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RESEARCH ARTICLE
A. Ludwikow
, M. Szczepaniak1,3 , N. Belter1 , E. Dominiak1 & J. Sadowski1,2
1 Department of Biotechnology, Faculty of Biology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Umultowska 89,
Poland
61-614, Poznan,
Poland
2 Institute of Plant Genetics, Polish Academy of Sciences, Strzeszynska
34, 60-479 Poznan,
3 Present address: International Institute of Molecular and Cell Biology, 4 Ks. Trojdena Street, 02-109 Warsaw, Poland.
Keywords
ABA signalling; Brassica oleracea;
chromosomal mapping; drought; gene
expression; genetic markers; PP2C group A.
Correspondence
J. Sadowski, Department of Biotechnology,
Faculty of Biology, Institute of Molecular
Biology and Biotechnology, Adam Mickiewicz
Abstract
We investigated the expression profiles and genomic organisation of the ABAresponsive genes encoding protein phosphatases 2C (PP2C, group A members)
in Brassica oleracea to better understand their functional and genetic relations.
Gene expression profiling of drought responsive genes in B. oleracea and
Arabidopsis thaliana revealed significant differences in the gene expression
pattern of a key regulator of ABA signalling ABI1 PP2C. This finding prompted
us to study genetic relations within the PP2Cs group A in the Brassica species.
Twenty homologous B. oleracea sequences were identified and characterised
as putative PP2C group A members. Phylogenetic analysis revealed that the
B. oleracea homologues were closely related to the particular members of
the A. thaliana PP2C. The genetic analysis corroborated the presence of two
to three gene copies in B. oleracea in comparison to the nine unique PP2C
genes in the A. thaliana genome. Gene expression analyses showed significant
differences in PP2C gene expression pattern in B. oleracea. Our results indicate
that PP2C-based drought stress signalling in B. oleracea has evolved distinctly.
Different reactions of particular B. oleracea PP2C genes to drought stress and
ABA treatment indicate low conservation of gene expression patterns and
functional divergence between B. oleracea and A. thaliana homologous genes.
Introduction
Studies on the expressions and functions of duplicated
genes in paleopolyploid plant species have shed more
light on the role of polyploidisation and diploidisation
in the structural and functional divergence of genes
involved in stress signalling. Although members of the
protein phosphatase 2C (PP2C) group A are important
stress response switches in Arabidopsis thaliana and Oryza
sativa (Xue et al., 2008; Umezawa et al., 2010), little is
known about the role of duplicated homologous genes
encoding these phosphatases in Brassica crops.
Evolutionary genomics-based studies demonstrated
the presence of additional copies of the ABI1 (ABAInsensitive) PP2C genes in Brassica species as compared
to the Arabidopsis model (both species are paleopolyploids
and Brassicaceae family members). ABI1 homologues were
124
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7
AtABI1
BolC.ABI1
AtABI2
BolC.ABI2
AtHAB1
BolC.HAB1
-1
-2
Drought
ABA 1.5 h
ABA 2.5 h
Drought
ABA 1.5 h
ABA 2.5 h
Drought
ABA 1.5 h
ABA 2.5 h
-3
7
6
5
AtPP2CA
BolC.PP2CA.a
6
5
0
ABA 1.5 h
ABA 2.5 h
0
Drought
ABA 1.5 h
ABA 2.5 h
8
AtHAI3
BolC.HAI3.a
ABA 1.5 h
ABA 2.5 h
AtHAI1
BolC.HAI1.a
Drought
7
AtHAI2
BolC.HAI2.b
AtAHG1
BolC.AHG1.a
BolC.AHG1.b
Drought
AtHAB2
BolC.HAB2.a
BolC.HAB2.b
BolC.HAB2.c
4
3
0
Drought
ABA 1.5 h
ABA 2.5 h
0
Drought
ABA 1.5 h
ABA 2.5 h
Drought
ABA 1.5 h
ABA 2.5 h
Figure 1 Gene expression proles of PP2C group A members in A. thaliana and B. oleracea. The qPCR results indicate the relative expression of PP2C
gene family members in drought or ABA-treated samples. The gene transcript levels were determined using three replicates and were normalised using
18S rRNA. Each quantication was repeated at least twice with similar results. The results were displayed in mean log2 fold change SE (n = 6) of two
independent experiments.
et al.
A. Ludwikow
line A12DHd of the mapping population. The genespecific primer pairs were designed from the Brassica
GSS sequences corresponding to the A. thaliana PP2C
genes. The primer sequences and numbers of the Brassica
GSS clones for individual genes are presented in Table
S4. DNA extraction, non-radioactive labelling, restriction
enzyme digestion and Southern analysis were performed
as described earlier (Babula et al., 2003).
