SnRK and CPK kinases for ABA activation of S-type anion channels could be that SLAC1 serves as coincidence detector through differential phosphorylation by protein kinases of the Ca2+-dependent and -independent branches. The amino acid residue serine 120 of SLAC1 has been shown to be required for OST1, but not for CPK23 activation of SLAC1 in Xenopus oocytes (Geiger et al., 2009, 2010). A different site, serine 59, has been shown to be required for SLAC1 activation by CPK6 (Brandt et al., 2012). Thus we investigated whether several CPKs can activate the SLAC1 S120A mutant in oocytes and whether the SLAC1 S59A mutant is activated by OST1 and other CPKs in oocytes. CPK5, CPK6, and CPK23 activation of SLAC1 S120A was similar to WT SLAC1 activation (Figure 6A–C and Figure 6—figure supplement 1A,B). In contrast, SLAC1 S59A activation by these CPKs was strongly impaired (Figure 6A–C and Figure 6—figure supplement 1A,B). Interestingly however, OST1 was able to activate SLAC1 S59A (Figure 6D–F), which was confirmed in multiple independent experimental sets under the imposed conditions. These results suggest that S59 is required for strong activation by protein kinases of the Ca2+-dependent CPK branch, while S120 represents a crucial amino acid for strong activation by the Ca2+-independent branch of the ABA signaling core. To avoid spurious phosphorylation by high protein kinase concentrations in oocytes, effects of co-expression of CPK6 and OST1 at low levels that do not fully activate SLAC1 were investigated. These experiments show a clear enhanced SLAC1 activation in oocytes when both kinases are co-expressed (Figure 6—figure supplement 2A–D). This enhancement of SLAC1 activation by OST1 became less clear when an inactive OST1 protein kinase (OST1 D140A) was analyzed (Figure 6—figure supplement 2E).10.7554/eLife.03599.017Figure 6.Ca2+-dependent protein kinase and OST1 protein kinase activation of SLAC1 in oocytes requires serine 59 or serine 120, respectively while in planta ABA-dependent S-type anion current activation and stomatal closing are only impaired in SLAC1 S59A/S120A double amino acid mutants.


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)

SLAC1 exhibits enhanced activity by co-expression of CPK6 and OST1 in Xenopus oocytes.(A–D) If SLAC1 (5 ng cRNA) is expressed alone or with non-BIFC OST1 (7.5 ng), no anion currents can be detected (C and D). If CPK6 (0.5 ng) is co-expressed, SLAC1-mediated currents can be seen (A, C, and D) which are enhanced when OST1 (7.5 ng) is added (B–D). Due to overlapping data of ‘SLAC1’ and ‘SLAC1 + OST1’ alternating data points are shown in (C). (E) This enhancement is almost completely impaired when the kinase inactive mutant OST1 D140A is co-injected with SLAC1 and CPK6. In (A and B) typical current responses are shown while in (C and E) average current–voltage relationships ±SEM and the number of the measured cells are presented. (D) Shows average currents at −140 mV ±SEM (*** indicates p = 0.005). Several error bars are not visible, as these were smaller than the illustrated symbols.DOI:http://dx.doi.org/10.7554/eLife.03599.020
© Copyright Policy
Related In: Results  -  Collection

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

fig6s2: SLAC1 exhibits enhanced activity by co-expression of CPK6 and OST1 in Xenopus oocytes.(A–D) If SLAC1 (5 ng cRNA) is expressed alone or with non-BIFC OST1 (7.5 ng), no anion currents can be detected (C and D). If CPK6 (0.5 ng) is co-expressed, SLAC1-mediated currents can be seen (A, C, and D) which are enhanced when OST1 (7.5 ng) is added (B–D). Due to overlapping data of ‘SLAC1’ and ‘SLAC1 + OST1’ alternating data points are shown in (C). (E) This enhancement is almost completely impaired when the kinase inactive mutant OST1 D140A is co-injected with SLAC1 and CPK6. In (A and B) typical current responses are shown while in (C and E) average current–voltage relationships ±SEM and the number of the measured cells are presented. (D) Shows average currents at −140 mV ±SEM (*** indicates p = 0.005). Several error bars are not visible, as these were smaller than the illustrated symbols.DOI:http://dx.doi.org/10.7554/eLife.03599.020
Mentions: In addition to possible direct cross-regulation of CPKs and SnRK2s, another non-mutually exclusive potential mechanism for the requirement of both SnRK and CPK kinases for ABA activation of S-type anion channels could be that SLAC1 serves as coincidence detector through differential phosphorylation by protein kinases of the Ca2+-dependent and -independent branches. The amino acid residue serine 120 of SLAC1 has been shown to be required for OST1, but not for CPK23 activation of SLAC1 in Xenopus oocytes (Geiger et al., 2009, 2010). A different site, serine 59, has been shown to be required for SLAC1 activation by CPK6 (Brandt et al., 2012). Thus we investigated whether several CPKs can activate the SLAC1 S120A mutant in oocytes and whether the SLAC1 S59A mutant is activated by OST1 and other CPKs in oocytes. CPK5, CPK6, and CPK23 activation of SLAC1 S120A was similar to WT SLAC1 activation (Figure 6A–C and Figure 6—figure supplement 1A,B). In contrast, SLAC1 S59A activation by these CPKs was strongly impaired (Figure 6A–C and Figure 6—figure supplement 1A,B). Interestingly however, OST1 was able to activate SLAC1 S59A (Figure 6D–F), which was confirmed in multiple independent experimental sets under the imposed conditions. These results suggest that S59 is required for strong activation by protein kinases of the Ca2+-dependent CPK branch, while S120 represents a crucial amino acid for strong activation by the Ca2+-independent branch of the ABA signaling core. To avoid spurious phosphorylation by high protein kinase concentrations in oocytes, effects of co-expression of CPK6 and OST1 at low levels that do not fully activate SLAC1 were investigated. These experiments show a clear enhanced SLAC1 activation in oocytes when both kinases are co-expressed (Figure 6—figure supplement 2A–D). This enhancement of SLAC1 activation by OST1 became less clear when an inactive OST1 protein kinase (OST1 D140A) was analyzed (Figure 6—figure supplement 2E).10.7554/eLife.03599.017Figure 6.Ca2+-dependent protein kinase and OST1 protein kinase activation of SLAC1 in oocytes requires serine 59 or serine 120, respectively while in planta ABA-dependent S-type anion current activation and stomatal closing are only impaired in SLAC1 S59A/S120A double amino acid mutants.

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