Volume 4, Issue 1, Pages 83-96 (January 2011) Calcium-Dependent Protein Kinase CPK21 Functions in Abiotic Stress Response in Arabidopsis thaliana  Franz Sandra , Ehlert Britta , Liese Anja , Kurth Joachim , Cazalé Anne-Claire , Romeis Tina   Molecular Plant  Volume 4, Issue 1, Pages 83-96 (January 2011) DOI: 10.1093/mp/ssq064 Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 1 Calcium-Dependent Electrophoretic Mobility Shift of CPK21 Wild-Type and EF-Hand Mutant Enzymes. (A) Scheme of CPK21 protein structure and generated EF-hand mutant variants. Conserved CDPK protein domains are indicated: variable domain (white box), kinase domain (dashed box), autoinhibitory junction domain (black box), and the calmodulin-like domain with four serial Ca2+-binding EF-hand motifs (gray boxes). Enzyme variants carrying point mutations in either EF-hand motifs (ΔEF) or in the active centre of the kinase (D204A) were generated by site-directed mutagenesis (black cross). (B) Western blot analyses of in N. benthamiana expressed and affinity-purified Strep-tagged CPK21 enzyme variants. Isolated protein was incubated in the presence of 1 mM Ca2+ or 5 mM EGTA in the loading buffer, separated by SDS gel electrophoresis and visualized by immunodetection using anti-StrepHRP conjugate. A Ca2+-dependent electrophoretic mobility shift typical for Ca2+-binding enzymes can be seen for CPK21 wild-type form and still for enzyme variants lacking either EF1 or EF2. Molecular Plant 2011 4, 83-96DOI: (10.1093/mp/ssq064) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 2 Calcium-Dependent Protein Kinase Activity of Purified CPK21 Wild-Type and EF-Hand Mutant Enzymes. Strep-tagged CPK21 and its enzyme variants were transiently expressed in N. benthamiana leaves, proteins were affinity-purified and immobilized to Strep-tactin beads, and aliquots were subjected to protein kinase assays. (A) In vitro kinase assay CPK21 wild-type and EF-hand mutant enzymes with syntide2 as substrate in the absence or presence of increasing Ca2+ concentrations (0.05, 0.5, and 5 μM CaCl2; indicated by the triangle) in the kinase buffer. Double estimates of phosphorylated peptide substrate evaluated by scintillation counting are shown. Kinase activity of CPK21 variants carrying mutations at their N-terminal EF-hand motifs EF1 and EF2 are more severely affected than in the C-terminal motifs. (B) Autoradiography of in vitro kinase assay of purified CPK21 wild-type and mutant enzymes with histone as substrate. Kinase assays were performed as described under (A) except that the reaction mixture was separated by SDS gel electrophoreses followed by autoradiography. Positions of CPK21 (auto-phosphorylation) and histone (substrate trans-phosphorylation) are indicated by arrows. (C) Western blot analysis of affinity-purified CPK21 wild-type and EF-hand mutant enzymes using an anti-STREP–HRP conjugate. Molecular Plant 2011 4, 83-96DOI: (10.1093/mp/ssq064) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 3 Identification of Atcpk21 Mutant and Localization of PCPK21::GUS. (A) Scheme of the exon–intron gene structure of AtCPK21 (At4g04720) indicating the T-DNA insertion site in the first exon. (B) PCR analysis of genomic DNA isolated from wild-type (Col-0) and homozygous mutant (SALK_029412) plants using primer specifically amplifying the T-DNA insertion (T-DNA) or the CPK21 wild-type allele (CPK21). (C) Transgenic Col-0 lines expressing PCPK21::GUS expression were generated. GUS staining of 4-week-old plants is shown. Molecular Plant 2011 4, 83-96DOI: (10.1093/mp/ssq064) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 4 CPK21 Regulates Gene Expression of Stress Marker Genes. The gene expression of CPK21, CPK23, and selected marker genes was analyzed by RT–PCR in seedlings grown on plates under control conditions or on plates containing 300 mM mannitol conditions comparing Col-0 wild-type, Atcpk21 mutant line, and a complementation line (PCPK21::CPK21). An accumulation of stress marker genes DREB1A, COR15A, RD29A, but also CPK23, closest homolog to CPK21, was observed in cpk21 mutant plants already in the absence of stress exposure in comparison to Col-0. Upon complementation with PCPK21::CPK21, again, a less prominent gene transcript accumulation was observed in the absence of stress exposure for DREB1A, COR15A, and CPK23. Growth under hyperosmotic conditions with 300 mM mannitol caused a stress marker gene induction in all three lines compared. Molecular Plant 2011 4, 83-96DOI: (10.1093/mp/ssq064) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 5 In Vivo Protein Kinase Activity of CPK21 Wild-Type Enzyme and Kinase Inactive Enzyme Variant D204A Expressed in A. thaliana in Response to Abiotic Stress Stimuli. Stable transformants of Strep-tagged CPK21 wild-type and the kinase-inactive CDPK21D204A variant were generated in the cpk21 mutant. For stress assays, plants were grown in liquid cultures. Leaf material was harvested before or at different time points after the stress stimulus (addition of NaCl or mannitol to the medium), CPK21 protein was affinity-purified, and aliquots were analyzed by Western blot (lower panel). Protein kinase activity was determined in immuno-complex kinase assays with syntide2 as substrate, and Ca2+-dependent protein kinase activity is shown (upper panel). (A) In vivo kinase activity of CPK21 and CPK21D204A in response of Arabidopsis plants to the addition of 100 mM NaCl. (B) In vivo kinase activity of CPK21 in response of Arabidopsis plants to hyperosmotic (addition of 200 mM mannitol) and hypo-osmotic (addition of two volumes of water) stress. Molecular Plant 2011 4, 83-96DOI: (10.1093/mp/ssq064) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions

Figure 6 CPK21 Functions as Negative Regulator of Osmotic Stress Signaling In Planta. (A) Assessment of growth performance on plates (1 MS, 3% sucrose) of Col-0 wild-type, Atcpk21, and a complementation line containing PCPK21::CPK21 under control conditions or in the presence of mannitol (200 and 300 mM), NaCl (100 and 150 mM) and PEG (with osmotic potential of –0.45 and –0.95 MPa). One-week-old seedlings were transferred on plates with the respective growth media and the performance of the plants was evaluated at 3.5 (–0.95 MPa PEG) and 4.5 weeks after the transfer. (B) Evaluation of the seedling growth assays on mannitol-containing plates. Seedling development was assessed on individual plates containing either 200 or 300 mM mannitol. The percentage of healthy green seedlings (green color), seedlings with growth arrest and chlorotic symptoms (pale green color), and dead seedlings with strong chlorotic symptoms (yellow color) are indicated. The statistical evaluation for five independent plates (in total representing 30–40 seedlings per line) assessing seedling growth under 300 mM mannitol is given in the panel to the right. The asterisk indicates a significant difference obtained from Student's t-test (P < 0.05). Data are shown as means ± SD. After 3.5 weeks' growth under hyperosmotic conditions, about 80% of the plants were chlorotic in Col-0, whereas significantly fewer seedlings of Atcpk21 mutant plants showed these stress symptoms. In the complementation line, chlorotic plants—indicative of restoring wild-type sensitivity—were observed. Molecular Plant 2011 4, 83-96DOI: (10.1093/mp/ssq064) Copyright © 2011 The Authors. All rights reserved. Terms and Conditions