Developmental Cell, Volume 22

Supplemental Information

Genetic Framework of Cyclin-Dependent

Kinase Function in Arabidopsis

Moritz K. Nowack, Hirofumi Harashima, Nico Dissmeyer, Xin’Ai Zhao, Daniel Bouyer,

Annika K. Weimer, Freya De Winter, Fang Yang, and Arp Schnittger

Inventory of supplemental data

Figure S1. Embryo development in cdka;1 and cdka;1 cdkb1;1 mutants.

Figure S2. Columella fate in cdka;1 mutants and rescue plants.

Figure S3. RBR1 ChIP of CDKB1;2 promoter fragments.

Figure S4. Temperature-dependent and dosage-sensitive overproliferation in the rbr1-2 allele.

Supplemental Experimental Procedures

Supplemental References

Figure S1. Embryo development in cdka;1 and cdka;1 cdkb1;1 mutants.

(A-E) Embryo development in the wild type from globular (A) over torpedo (B, C) to mature

green stage (D,E).

(F-J) Embryo development in a heterozygous cdka;1 mutant showing a class of embryos with

fewer and larger cells from globular (F) over torpedo (G, H) to mature green stage (I, J).

Note the increased cell size in cdka;1 mutant embryos (compare C and H, and E and J). Seed

size and endosperm proliferation are not affected in the seeds containing homozygous mutant

offspring. Seeds shown in A and F are developing in homozygous cdka;1-/-

mutants rescued

by a hemizygous PROCDKA;1:CDKA;1:YFP tracer construct, mimicking the heterozygous

cdka;1+/-

mutant phenotype. Segregating homozygous mutant embryos and endosperms can

be recognized by the absence of a YFP signal.

(K-P) Whole-mount seeds at different stages of development. Wild type at 21 days after

flowering (daf, K), early arrested embryos in cdka;1 cdkb1;1 mutant seeds at 7 daf (L-M),

later arrested embryo in cdka;1 cdkb1;1 mutant seeds at 10 daf (N), embryos in cdka;1

cdkb1;1 mutant seeds at 14 daf (O) and 21 daf (P). The arrowhead points at the embryos

inside the seeds.

(Q-V) Isolated embryo of the wild type (Q) or close-up of arrested embryos in cdka;1

cdkb1;1 mutant seeds, and an isolated cdka;1 cdkb1;1 mutant embryo at 21 days after

fertilization (V). (R-U) each are details of (L-O), respectively.

(W-X) PCR-based genotyping of mature embryos segregating from a heterozygous cdka;1+/-

mutant mother plant. Embryos with a wild-type phenotype were either CDKA;1+/+

wild type

or cdka;1+/-

heterozygous mutants (W, boxed lanes). Embryos with a cdka;1 phenotype did

not yield any CDKA;1 wild-type PCR product, and most showed a cdka;1 mutant PCR

product (X, boxed lanes). Note that the PCR sometimes failed, probably due to the low

amount of genomic DNA delivered by the few celled mutant embryos.

Scale bars, 100 µm (A-B, D, F-G, I, K-Q, V), 50 µm (E, J), and 20 µm (C, E, H, J, R-U).

Figure S2. Columella fate in cdka;1 mutants and rescue plants.

(A-D) Initially correctly specified tissue identity in cdka1 mutant root tips despite the severely

disturbed cellular pattern. J2341 and J1092 mark columella initials, and columella and lateral

root cap in the wild type (A, C) and in the cdka;1 mutant (B, D), respectively.

(E-H) Purple Lugol staining of starch grains revealing correctly specified columella cells in

the wild type (E), while starch grains are absent from arrested cdka;1 root tips (F). cdka;1

mutants rescued by co-depletion of RBR in a double homozygous cdka;1 rbr1-2 mutant (G)

or complemented with a PROCDKA;1:CDKB1;1 construct (H) show a reconstitution of lastingly

maintained columella fate.

Scale bars, 20 µm.

Figure S3. RBR1 ChIP of CDKB1;2 promoter fragments.

(A) Different pairs of overlapped primers (1 to 8) designed at approximately 1.14 kb upstream

of the region of the CDKB1;2-coding site because of the lack of predicted E2F-binding motif.

