Supplemental methods:
Cloning:
pCPK28::GUS (pI2-pCPK28-GUS-mcs2): An S3 terminator was amplified by PCR from
plasmid pPLEX3001 (Schünmann et al. 2003) using primers 401 and 402 and the ends of
the resulting fragment were cut with XbaI and NotI. The 35S terminator of vector pam-PATMCS (accession number AY436765) was replaced by the S3 terminator using the vector’s
XbaI and NotI restriction sites, generating the construct pI1. From genomic DNA of A.
thaliana, ecotype Col-0, a promoter fragment of 1803 bp of AtCPK28 was amplified by PCR
with primers 366 and 525. The promoter fragment was cut at the ends with AscI and EcoRI
and used to replace the 35S promoter of pI1, released via AscI and EcoRI. This cloning step
generated pI2-pCPK28. A multiple cloning site (mcs2) made from annealed primers 508 and
509 was cloned into pI2-pCPK28 opened with NotI and BstXI, generating pI2-pCPK28-mcs2.
A β-Glucuronidase (GUS) gene was amplified with primers 403 and 438 from a GUS-vector
introducing EcoRI and SmaI restriction sites at the ends. These sites were used to clone the
GUS gene 3’ of the CPK28 promoter in pI2-pCPK28-mcs2 generating pI2-pCPK28-GUSmcs2.
VK variants of CPK28 (pXCS-CPK28-VK-YFP and pXCS-CPK28-VK-D188A-YFP): The VK
region of CPK28 was amplified with specific VK-primers from pXCSG-CPK28-YFP and
pXCSG-CPK28-D188A-YFP and subcloned into pJET1.2 (Fermentas). Coding sequences
were confirmed by sequencing and transferred to the binary expression vector pXCS–YFP
(Feys et al. 2005) opened with HindIII and SmaI.
pXCSG-pSUC2 and pXCSG-pKNAT1: From genomic DNA of A. thaliana, ecotype Col-0
promoter fragments of SUC2 and KNAT1 were amplified by PCR (An et al. 2004) (Primers
see Supplemental Table S1). Promoter fragments were cut at the ends with AscI and HindIII
and used to replace the 35S promoter of pXCSG-YFP, released via AscI and HindIII. This
cloning step generated pXCSG-pSUC2-YFP or pXCSG-pKNAT1-YFP, respectively.
Transient protein expression in Arabidopsis protoplasts:
The preparation and transfection of Arabidopsis protoplasts was carried out as described
(Wu et al. 2009). To exclude phosphorylation by endogenous CPK28, approximately 2 × 106
protoplasts derived from cpk28-1 plants were transfected with 100 μg DNA of each
expression construct, harvested 16 – 18 h after transfection, and frozen in liquid nitrogen. For
protein extraction, frozen pellets were resuspended by vortexing in extraction buffer [100 mM
Tris-HCl (pH 8,0), 200 mM NaCl, 20 mM DTT, 10 mM NaF, 10 mM NaVO4, 10 mM β-
glycerole-phosphate, 0,5 mM AEBSF, 100 μg/ml avidin and 1 × protease inhibitor mixture
(Sigma)]. Crude extracts were centrifuged at 20,000 × g for 5 min at 4°C. The supernatant
was transferred to fresh tubes and incubated with Strep-Tactin MacroPrep (IBA) for 30 min at
4 °C. Immobilised protein was washed five times with Wash buffer (100 mM Tris-HCl (pH
8,0), 200 mM NaCl, 2 mM DTT, 10 mM NaF, 10 mM NaVO 4, 10 mM β-glycerole-phosphate,
0,05 % TritonX-100) and, after acetylation of cysteins with iodacetamide, subjected to trypsin
(Promega) digestion in 50 mM ammonium bicarbonate overnight at 37°C. Tryptic digest
solutions were separated from the Streptactin matrix by centrifugation, transferred to new
reaction tubes and vacuum-dried at 30°C.
Mass spectrometry:
Tryptic peptide mixtures were analysed by selected reaction monitoring using nanoflow
HPLC (Proxeon Biosystems, www.proxeon.com) and a triple quadrupole mass spectrometer
(TSQ Quantum Discovery Max, Thermo Scientific) as mass analyzer. Peptides were eluted
from a 75 µm analytical column (Reprosil C18, Dr. Maisch, www.dr-maisch.com) on a linear
gradient running from 10 % to 30 % acetonitrile in 50 minutes and were ionized by
electrospray directly into the mass spectrometer. Specifically, phosphorylated and nonphosphorylated
forms
of
the
peptides
FHDIVGSAYYVAPEVLK,
ISLHEFR,
and
LTAAQALSHAWVR were analysed. Selected reaction monitoring (SRM) was used to
quantify the abundance of phosphorylated and unphosphorylated forms of the respective
peptides within each sample. The quadrupole Q1 was set as a mass filter for the parent ion,
while Q3 was set to monitor specific fragment ions. Mass width for Q1 and Q3 was 0.7 Da,
scan time was set to 5 milliseconds. Suitable fragment ions for SRM were selected
experimentally determined and optimised using the software Pinpoint (Thermo Scientific).
For each peptide, at least three fragment ions were used. Data analysis merging of fragment
ion information to peptide average was also done using the Pinpoint software. For each
phosphorylated and unphosphorylated peptide, intensities sums of three to five most intense
fragment ions were displayed.
Additional hormone treatments:
Plants were grown on soil under long-day conditions (16 h light, 8 h dark) at 22°C unless
otherwise stated. Hormone treatments were performed by spraying plants with either 0,95 %
EtOH (mock), 100 µM Epibrassinolide (Sigma) or 100 µM IAA (Sigma) every other day.
