IEF
pI
3
10
kDa
1
100
2
3
30
55
26
28
12 13
14
10
72
49
9
45
4
5 1617
18
21
22
8
6 7 46
50
11
15
18
19
23 20 27
51
53
24 25 26
31
48
SDS -PAGE
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
29
34
35
35
33
32
38
36
41
17
37
3
39
52
40
10
43
44 45
42
46
47
Supplemental Figure 1. Two-dimensional separation of the highly enriched transcriptionally
active chromosome (TAC-II) isolated from spinach chloroplasts. After separation by 2-DE
Coomassie Brilliant Blue staining revealed a pattern consisting of 85 spots. All visualized
spots on the gel were cut out. After trypsin digestion the corresponding peptides were
analyzed with MALDI-TOF. Proteins belonging to 53 spots could be identified and are listed
in Supplemental Data Set 1.
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
Supplemental data. Melonek et al. (2012). Plant Cell.
SWIB-5:GFP
MitoTracker
SWIB-5:GFP+MitoTracker
SWIB-6:GFP
MitoTracker
SWIB-6:GFP+MitoTracker
Supplemental Figure 2. MitoTracker Orange labeling of onion epidermal cells transiently expressing
SWIB-5:GFP and SWIB-6:GFP constructs. White arrows indicate plastids. Scale bar: 6 µm.
Transient transformation of onion epidermal cells was performed as described (Krause et al., 2005).
After 18 h of incubation at 22 °C in darkness, the onion cells were labeled with MitoTracker Orange
(Invitrogen GmbH) and analyzed by laser scanning microscopy (Leica TCS SP5, Leica Microsystems
CMS GmbH). Images were scanned sequentially. For MitoTracker Orange the excitation of 543 nm and
emission ranging from 570 to 620 nm and for GFP excitation of 488 nm and emission ranging form 500
to 540 nm were used.
Krause, K., Kilbienski, I., Mulisch, M., Rödiger, A., Schäfer, A., and Krupinska, K. (2005). DNA-binding proteins of the
Whirly family in Arabidopsis thaliana are targeted to the organelles. FEBS Lett. 579: 3707-3712.
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
B
A
SWIB-4 KKPAAKPKA-KAKPKPKAKSDSP-AK 24
HistoneH1 KKPAAKPKATKAKPKPKPKTVAPKAK 26
CND41
KKKDKKSSN-KKKSVKDSKANLP-AQ 24
Supplemental Figure 3. Sequence alignment of the histone H1 motif of the SWIB-4 protein
and in vitro import assays with pea chloroplasts. (A) Alignment of the lysine-rich region of
SWIB-4 with partial sequence of histone H1 from tobacco and an analogous lysine-rich region
of the CND41 (chloroplast nucleoid DNA binding protein 41 kDa) protein. (B) The translocation
of the SWIB-4 protein into chloroplasts shown by in vitro import of the radiolabelled in vitro
translation product with isolated pea chloroplasts. While the precursor protein was sensitive
towards thermolysin, the mature SWIB-4 protein was protected from thermolysin treatment (+).
Plant material
Pea (Pisum sativum L. cv. Kleine Rheinländerin) plants used for preparation of chloroplasts for
in vitro import assays were grown on vermiculite for 10-14 days with a 16 hour photoperiod.
In vitro import assays with pea chloroplasts
For in vitro import assays, the complete coding sequence of the At3g03590 gene was cloned
behind the T7 RNA polymerase promoter into a modified pBluescript vector containing a sequence
encoding a methionine/serine tag (MSMSMS) at C-terminal end of the protein. Import assays
were performed with isolated pea chloroplasts as described (Krause et al., 2005).
Krause, K., Kilbienski, I., Mulisch, M., Rödiger, A., Schäfer, A., and Krupinska, K. (2005). DNA-binding proteins
of the Whirly family in Arabidopsis thaliana are targeted to the organelles. FEBS Lett. 579: 3707-3712.
Murakami, S., Kondo Y., Nakano, T., Sato, F. (2000). Protease activity of CND41, a chloroplast nucleoid DNA-binding
protein, isolated from cultured tobacco cells. FEBS Lett. 468: 15-18.
Nakano, T., Murakami, S., Shoji, T., Yoshida, S., Yamada, S., Sato, F. (1997). A novel protein with DNA-binding activity
from tobacco chloroplast nucleoids. Plant Cell 9: 1673-1682.
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
TP
N
C
M
S
SWIB-4
PsbE
Pol I
CBB
Supplemental Figure 4. Immunological detection of SWIB-4 in chloroplasts and nuclei prepared
from leaves of Arabidopsis thaliana. Protein fractions isolated from Arabidopsis rosette leaves:
total leaf protein (TP), nuclei (N), chloroplast (C), membranes (M) and stroma (S) were resolved
by SDS-PAGE and transferred onto nitrocellulose membrane. The peptide antibody directed
towards the SWIB-4 protein recognized two proteins of 19 and 20 kDa in the nuclei and two of 17
and 18 kDa molecular weights in chloroplasts. For control of the protein fractions’ purity
immunoassays were performed with antibodies directed towards cytochrome b559 (PsbE) and the subunit
A12.2 of nuclear RNA polymerase I (Pol I). Equal loading of the fractions is shown by staining with
Coomassie Brilliant Blue (CBB).