(B) ChIP PCR showing the interaction between the RBR1 protein and different fragments of

the CDKB1;2 promoter. (C) Potential E2F-binding motifs found in the CDKB1;2 promoter

region. Degenerated motifs WTTSSSSS and WCTSSSSS (W being A or T and S being C or

G) were not found in CDKB1;2 promoter, except one and two SSSSS motifs located within

the fragment 5 and 7, respectively. The fragments containing the potential E2F-binding motif

and the corresponding motifs are highlighted in red.

Figure S4. Temperature-dependent and dosage-sensitive overproliferation in the rbr1-2 allele.

The overproliferation phenotype and the degree of cdka;1 mutant rescues caused by the rbr1-

2 allele is temperature- and dosage-dependent. (A-C) Grown at 22°C, wild type (A) and rbr1-

2-/-

mutant plants (B, C) are hardly distinguishable. Only small areas of overproliferating cells

can be seen in rbr1-2-/-

mutants (C, arrowhead). (D-F) Grown at 17°C, wild type plants (D)

are largely similar to wild type plants grown at 22°C (compare A and D). rbr1-2-/-

mutants,

however, show areas with excessive epidermal overproliferation on their leaves,

macroscopically visible as white patches (E, arrowheads). Under the raster-electron

microscope the white patches resolve into areas with densely over-proliferated epidermal cell

clusters, separated by large epidermal pavement cells (F).

(G-I) Dosage dependent cdka;1 rbr1-2 rescue phenotype. When compared to the wild type

(G), a double homozygous cdka;1-/-

rbr1-2-/-

mutant grown at 17°C shows small serrated

leaves, islands of strongly over-proliferating cells (H, arrowheads, see inset for detail) and a

generally slightly smaller plant body (H). The homo-heterozygous cdka;1-/-

rbr1-2+/-

double

mutant, however, remains much smaller than the wild type or the double homozygous rescue,

and shows hardly any over-proliferation in epidermal cells (I).

Scale bars, 750 µm (A, C, D, F), 5 mm (B, E), 1 cm (G-I).

Supplemental Experimental Procedures

Isolation of cdkb1 mutant alleles:

The following alleles were isolated for CDKB1;1 (At3g54180): cdkb1;1-1: SALK_073457

(N573457); cdkb1;1-2: SAIL_540_E02 (N879507 and CS879507); cdkb1;1-3:

SALK_044765 (N544765); and cdkb1;1-4 (SAIL_871_C10; N522275 and CS877559). For

CDKB1;2 (At2g38620), the following alleles were isolated: cdkb1;2-1: SALK_133560

(N633560); cdkb1;2-2: SALK_034570 (N534570). The resistances against selective agents

are silenced for cdkb1;1-3, cdkb1;2-1, and cdkb1;2-2, and partially silenced for cdkb1;1-1.

cdkb1;1-4 could not be germinated and was withdrawn from both NASC and ABRC

collections.

Primer sequences for genotyping T-DNA mutant insertions

primer sequence 5’ > 3’