Plants were evaluated for the cpk28 phenotype 45 dag.
Germination assay:
Surface-sterilized seeds were plated on 0,5x Murashige and Skoog (MS) medium (pH 5.8)
(Duchefa, www.duchefa.com) supplemented with 0.8% agar. To examine germination, seeds
were generally first incubated for 2 d at 4°C and then grown at 22°C under LD conditions.
Seed germination was quantified by scoring radicle emergence at the time points indicated in
the figures. Presented are the averaged results of three independent seed batches (for each
batch, WT and cpk28 mutant seeds were harvested at the same time, respectively) (n ≥ 36
per batch and genotype).
Hypocotyl elongation assay:
Surface-sterilized seeds were sown on 0,5x MS medium containing 0.8% agar supplemented
with or without GA3 or PAC. Plates were incubated for 2 d at 4° C and then grown at 22°C in
the dark or under LD conditions for 6 days. Hypocotyl lengths were measured with the
ImageJ software.
Protein extraction and Western Blot:
Leaf material of transgenic lines was harvested and ground in liquid nitrogen, and proteins
were extracted with extraction buffer (Franz et al. 2011). Proteins were separated on a 12%
SDS/PAGE gel and analysed by Western blot. YFP-fusion protein detection was performed
by anti-GFP (Roche, www.roche-applied-science.com) and an anti-mouse AP-conjugated
antibody (Sigma, www.sigmaaldrich.com).
Protein purification:
Tagged CPK28 variants were transiently expressed in N. benthamiana as described
previously (Witte et al. 2004). 0,5 g of N. benthamiana leaf material was ground in liquid
nitrogen and resuspended in 1 ml extraction buffer (100 mM Tris/HCl pH 8.0, 5 mM EGTA, 5
mM EDTA, 100 mM NaCl, 20 mM DTT, 0.5 mM AEBSF, AL-Mix (2µg/ml antipain and 2µg/ml
leupeptin), 2 µl/ml protease inhibitor cocktail (Sigma), 10 mM NaF, 10 mM Na3VO4, 10 mM
-glycerolphosphate, 100 µg/ml avidin and 0.5% (v/v) Triton X-100). After removing insoluble
debris, the supernatant was incubated with 40 µl Strep-tactin beads (IBA, www.ibalifesciences.com) and tagged proteins were immobilised by gentle end-over-end-rotation at
4°C for 30-60 min. Proteins were purified by washing 3-5 times with washing buffer (100 mM
Tris pH 8,0, 150 mM NaCl, 1 mM EDTA, 2 mM DTT). Immobilised proteins were stored in
washing buffer and transferred to buffer E (50 mM HEPES pH 7,4, 2 mM DTT, 0,1 mM
EDTA) before the in vitro kinase assay.
Kinase activity assay:
Equal protein amounts used in kinase assays were determined by prior SDS-Gel
electrophoresis and Western Blot analysis via Strep-Tactin-HRP or -AP conjugate (IBA).
Protein purity was determined by detection of a single protein band on a Coomassie gel.
Phosphorylation of Syntide 2 was assessed by the P81-filter-binding method as described
(Romeis et al. 2001). The final reaction mixture (30 µl) contained 35 mM HEPES pH 7.4,
10 mM MgCl2, 1,4 mM DTT, 0.07 mM EDTA, 10 µM Syntide 2, 10 µM ATP, 3 µCi [γ-32P]ATP and different concentrations of CaCl2 or 2 mM EGTA as indicated. The reaction was
carried out for 20 min at 30°C and was stopped by adding 3 µl of 10% phosphoric acid.
Subsequently, 20 µl of each reaction was dropped on P81 paper and dried at room
temperature. Three washing steps with 1% phosphoric acid removed free [γ-32P]-ATP and
incorporated 32P was determined by liquid scintillation counting.
An, H., Roussot, C., Suarez-Lopez, P., Corbesier, L., Vincent, C., Pineiro, M., Hepworth,
S., Mouradov, A., Justin, S., Turnbull, C. and Coupland, G. (2004) CONSTANS
acts in the phloem to regulate a systemic signal that induces photoperiodic flowering
of Arabidopsis. Development, 131, 3615-3626.
Feys, B.J., Wiermer, M., Bhat, R.A., Moisan, L.J., Medina-Escobar, N., Neu, C., Cabral,
A. and Parker, J.E. (2005) Arabidopsis SENESCENCE-ASSOCIATED GENE101
stabilizes and signals within an ENHANCED DISEASE SUSCEPTIBILITY1 complex
in plant innate immunity. Plant Cell, 17, 2601-2613.
Franz, S., Ehlert, B., Liese, A., Kurth, J., Cazale, A.C. and Romeis, T. (2011) Calciumdependent protein kinase CPK21 functions in abiotic stress response in Arabidopsis
thaliana. Mol Plant, 4, 83-96.
Romeis, T., Ludwig, A.A., Martin, R. and Jones, J.D.G. (2001) Calcium-dependent protein
kinases play an essential role in a plant defence response. EMBO J, 20, 5556-5567.
Schünmann, P.H.D., Llewellyn, D.J., Surin, B., Boevink, P., Feyter, R.C.D. and
Waterhouse, P.M. (2003) A suite of novel promoters and terminators for plant
biotechnology. Functional Plant Biology, 30, 443-452.
Witte, C.P., Noel, L.D., Gielbert, J., Parker, J.E. and Romeis, T. (2004) Rapid one-step
protein purification from plant material using the eight-amino acid StrepII epitope.
Plant Mol Biol, 55, 135-147.
Wu, F.H., Shen, S.C., Lee, L.Y., Lee, S.H., Chan, M.T. and Lin, C.S. (2009) TapeArabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Plant Methods, 5.