Plant material
Arabidopsis thaliana wild type Columbia (Col-0) plants were cultivated at 22°C in soil under long
day conditions (16 h light/8 h dark). For isolation of protein extracts rossette leaves were used .
Protein isolation and immunoblot analysis
For extraction of total proteins, Arabidopsis rosette leaves were ground in liquid nitrogen and
nuclei were isolated as described (Desveaux et al., 2004). For an efficient separation of proteins
having low molecular weight, 16 % (w/v) polyacrylamide gels were used and proteins were blotted
onto nitrocellulose (Schleicher & Schuell). The polyclonal anti SWIB-4 antiserum raised in rabbit
was diluted 1:1000 with buffer consisting of 50 mM Tris/HCl pH 7.4 and 150 mM NaCl. As controls,
immunoblot assays with antibodies directed towards the cytochrome b559 apoprotein A of 9.5 kDa
(Vallon et al, 1987) as well as the polymerase I subunit A12.2 from rabbit (Agrisera AB) were used.
Immunoblotting and detection was done as described (Grabowski et al., 2008).
Desveaux, D., Subramanian, R., Després, C., Mess, J., Lévesque, C., Fobert, P., Dangl, J. (2004). A Whirly transcription
factor is required for salicylic acid-dependent disease resistance in Arabidopsis. Dev. Cell 6: 229-240.
Grabowski, E., Miao, Y., Mulisch, M., Krupinska, K. (2008). Single-stranded DNA-binding protein Whirly1 in barley leaves is
located in chloroplasts and nuclei of the same cell. Plant Physiol. 147: 1800-1804
Vallon, O., Hoyer-Hansen, G., Simpson, D.J. (1987). Photosystem II and cytochrome b-559 in the stroma lamellae of barley
chloroplasts. Carlsberg Res. Commun. 52: 405-421
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
A
swib-4-1
swib-4-3
LBb1-3
LB1
3’UTR
5’UTR
ATG
STOP
o2588
100 bp
swib4-2
1
2
1/- 4-1/4
ib- ibCol-0 sw sw
2
1
2/- 4-2/4
ib- ibCol-0 sw sw
RP+LP
RP+LB1
1
2
3/- 4-3/4
ib- ibCol-0 sw sw
RP+o2588
LP+RP
RP+LP
RP+LBb1-3
B
3d
5d
7d
4.5
swib-4-1/-1
swib-4-1/-1
cotyledon area (mm2)
Col-0
4
3.5
*
3
2.5
2
1.5
*
1
0.5
0
3d
5d
7d
4.5
5d
7d
swib-4-1/-2
cotyledon area (mm2)
swib-4-1/-2
Col-0
4
3.5
3
*
2.5
2
1.5
1
*
0.5
0
5d
7d
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
3d
5d
7d
4.5
swib-4-2/-1
cotyledon area (mm2)
Col-0
4
3.5
3
2
1.5
*
0.5
0
5d
7d
4.5
5d
7d
swib-4-2/-2
Col-0
4
swib-4-2/-2
cotyledon area (mm2)
3.5
3
*
2.5
2
*
1.5
1
0.5
0
3d
5d
7d
4.5
5d
7d
swib-4-3/-1
swib-4-3/-1
cotyledon area (mm2)
Col-0
4
3.5
3
2.5
2
1.5
1
0.5
0
3d
5d
7d
4.5
5d
7d
swib-4-3/-1
4
cotyledon area (mm2)
Col-0
*
2.5
1
3d
swib-4-3/-2
swib-4-2/-1
3.5
3
2.5
2
1.5
1
0.5
0
5d
7d
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
C
25
SWIB-4
22
relative change of expression (n)
20
15
11
10
8
5
5
1
0.31
0
Col-0
0.31
swib-4-1/-1 swib-4-1/-2 swib-4-2/-1 swib-4-2/-2 swib-4-3/-1 swib-4-3/-2
5d
100
64
48
61
63
101
102
7d
100
77
60
66
67
93
91
Supplemental Figure 5. Molecular characterization of SWIB-4 T-DNA insertion mutants.