T-DNA insertion

cdka;1-1 M212 TGTACAAGCGAATAAAGACATTTGAAGTACC

J504 GCGTGGACCGCTTGCTGCAACTCTCTCAGG

cdka;1-2 N031 CTACACTGAATTGGTAGCTCAAACTGTC

N034 CCAGATTCTCCGTGGAATTGCG

cdkb1;1-1 N185 TGGTTCACGTAGTGGGCCATCGCCCTGATA

N177 TGTCTTTGAGCAGCCATCTGTGTTG

cdkb1;2-1 N185 TGGTTCACGTAGTGGGCCATCGCCCTGATA

N178 TTTTTGTACTCAGGGCCGGCTTTAC

rbr1-2 M206 CTTCCACAGCCCGGTCGTTTC

J504 GCGTGGACCGCTTGCTGCAACTCTCTCAGG

rbr1-3 M211 CAGTACCAATTCAGCTGAGCA

M125 CCCATTTGGACGTGAATGTAGACAC

Wild type allele

cdka;1-1 N048 CAGATCTCTTCCTGGTTATTCACA

N049 TGTACAAGCGAATAAAGACATTTGA

cdka;1-2 N034 CCAGATTCTCCGTGGAATTGCG

N035 GGAGATCGACTCCATCGGGATC

cdkb1;1-1 N406 GCTTACCAATTGAGAACAACTGATTC

N177 TGTCTTTGAGCAGCCATCTGTGTTG

cdkb1;2-1 N178 TTTTTGTACTCAGGGCCGGCTTTAC

N278 GGTTCAAAACAAATTATCATCAACTAGG

rbr1-2 M206 CTTCCACAGCCCGGTCGTTTC

M207 GATTACCGCAGCATTCTAGTTGAACGC

rbr1-3 M206 CTTCCACAGCCCGGTCGTTTC

M217 TCTTCTCGCTTTGGTGAGTGT

Generation of cdk double and triple mutants:

The challenge to generate double and triple mutants of CDKA;1 and CDKB1 deficient plants

was the close linkage of CDKA;1 and CDKB1;1, and the lowered transmission of mutant

alleles through the male germ line. First, we generated a double heterozygous cdka;1+/-

cdkb1;1+/-

mutant by crossing a heterozygous cdka;1+/-

mutant as a seed parent with a

homozygous cdkb1;1-/-

mutant as a pollen parent. The such generated cdka;1+/-

cdkb1;1+/-

F1

plants carried the cdka;1 allele and the cdkb1;1 allele on different homologous chromosomes

(in trans). Next, we crossed these cdka;1+/-

cdkb1;1+/-

F1 plants as seed parents with wild-

type plants as pollen parents. In the resulting F2 generation, we selected for heterozygous

cdka;1+/-

cdkb1;1+/-

plants to identify individuals that, due to cross-over events, now carried

the cdka;1 allele and the cdkb1;1 allele on the same homologous chromosome (in cis). These

plants we then crossed as seed parents with homozygous cdkb1;1-/-

mutants as pollen parents

to obtain homo-heterozygous cdka;1+/-

cdkb1;1-/-

mutants in the F3 generation.

To obtain the triple mutant, we crossed these homo-heterozygous cdka;1+/-

cdkb1;1-/-

mutants as seed parents with double homozygous cdkb1;1-/-

cdkb1;2-/-

mutants as pollen

parents. In the F1 generation, we selected for triple cdka;1+/-

cdkb1;1-/-

cdkb1;2+/-

mutants.

Finally, we allowed these F1 plants to selfpollinate and obtained the triple hetero-homo-

homozygous cdka;1+/-

cdkb1;1-/-

cdkb1;2-/-

mutants in their progeny.

DNA work:

The coding sequences of CDKB1 and CDKB2 kinase genes were cloned via Gateway

Technology (Invitrogen) into a pAMPAT vector carrying a PROCDKA;1 promoter fragment

(Nowack et al., 2006).

Primer sequences for cloning of CDKB constructs

RNA work:

1. Semi-quantitative RT-PCR

For transcript analysis, total RNA was prepared with the RNeasy Mini Kit (Qiagen) and

cDNA synthesis was performed with SuperScript III RNase H reverse transcriptase

(Invitrogen) according to the manufacturer’s instructions.

Primer sequences for semi-quantitative RT-PCR

primer sequence 5’ > 3’

Generic cDNA primers J750 ATTCTAGAGGCCGAGGCGGCCGCCATG-

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVA

J751 ATTCTAGAGGCCGAGGCGGCCGCCATG-

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVC

J752 ATTCTAGAGGCCGAGGCGGCCGCCATG-

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVG

J753 ATTCTAGAGGCCGAGGCGGCCGCCATG-

TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVT

Gene primer sequence 5’ > 3’

CDKB1;1

(AT3G54180.1)

N315 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCCACCATGGA

GAAGTACGAGAAGCTAGAG

N225 GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGAACTGAGACTTG

CDKB1;2

(AT2G38620.2)

N324 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCCACCATGGAGAAAT

ACGAGAAGCTCG

N227 GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGAACTGAGATTTG

CDKB2;1

(AT1G76540.1)

N333 GGGACAAGTTTGTACAAAAAAGCAGGCTTCCCACCATGGACGAGGG

AGTTATAGCA

N229 GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAGAGAGAGGACTTTT

CTGGC

CDKB2;2

(AT1G20930.1)

