Calcium specificity signaling mechanisms in abscisic acid signal transduction in Arabidopsis guard cells
- University of California, San Diego, United States
Figures
Figure 1 with 1 supplement
Calcium-dependent protein kinase (CPK) quadruple loss of function mutants show abscisic acid (ABA) and Ca2+ insensitive S-type anion current activation and are impaired in stomatal closing.
(A–D) Intracellular Ca2+-activation of S-type anion channels enabled by pre-exposure to ABA (A and C) or high external Ca2+ pre-shock (Allen et al., 2002) (B and D) is strongly impaired in cpk5/6/11/…
Figure 1—figure supplement 1
CPK5 activates SLAC1 in Xenopus oocytes and ABA-activation of S-type anion currents in cpk5 single mutant is not impaired.
(A and B) Whole cell currents were measured in Xenopus oocytes expressing SLAC1 together with CPK5 and, as a control, CPK6. Large Cl− currents show that CPK5 is capable of activating SLAC1. (C and D)…
Figure 2 with 1 supplement
In protein phosphatase 2C (PP2C) quadruple mutant plants, Ca2+ activation of S-type anion currents is constitutively primed.
(A and C) 2 μM [Ca2+]cyt activates S-type anion currents in WT if the guard cells were pre-exposed to ABA. (B and D) In PP2C quadruple mutant guard cells ABA pre-exposure is not required for 2 μM …
Figure 2—figure supplement 1
Analysis of ABA activation of S-type anion currents in PP2C quadruple mutant guard cells at low [Ca2+]cyt.
(A and B) ABA application in WT and abi1-2/abi2-2/hab1-1/pp2ca-1 guard cells with [Ca2+]cyt buffered to a resting level of 0.1 μM does not result in large S-type anion current activation. Typical …
Figure 3 with 5 supplements
CPK activity is not changed by ABA or hyper-activated in pp2c quadruple mutants at defined Ca2+ concentrations.
(A and B) In-gel kinase assays with Histone-III as substrate for whole plant protein extracts show (B) 3 μM Ca2+-activated trans-phosphorylation kinase activities independent of application of 50 μM …
Figure 3—figure supplement 1
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 …
Figure 3—figure supplement 2
Protein kinase activities are not altered by ABA-application at 150 nM and 400 nM free Ca2+.
(A and B) Whole plant protein extracts were analyzed in in-gel protein kinase assays with the free Ca2+ concentration buffered to either 150 nM or 400 nM. No differences in the band pattern could be …
Figure 3—figure supplement 3
Signals in in-gel kinase assays are largely derived from kinase trans-phosphorylation activities.
(A and B) In-gel kinase assays with recombinant (A) CPK6 and (B) OST1 protein kinases in gels with (left lanes in A and B) and without (right lanes in A and B) the kinase substrate Histone-III were …
Figure 3—figure supplement 4
CPK6 is de-phosphorylated by the PP2Cs ABI1, ABI2, and PP2CA.
In in vitro protein kinase assays, recombinant CPK6 was incubated in the presence of 5 μM free Ca2+ which results in auto-phosphorylation signals (lanes 1 and 5). After the initial …
Figure 3—figure supplement 5
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 …
Figure 4 with 1 supplement
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.
(A) Bimolecular fluorescence complementation (BiFC) experiments in Nicotiana benthamiana leaves show YFP-derived fluorescence signals of YC-SLAC1 co-expressed with CPK6-YN and YN-ABI1. (B) …
Figure 4—figure supplement 1
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 …
Figure 5 with 1 supplement
Both, ABA- and high external Ca2+-activation of S-type anion currents at elevated [Ca2+]cyt and imposed Ca2+-oscillation-triggered stomatal closure are impaired in snrk2.2/2.3/ost1 triple mutant guard cells while the ABA-activation of ICa currents is intact.
(A–D) Whole-cell patch-clamp experiments reveal that [Ca2+]cyt-activation of S-type anion currents is disrupted in snrk2.2/2.3/ost1 triple mutant guard cells even if pre-incubated with high external …
Figure 5—figure supplement 1
snrk2.2/2.3/ost1 triple mutant guard cells show intact ABA activation of Ca2+-permeable ICa currents.
(A and B) Whole-cell patch-clamp experiments of WT (A) and snrk2.2/2.3/ost1 triple mutant (B) guard cells in the absence of ABA (top traces). ABA activation of Ca2+-permeable ICa currents was …
Figure 6 with 5 supplements
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.
(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 …
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Figure 6—source data 1
Statistical data and number of repeats (n) for the (Table 1) patch clamp measurements shown in Figure 6G and Figure 6—figure supplement 3A and (Table 2) for measurements of stomatal apertures presented in Figure 6H and Figure 6—figure supplement 3B (n = 3 experiments and >45 total stomata per group).
The Student's t-test was used to calculate all p-values.
- https://doi.org/10.7554/eLife.03599.018
Figure 6—figure supplement 1
SLAC1 serine 59 but not serine 120 is required for CPK5 or CPK23 activation in Xenopus oocytes.
(A and B) SLAC1 activation by CPK5 (A) and CPK23 (B) is comparable to WT when serine 120 is substituted by alanine (S120A) while the CPK5 and CPK23 activation of SLAC1 S59A was strongly impaired. …
Figure 6—figure supplement 2
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,…
Figure 6—figure supplement 3
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 …
Figure 6—figure supplement 4
Analysis of expression and subcellular localization of SLAC1-WT, SLAC1S59A, S120A, and S59A/S120A in slac1-1 complementation lines.
Confocal laser microscopy of guard cells of slac1-1 mutant lines expressing SLAC1-WT-mVenus, SLAC1S59A-mVenus, SLAC1-S120A-mVenus or SLAC1-S59A/S120A-mVenus shows membrane-localized expression of …
Figure 6—figure supplement 5
BiFC fluorescence intensities are altered for CPK6 and ABI1 co-expression with SLAC1-WT, SLAC1S59A, S120A, and S59A/S120A.
Quantitative BiFC experiments showed that CPK6-YN + YC-SLAC1-WT derived fluorescence signals are significantly decreased when CPK6-YN was expressed with YC-SLAC1-S59A, YC-SLAC1-S120A, or …
Figure 7
Simplified schematic model for Ca2+-specificity mechanism within ABA-dependent SLAC1 activation in guard cells.
(A) Without ABA, Ca2+ elevations that can also function in stomatal opening responses (Irving et al., 1992; Shimazaki et al., 1992; Curvetto et al., 1994; Shimazaki et al., 1997; Cousson and …