(A) Schematic diagram of the SWIB-4 gene showing the location of the T-DNA insertions
in the mutant lines swib-4-1 (SAIL_1156_C12), swib-4-2 (GABI_420_H09), swib-4-3
(SALK_053441.46.80.x). All genotypes are in the Columbia ecotype. Protein-coding exons
are represented by black boxes and untranslated regions by white boxes, the only intron
is represented by a thin line between the black boxes. Positions of T-DNA insertions are
indicated. ATG, translation initation codon; Stop, translation termination codon. Genomic
DNA was extracted from wild type and mutant plants (swib-4-1, swib-4-2, swib-4-3) and
analyzed by PCR. Appropriate T-DNA and SWIB-4-gene-specific primers were employed
in order to estimate the homo- and heterozygous plants. (B) Phenotype analysis of the
SWIB-4 mutant lines. The seeds were sterilised as described in Aronsson & Jarvis (2011),
sown on MS-plates supplemented with 3 % sucrose and stratificated at 4°C for 3 days.
The seedlings were grown in long-day conditions (16h light/8h dark) at 40 µmol m-2 s-1 light
intensity. Cotyledon areas of 5- and 7-day-old (5d, 7d) seedlings (n ≥ 20) were measured
using the Cell^F Imaging software for Life Science Microscopy (Olympus). Statistically
significant differences between mutant lines and the wild type are indicated by asterisks
(Student’s t test, P<0.0001). Scale bar: 10 mm (C) Levels of SWIB-4 mRNA in seedlings
of wild type (Col-0), swib-4-1, swib-4-2, swib-4-3 as determined by qRT-PCR. RNA was
extracted from whole seedlings and used in SYBR-Green real-time PCR analysis employing
the QuantiFast SYBR Green PCR Kit (Qiagen) according to the manufacturer’s instructions.
Primers used for these analyses are listed in Supplemental Table 1. Each reaction was
repeated three times. Data analysis was accomplished by the 7300 System software (Applied
Biosystems). Relative quantification of transcript levels was performed using the “Delta-delta
CT method” as recommended by PE Applied Biosystems (Perkin Elmer). The table below
the graph again shows relative changes in the area of cotyledons of mutant seedlings
measured after 5 days (5d) and 7 days (7d) of growth as shown in B. The area of
cotyledons from wild-type seedlings was set at 100%. The swib-4-1 and swib-4-2 mutants
having enhanced levels of the SWIB-4 transcript showed delayed post-germination
development compared to the wild type.
Aronsson, H., Jarvis, R.P. (2011) Rapid isolation of Arabidopsis chloroplasts and their use for in vitro protein import
assays. Methods Mol. Biol. 774: 281-305.
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
Supplemental data. Melonek et al. (2012). Plant Cell.
MEDIUM (=IQR)
SWIB-3
SWIB-4
SWIB-6
13
12.5
12
11.5
11
10.5
10
9.5
9
8.5
8
LOW
Level of expression (signal intensity on Array ATH1: 22k array)
HIGH
SWIB-2
7.5
7
6.5
6
5.5
5
Number of samples:
299
247
591
862
225
90
s
ue
liq
s
si
ue
e
liq
ur
si
at
d
m
an er
s
w
er
w
flo
d
flo
pe
lo
ve
er
w
de
flo
g
un
yo
tte
se
ng
lti
ro
d
bo
pe
lo
ve
tte
se
de
ro
g
un
yo
d
g
lin see
ed
d
se ate
in
rm
ge
Stage of development:
261 1734 630
Supplemental Figure 6. Development-dependent expression of genes encoding SWIB-2
to -6 proteins from the group 4 of the SWIB domain family of Arabidopsis. Expression
values are based on Genevestigator. All 22k arrays from Arabidopsis were selected,
resulting in the maximum number of annotated development categories and the maximum
number of arrays per category. The left axis represents the change of gene expression using
base alogarithm. The numbers on the bottom axis represent the number of arrays per
developmental stage.
Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., Gruissem, W. (2004). GENEVESTIGATOR.
Arabidopsis microarray database and analysis toolbox. Plant Physiol. 136: 2621-2632
Supplemental Data. (2012). Melonek et al. Plant Cell 10.1105/tpc.112.099721
Supplemental Table 1. List of primers used for molecular characterization of swib-4 mutants.
T-DNA line
SAIL_1156_C12
GABI_420_H09
SALK_053441.46.80.x
mutant
name
swib4-1
swib4-2
swib4-3
Primers used for qRT-PCR analysis
Actin
SWIB-4
LP-primer sequence (5’-3’)
CTTAGTGGTTTGATCGGCTTG
CTTAGTGGTTTGATCGGCTTG
GAGAAGCAGCCATGAATTTTG
RP-primer sequence (5’-3’)
TCGTGGGACTTGATGTAGGTC
ACCTAATTTTTCCGGCAAATG
TACCACATCGAGCCTCTCAAC
T-DNA-specific-primer sequence (5’-3’)
GCCTTTTCAGAAATGGATAAATAGCCTTGCTTCC
CGCCAGGGTTTTCCCAGTCACGACG
ATTTTGCCGATTTCGGAAC
Forward (5’-3’)
Reverse (5’-3’)
AAGCTCTCCTTTGTTGCTGTT
AGCCACCTCTTCTTTGGTTT
GACTTCTGGGCATCTGAATCT
TCGTGGGACTTGATGTAGGT