N341 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCCCACCATGGACAACAAT

GGAGTTAAACC

N231 GGGGACCACTTTGTACAAGAAAGCTGGGTCTCAGAGAGAGGACTTG

Gene

CDKB1;1 N358 GATGTTATCAACATCGATCTATGTTG

N376 GATATGAAGCAATTGCTGAAACTCAG

N496 GTTCATTGCTGTATCTGTTGTCATCG

N497 CATCTCATTGCTACCACACCATTGTG

CDKB1;2 N361 CTTGCCTCCTAATCATCTCGGC

N362 GAAATACGAGAAGCTCGAAAAGG

N407 CCTTTTCGAGCTTCTCGTATTTC

N424 AGCGTGCGAGGGATTCAAATGTTAAACG

EF1 N373 ATGCCCCAGGACATCGTGATTTCAT

N374 TTGGCGGCACCCTTAGCTGGATCA

ACT2 N628 GGCTCCTCTTAACCCAAAGGC

N629 CACACCATCACCAGAATCCAGC

TUB2 N481 GAGCCTTACAACGCTACTCTGTCTGTC

N482 ACACCAGACATAGTAGCAGAAATCAAG

CDKA;1 full length M153 ATAGAATGAAGGAGATTACTGGTTTTATGCC

M152 GTGTGGAGTTTACTTCAGCTTTATTATTCAGG

Fragment 5' of the T-DNA insertion M152 GTGTGGAGTTTACTTCAGCTTTATTATTCAGG

M151 GCACCACATCCTGCAATTTGACAATG

Fragment 3'of the T-DNA insertion M153 ATAGAATGAAGGAGATTACTGGTTTTATGCC

M150 CTTCAAGATTTTCAGAATCATGGGAACTC

2. qRT-PCR and expression analysis

RNA extraction was performed using RNeasy Mini Kit (Qiagen). For qRT-PCR analysis at

least three biological replicates were used. RNA-concentration and purity was tested using

nanodrop-photometric quantification (Thermo Scientific) and RNA-integrity was verified by

running 0.5 µg of total RNA on 1.5% agarose TBE-gels to detect the 28S and 16S rRNA

bands. For each replicate 0.5 μg RNA was treated with DNAseI (MBI Fermentas) according

to the manual to avoid contamination of genomic DNA and subsequently processed to obtain

cDNA using poly T-primer and SuperScript III reverse transcriptase (invitrogen) according to

the manufacturer’s instructions. Remaining RNA was digested using RNAseH (Fermentas).

For negative control, all steps were followed in the same manner without adding the reverse

transcriptase. The resulting cDNA was used for Reverse Transcription (RT)-PCR or

quantitative Real Time-PCR (qRT-PCR) using the Roche LightCycler 480 system.

Oligonucleotides were designed using either Primer3Plus-design tool

(http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) or QuantPrime (qPCR

primer design tool: http://www.quantprime.de/main) and used in final concentration of 0.25

μM each. 3-4 biological replicates with 2-3 technical replicates per biological replicate were

analyzed in the qRT-PCR-experiments. Cq calling was done using the Second Derivative

Maximum method. Target specific efficiencies were calculated as the mean of all reaction

specific efficiencies for a given target. Reaction specific efficiencies were deduced using

LinRegPCR 7.4 (http://LinRegPCR.nl (Ramakers et al., 2003; Ruijter et al., 2009)). Data was

quality controlled, normalized against 3 reference genes (ACT7 (AT5G09810), EXP

(AT4G26410) , TIP41-like (AT4G34270) and statistically evaluated (One-Way Anova Test)

using qbasePLUS 2.1 (http://www.biogazelle.com/products/qbaseplus (Hellemans et al.,

2007)).

