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. 2010 Aug;63(3):484-98.
doi: 10.1111/j.1365-313X.2010.04257.x. Epub 2010 May 20.

The Ca(2+) -dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis

Norbert Mehlmer  1 , Bernhard Wurzinger, Simon Stael, Daniela Hofmann-Rodrigues, Edina Csaszar, Barbara Pfister, Roman Bayer, Markus Teige
Affiliations
Free PMC article

The Ca(2+) -dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis

Norbert Mehlmer et al. Plant J. 2010 Aug.
Free PMC article

Abstract

Plants use different signalling pathways to respond to external stimuli. Intracellular signalling via calcium-dependent protein kinases (CDPKs) or mitogen-activated protein kinases (MAPKs) present two major pathways that are widely used to react to a changing environment. Both CDPK and MAPK pathways are known to be involved in the signalling of abiotic and biotic stresses in animal, yeast and plant cells. Here, we show the essential function of the CDPK CPK3 (At4g23650) for salt stress acclimation in Arabidopsis thaliana, and test crosstalk between CPK3 and the major salt-stress activated MAPKs MPK4 and MPK6 in the salt stress response. CPK3 kinase activity was induced by salt and other stresses after transient overexpression in Arabidopsis protoplasts, but endogenous CPK3 appeared to be constitutively active in roots and leaves in a strictly Ca(2+) -dependent manner. cpk3 mutants show a salt-sensitive phenotype comparable with mutants in MAPK pathways. In contrast to animal cells, where crosstalk between Ca(2+) and MAPK signalling is well established, CPK3 seems to act independently of those pathways. Salt-induced transcriptional induction of known salt stress-regulated and MAPK-dependent marker genes was not altered, whereas post-translational protein phosphorylation patterns from roots of wild type and cpk3 plants revealed clear differences. A significant portion of CPK3 was found to be associated with the plasma membrane and the vacuole, both depending on its N-terminal myristoylation. An initial proteomic study led to the identification of 28 potential CPK3 targets, predominantly membrane-associated proteins.

Keywords: Ca2+-dependent protein kinase; MAP kinase; N-myristoylation; crosstalk; protein phosphorylation; salt stress adaptation.

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Figures

Figure 1
Figure 1
CPK3 kinase activities and salt-sensitive phenotype of cpk3 mutants.(a) Activation of transiently expressed HA-epitope tagged CPK3 in response to different stresses in Arabidopsis protoplasts. Immunocomplex kinase assays were performed as described in Experimental procedures. The top panel shows the incorporation of 32P into the generic substrate Histone IIIS, and the bottom panel displays CPK3 protein levels: 0, mock treatment; cold, 4°C; NaCl, 150 mm; heat, 37°C; H2O2, 1 mm; flagellin, 15 nm; laminarin, 1 mm.(b) Endogenous CPK3 kinase activity in response to salt stress in roots of cpk3-2 knock-out and Col-0 plants measured in immunocomplex kinase assays. Plants were treated with 150 mm NaCl for 0, 10 and 30 min. Immunocomplex kinase assays were performed in the absence or presence 200 μm EGTA.(c) CPK3 transcript levels in response to salt stress.(d) CPK3 protein levels in Col-0 and three independent T-DNA insertion lines.(e) Germination rates of Col-0 and the T-DNA insertion lines on quarter-strength Hoagland + 150 mm NaCl. Error bars indicate SEM (n = 10). Statistically significant differences from Col-0 calculated by a two-tailed Student’s t-tests: *P ≤ 0.05; **P ≤ 0.001.
Figure 2
Figure 2
Localization and N-myristoylation of CPK3.(a) Tissue-specific expression of CPK3 in plants: root, stem, flower, young (20 days post germination) and old (40 days post germination) leaves.(b) Endogenous CPK3 in subcellular fractionation from wild-type plants. Lanes from left to right: total cell extract, protein from the 10 000 g (10 k) supernatant (S) and pellet (P), as well as the 100 000 g (100 k) supernatant (S) and pellet (P).(c) In vitro myristoylation of CPK3 and CPK2 as a positive control. Wild-type (WT) and non-myristoylable G2A mutants of CPK2 and CPK3 were in vitro translated in the presence of either 3H-labelled myristic acid or 35S-labelled methionine, and incorporation of the label was scored by autoradiography.(d) Fractionation of YFP-tagged CPK3 (WT) and G2A mutants (G2A) from infiltrated tobacco leaves using a GFP antibody. S, supernatant; P, pellet; 10 k, centrifugation for 10 min at 10 000 g; 100 k, centrifugation for 1 h at 100 000 g.
Figure 3
Figure 3
Co-localization of wild-type and non-myristoylated CPK3 with the vacuolar and the plasma membrane in tobacco leaf epidermal cells. The green channel shows the CPK3-YFP signal, the red channel displays chlorophyll autofluorescence and the magenta channel displays the mCherry fusion proteins. Co-localization can be deduced from a white signal in the merged images.(a–c) Co-localization of CPK3WT-YFP and TPK1-mCherry at vacuolar membranes. The nucleus (N) is marked by an arrow.(d–f) Co-expression of CPK3G2A-YFP and TPK1-mCherry.(g–i) Co-localization of CPK3WT-YFP and CPK9-mCherry at the plasma membrane.(j–l) Co-expression of CPK3G2A-YFP and CPK9-mCherry. Scale bars: 10 μm.(m) Co-partitioning of endogenous CPK3 with different membranes from Arabidopsis leaves in phase-partitioning experiments. The upper phase (U) contains purified plasma membrane; the lower phase (L) contains plasma, mitochondrial, vacuolar and endoplasmic reticulum membranes. Different membranes were detected via western blotting using the indicated markers.
Figure 4
Figure 4
Crosstalk between MAPK and CPK3 kinase activities, and induction of marker genes, upon salt stress.(a) Salt-triggered induction of known salt stress-responsive marker genes was compared among the wild type (Col-0), cpk3-2, and the two CPK3 overexpressor lines by RT-PCR, and compared with Actin (ACT3) as an internal control. Gene identifiers and sequences of the used primers are listed in Table S1. Fourteen-day-old seedlings were treated with 150 mm NaCl for the indicated time periods, as described in the Experimental procedures.(b) Salt-triggered activation of MPK6 in wild type (Col-0), cpk3-2 knock-out, and two independent CPK3 overexpressor lines towards myeline basic protein (MBP) as generic substrate. Kinase activities were measured in immunocomplex kinase assays upon salt treatment of 14-day-old seedlings for 15 min, as described in the Experimental procedures.
Figure 5
Figure 5
Comparison of threonine phosphorylation patterns in the roots of 6-week-old Col-0 and cpk3-2, hydroponically grown in half-strength Hoagland. (a) or half-strength Hoagland supplemented with 150 mm NaCl for 30 min before protein isolation (b). Spot patterns were obtained by western blot analysis with an anti p-Thr antibody. Numbers in the figures indicate spots showing different intensities between Col-0 and cpk3-2 root material throughout three independent replications of the experiment.
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