Primer sequences for quantitative RT-PCR and quantitative PCR of the RBR1 targets

qRT PCR RBR1 targets

Gene identifier sequence 5’ > 3’

ACT7 AT5G09810.1 forward GTTGCCATTCAGGCCGTTCTTTC

reverse CAGAATCGAGCACAATACCGGTTG

TIP41-like

AT4G34270.1 forward TGAACTGGCTGACAATGGAGTG

reverse CATGAGCTTGGCATGACTCTCAC

EF1A AT5G60390.1 forward TGTAACAAGATGGATGCCACCAC

reverse TCCCTCGAATCCAGAGATTGGC

PCNA1 AT1G07370.1 forward CGGTGACATTGGAACCGCTAAC

reverse TCACAATTGCATCTTCCGGCTTG

MCM2 AT1G44900.1 forward GATAGAGGAACTGCAGACAAAGGC

reverse TCGGCCATGATCCAACTCGAAG

MCM5 AT2G07690.1 forward AGCTACAGGAGAATCCGGAGGATG

reverse AGATGCCGATCAACTGAGAGAAGC

ORC1A AT4G14700.1 forward TCGGTTGTGATCTTGGTGAATGC

reverse GCTGCAGCTTCTGCAATCTGTG

ORC3 AT5G16690.1 forward ACTTGACCCAGAAGCACGTTACC

reverse TGTGAAGGTGAAAGATCCCTGAGC

CDKA;1 AT3G48750.1 forward ACTGGCCAGAGCATTCGGTATC

reverse TCGGTACCAGAGAGTAACAACCTC

CDKB1;1 AT3G54180.1 forward CAACTGGTGTTGACATGTGGTCTG

reverse TCAGTTGGTGTTCCTAGCAACCTG

CDKB1;2 AT2G38620.2 forward TGCCGAGATGATTAGGAGGCAAG

reverse GGGCTAAAGTCCTAAACTGGTTCC

CDC6 AT2G29680.1 forward AGGCTCTATGTGTCTGCAGGAG

reverse ACCACTTGACACTCTGGAACTGG

Primer sequences for quantitative RT-PCR and quantitative PCR for ChIP qPCR

qPCR ChIP

Gene transcript_identifier sequence 5’ > 3’

PCNA1 AT1G07370.1 forward TCTTAAAACGATTGAGGCCG

reverse AATCGTTTGCGGCTATTTTG

CDKB1;1 AT3G54180.1 forward GTTGCAGTTGTCACGAACGA

reverse CCACGTCACTTCGTTTTTCC CDKB1;2 AT2G38620.1 (ChIP

PCR) forward CTTTCCCCTTTACATAGAAC

reverse TCATTCCAATTTCATGTATGG

(ChIP PCR) forward CCATACATGAAATTGGAATGA

reverse GGACTGTGAACTCTTACTCA

(ChIP PCR) forward GAAACAACCCACCATTAATG

reverse TAATGTTCAATGTTTGGCAC

(ChIP Q-PCR) forward GTGCCAAACATTGAACATTA

reverse CTTGAAGAGTCGATCATTTC

(ChIP PCR) forward GCTCGAAGTTTCAACCAATT

reverse TTCAATTCATTATCCATGGTG

(ChIP PCR) forward GCTTTACATCAAAGATCATGC

reverse TTAACATTTGAATCCCTCGC

(ChIP PCR) forward TTATTGGTACGTGTGAAGCG

reverse TCGTATTTCTCCATTGATGG

(ChIP PCR) forward GACCCAAACAAAAAACATTCA

reverse GTGGTATACCTTCTTCGTCC

MCM2 AT1G44900.1 forward TAGTGAAAGCCCATACGATG

reverse CTATCTGTGTATTGTTTCCG

MCM5 AT2G07690.1 forward GTAGCGGGAAATAGCAATGG

reverse TCCATTTACCCGTCCATAGG

ORC1A AT4G14700.1 forward CTCTTACCCAAATTCATTTCC

reverse TGGAAGAAGAGGGATTTTAC

ORC3 AT5G16690.1 forward CCAATTCCGGTCTAGTCTGG reverse TGGAGCAATCGAAAACGACG LB25 AtenSAT

(Chr4 1673860-1674024)

forward GAGGACCCGATCTCCTTCAT

reverse CGAGAACACCCCTGATAACG

RB32.5 AtenSAT (Chr4 1774813-1774954)

forward CGAACACACGGATATGTTGC

reverse TGGTGATGTACTCGCTGTCAA

RB35 AtenSAT forward CACGATAGATCAATAGGATTGAGG

(Chr4 1777125-1777324)

reverse CACGATCGTATGAGTTAGCAACTTT

AtCDC6

AT2G29680.1 forward TTGTTCAAACCATAATGCGG

reverse CAAAGAGGGAAAAATGGAAG

3. Generation of amiRNA lines

Artificial micro-RNAs (amiRNAs) against CDKB1 genes were designed and cloned as

described (Schwab et al., 2006).

amiRNA sequences against CDKBs and primer sequences for amiRNA cloning

gene / construct amiRNA sequence sequence I miR-s 5’ > 3’ sequence II miR-a 5’ > 3’

CDKB1;1 (AT3G54180.1)

cdkb1;1-A (MN67)

TAAGAGTTAAGTCTTGCGGGT

gaTAAGAGTTAAGTCTTGCGGGTtctctcttttgtattcc

gaACCCGCAAGACTTAACTCTTAtcaaagagaatcaatga

cdkb1;1-B (MN68)

TCTTAGGGTAAACATGCCACT

gaTCTTAGGGTAAACATGCCACTtctctcttttgtattcc

gaAGTGGCATGTTTACCCTAAGAtcaaagagaatcaatga

cdkb1;1-C (MN69)

TCTGTCAAAATATGGGTGATG

gaTCTGTCAAAATATGGGTGATGtctctcttttgtattcc

gaCATCACCCATATTTTGACAGAtcaaagagaatcaatga

CDKB1;2 (AT2G38620.2 )

cdkb1;2-A (MN70)

TACGTGATAAGTCTTGCGCCT

gaTACGTGATAAGTCTTGCGCCTtctctcttttgtattcc

gaAGGCGCAAGACTTATCACGTAtcaaagagaatcaatga

cdkb1;2-B (MN71)

TATGCCATTACACCGGGCGAT

gaTATGCCATTACACCGGGCGATtctctcttttgtattcc

gaATCGCCCGGTGTAATGGCATAtcaaagagaatcaatga

cdkb1;2-C (MN72)

TAGACAATAAACTTGGGCCGT

gaTAGACAATAAACTTGGGCCGTtctctcttttgtattcc

gaACGGCCCAAGTTTATTGTCTAtcaaagagaatcaatga

both CDKB1s together

cdkb1-A (MN73) TGAAATTCTTTCGA

CTGGATT gaTGAAATTCTTTCGACTGGATTtctctcttttgtattcc

gaAATCCAGTCGAAAGAATTTCAtcaaagagaatcaatga

cdkb1-B (MN74) TAAACGAGATAGACATTGGAT

gaTAAACGAGATAGACATTGGATtctctcttttgtattcc

gaATCCAATGTCTATCTCGTTTAtcaaagagaatcaatga

cdkb1-C (MN75) TTGAAACTCAGAATAACCAGG

gaTTGAAACTCAGAATAACCAGGtctctcttttgtattcc

gaCCTGGTTATTCTGAGTTTCAAtcaaagagaatcaatga

4. RNA in situ hybridization

Sample preparations and in situ hybridizations were performed as described previously

(Müller et al., 2006). The STM and WUS probes contained the nucleotide sequences 78 to

1122 and 249 to 887, respectively, relative to the ATG. The sequences were cloned into

pGEM vectors in antisense orientation relative to the T7 promoter. Linearized plasmids were

used as templates for probe synthesis using T7 RNA polymerase according to the

manufacturer’s instructions (Ambion). Probes were not hydrolyzed. After the color reaction,

slides were mounted in 30% glycerol and photographed using DIC microscopy on a Zeiss

Axiophot microscope.

Protein work

1. p9ckshs1-sepharose, p10Cks1At-sepharose

To express and purify p10Cks1At, a fusion protein with the maltose-binding protein (MBP)

was generated. p10Cks1At was amplified with primers #263 and #192 followed by primers

#264 and #133 to conjugate a TEV recognition sequence and attB recombination sites,

respectively. The PCR product was cloned into the GATEWAY entry vector pDONR201

(Invitrogen) using BP Clonase II (Invitrogen). A recombination reaction was performed

between the entry clone and the destination vector pDEST-periHisMBP (Nallamsetty et al.,

2005) by using LR Clonase II (Invitrogen) and E. coli BL21-AI cells (Invitrogen) were

transformed with the resulting vector, pDEST-HisMBP-p10Cks1At. E. coli cells were grown

until OD600=0.6 and the production of the fusion protein was induced by adding 0.3 mM

IPTG and 0.2% arabinose for 6 h at 37ºC. Cells were harvested by centrifugation and re-

suspended in Ni-NTA binding buffer (50 mM NaH2PO4, 100 mM NaCl, 10% (v/v) glycerol,

25 mM imidazole, pH 8.0), and lysed by sonication. After addition of Triton X-100 to 0.2%,

the cell slurry was incubated at 4ºC and clarified by centrifugation. The supernatant was

passed through a column packed with Ni-NTA resin (Qiagen), which was washed sequentially

with Ni-NTA binding buffer, and eluted with HIS elution buffer (Ni-NTA binding buffer

containing 225 mM imidazole). The eluate was dialysed against TEV reaction buffer (50 mM

Tris-HCl, pH 8.0, 0.5 mM EDTA, 1 mM DTT). p10Cks1At was cleaved from HisMBP using

TEV protease which was purified as described (Kapust et al., 2001). The fragments were

separated by Ni-NTA column. The flow through fractions which contain cleaved p10Cks1At

proteins were concentrated with Ultra-15 (Millipore) and the buffer was exchanged to

coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3) with a PD-10 column (GE Healthcare).

Purified p10Cks1At was coupled to CNBr-activated Sepharose 4B (GE Healthcare) at a

concentration of 3.5 mg/ml of gel according to the manufacturer’s instructions. p9ckshs1-

beads were prepared as described (Azzi et al., 1994).

2. GST-AtRBR1-His6

To express full-length AtRBR1, pGEX-4T-1 (GE Healthcare) was amplified with primers

#294 and #295 and self-ligated to introduce a hexa-histidine sequence (pGEX-His6). A full-

length AtRBR1 was amplified with primers #194 and #293. PCR products were digested with

BamHI and XhoI, and ligated into BamHI - XhoI sites of pGEX-His6. E. coli BL21-AI cells

were transformed with the resulting vector, pGEX-AtRBR-His6, and grown until OD600=0.6

at 37ºC. The culture was transferred to 18ºC and grown for 30 min. The production of the

fusion protein was induced by adding 0.3 mM IPTG and 0.2% arabinose overnight at 18ºC.

Cells were harvested by centrifugation and re-suspended in phosphate-buffered saline (PBS)

buffer (140 mM NaCl, 2.7 mM KCl, 10.1 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3), and

lysed by sonication. After addition of Triton X-100 to 0.2%, the cell slurry was incubated at

4ºC then clarified by centrifugation. The supernatant was passed through a column packed

with Glutathione-agarose (Sigma), which was washed sequentially with PBS, and eluted with

GST elution buffer (50 mM Tris-HCl, pH 8.0, 10 mM Glutathione). The eluate was

sequentially purified with a column packed with Ni-NTA resin. GST-AtRBR1-His6 was

eluted with HIS elution buffer and the buffer was exchanged to kinase buffer (50 mM Tris-

HCl, pH 7.5, 10 mM MgCl2, 1 mM EGTA) with a PD-10 column.

3. Preparation of CDK-cyclin complexes in E. coli

CYCA2;3 and CYCD3;1 in a Gateway entry vector pDNOR201 were kindly provided by

Lieven De Veylder. A recombination reaction was performed between the entry clone and the

destination vector pHGGWA (Busso et al., 2005) by using LR Clonase II to fuse a GST-tag.

CDK-CYCA2;3 and CDK-CYCD3;1 complexes were produced in E. coli co-expressing GST

fused to Saccharomyces cerevisiae Civ1p. All three proteins were co-expressed by

transforming an E. coli SoluBL21 strain with both plasmids, pCDFDuet-1, containing

StrepIII-CDK and GST-Civ1p, and pHGGWA, containing the respective cyclin. Expression

and purification of active cell cycle complexes was performed as described in detail in

Harashima and Schnittger (Harashima and Schnittger, submitted). In short, cells were grown

in 50 ml of LB medium containing ampicillin and spectinomycin to OD600 of 0.6, and

incubated for 30 min at 18ºC. The production of proteins was induced by adding IPTG to

0.3mM, cells were then cultured overnight at 18°C before being harvested by centrifugation.

The cell pellet was re-suspended in 2.5 mL Ni-NTA binding buffer and lysed by sonication.

After Triton X-100 was added to 0.2%, the cell slurry was sonicated followed by

centrifugation. The supernatant was passed through a column packed with 300 µL Ni-NTA

resin, which was washed sequentially with 3 ml Ni-NTA binding buffer, and eluted with 600

µL HIS elution buffer. The buffer was exchanged to kinase buffer containing protease

inhibitors cocktail (Roche) and Phos-STOP (Roche) with a PD Mini Trap G-25 column (GE

Healthcare).

4. Western Blot

cdka;1 mutants were grown in liquid half MS medium containing 1% sucrose, pH 5.8, then

harvested and frozen in liquid nitrogen. Plants were ground in the liquid nitrogen with a

mortar and a pestle. The resulting fine powder was suspended in either IP complete buffer

(Harashima et al., 2007), or IEF buffer (8 M Urea, 2 M Thiourea, 4% Chaps, 20 mM Tris-

HCl, pH 8.5). Cell debris was pelleted by centrifugation and protein concentration of the

supernatant was measured with a Bradford kit (Bio-Rad). After separating the proteins on the

SDS-PAGE gel, proteins were transferred onto the PVDF membrane in the Towbin buffer

with a wet blotting system (Bio-Rad), the membrane was then blocked with 5% non-fat dry

milk in TBST. To detect CDKA;1 proteins, the membrane was probed with a 1:5000 dilution

of anti-PSTAIRE monoclonal antibody (Sigma) and 1:10000 HRP-conjugated anti-mouse

antibody (KPL) in TBST. Enhanced chemoluminescent detection was performed with HRP

substrate (Millipore). To detect Strep-tag III fused CDK, the membrane was probed with a

1:250000 dilution of Strep-Tactin HRP (IBA) in TBST. To detect HisGST fused cyclins, the

membrane was probed with a 1:250000 dilution of anti-poly His-HRP (Sigma) in TBST.

5. Kinase assay

CKS-associated proteins purified from 300 µg plant extracts were processed for kinase assays

as described previously (Harashima et al., 2007) with Histone H1 (Millipore) or GST-

AtRBR1-His6 as a substrate. In the case of recombinant kinases, the equal amount of

CDKA;1 and CDKB1;1 were used by quantifying them by western blot with Strep-Tactin

HRP.

Primer sequences for cloning in protein work

gene primer sequence 5’ > 3’

p10Cks1At

133 GGGGACCACTTTGTACAAGAAAGCTGGGTTTACTTAACAAGCATGTTCTGAGCT

p10Cks1At

192 GGACTAGTTTACTTAACAAGCATGTTCTGAGCT

AtRBR1 194 GCGGGATCCATGGAAGAAGTTCAGCCTCCAGTG

p10Cks1At

263 GAGAATCTTTATTTTCAGGGGATGGGTCAGATCCAATACTCCGAGAAAT

p10Cks1At

264 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAGAATCTTTATTTTCAGGGG

AtRBR1 293 CCGCTCGAGTGAATCTGTTGGCTCGGTTTTAAGG

pGEX-4T-1 294 CATCATCACCGTGACTGACTGACGATCTGC

pGEX-4T-1 295 ATGATGATGCGGCCGCTCGAGTCG

6. Root growth on Roscovitine

To determine the root growth, dry seeds were sterilized with chlorine gas from 75% (v/v) Eau

de Javel (Floreal, Hagen, Germany) and 25% (v/v) hydrochloric acid and were sown directly

on MS containing different concentrations of Roscovitine (98%, Sigma Aldrich). Roscovitine

powder was dissolved in MeOH. The elongation of the roots was marked daily and measured

with ImageJ as performed previously (Dissmeyer et al., 2009). Two biological replicates with

each two technical replicates each containing 15 plants were analyzed.

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