SSR markers
SSR markers were developed from the Brassica GSS
clones or A. thaliana genome sequences with sequence
homology to the A. thaliana ABI1, HAB1 and HAB2. Primer
pairs flanking the SSR motifs for the ABI1, HAB1 and
HAB2 genes were designed from the Brassica sequences
using the following criteria: the size of amplified DNA
fragments were in the range of 200450 bp, the primer
Tm ranged from 55 C to 63 C, and the GC contents were
greater than 40%. Polyacrylamide gel electrophoresis
and silver staining of PCR products were performed
according to the Jamli and Heslop-Harrison protocols
(http://www.le.ac.uk/biology/phh4/acryl.htm).
Map construction
A linkage analysis was achieved using the JoinMap ver.
3.0 software (van Ooijen & Voorrips, 2001). The new
loci were included to the existing linkage groups on
the basis of a minimum logarithm of the odds (LOD)
with a threshold of 3.0 and a maximum recombination
fraction of 0.5. The genetic map distances were indicated
in centiMorgans using the Kosambi mapping function
(Kosambi, 1944). The conserved regions in the B. oleracea
and A. thaliana genomes were defined and based on the
method described earlier (Parkin et al., 2005).
Results
Drought-induced expression profiles for selected B.
oleracea genes via macroarray approach
To investigate the conservation of the drought stress
signalling pathways between a model plant A. thaliana
and its crop relative B. oleracea, a custom-made Arabidopsis cDNA macroarray was produced and used for the
identification of drought-induced gene expression profiles. To this end, within the gene set under study, 39
genes in B. oleracea and 26 genes in A. thaliana were
found to be differentially expressed in drought conditions compared to control conditions. Out of the 39
differentially expressed genes identified in B. oleracea,
127
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A. Ludwikow
et al.
A. Ludwikow
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A. Ludwikow
C03
C01
0
AC-CTCE02
A4
9
10
34410b
pN186E1NP
16
18
20
22
CB10097a
pW239E2
pO173E1
pO43E3
pO52E3
pN52E3NP
23
25
28
29
30
31
32
33
35
36
38
44
pN107J2
pW105E1NM
D10cdb
EIN3a
pN152E1
pN53E2
pO70J1
AC-CACE15
AC-CAAE15
pN97J1
pO118E1
AC-CAAJ01
pR36E3
AC-CATE13
pN129E1NM
pC2E1
Na12C08a
pO168E1
Na10H06a
EIN3c
C8bmk
Ol10F11a
pW116J1
pN3E2
pW200E2
BRMS330a
AC-CAGE13
pW153J1
14
15
16
18
20
21
22
fBolC.ABI1.a
25
26
27
28
29
30
31
32
33
36
37
38
39
A3
40
41
42
45
46
47
48
49
51
52
53
54
50
fBolC.PP2CA.a
55
56
55
56
AC-CACE08
AA-CATE17
58
59
pN121E2
pO12E2
61
pW216J1
63
64
65
66
67
Ni4B10a
AC-CACJ01
pN13E1
R85E1
pO70E1
Na10G10a
57
59
61
62
63
64
65
67
68
70
72
73
74
75
76
78
83
84
86
89
LEW6G7E1
pO111E1
c339E1
pN102E1
pO160J1
pN105E1
AC-CATE03
AA-CATJ07
pN180E3
CB10036a
pW111J1
pW152J1
AC-CACE09
fBolC.ABI2.b
pO170E1
mBN12AJ1
FITO262a
AC-CTAJ01
pR116E1NM
SSR18a
AA-CATJ06
pO98E2
pN22E1
At2g36100b
pW112E1
pN120J2
pW133E2
pO171E2
AC-CATJ02
C05
0
5
4
5
10
12
13
16
18
19
20
21
pN23E3
pO92J1
ACO2b
pN52E1
pO152J1
pO105J1
ACO2a
FITO204a
pW197E1
pW123E1
AC-CTCE06
pN123E2
pW145E1
pN216J1
pW164E1
pW138E1
7
8
9
A1
10
11
12
13
14
fBolC.HAB2.b
22
23
25
fBolC.AHG1.a
pW143J1
pO126E1
pN167E1NM
pN53E3NM
AC-CAAE14
AC-CAGE06
AC-CAGE05
SAM1
pN213J2
pW148E1
SN12574a
pR85E2
Na10D03a
pW172E2
D4amk
pO12E1
Bras069a
pR116E2NP
AA-CATJ01
AC-CACE01
SSR03a
AA-CATJ04
pW142J1
pW169E1
AC-CAGE03
pN148E2
pN207E1
pN20E1
pW188E1
pO10E3NM
pN154E1
AC-CAGE01
AC-CTAE14
AC-CAAE02
pW106E2
pW102J1
pO123E2NP
pN99E2NP
pR6E1
AC-CATE16
pR64E3NP
pO172J1
pO142E3NP
pR93E2NP
pW146J1
pO87J1
Rap2.3
pN47E2NM
pN96E1
CALSSRa
AA-CATJ11
D8emk
pO52E1
flower
AC-CAGJ03
AC-CAGJ02
pN107J3
AC-CACE05
AC-CAAE11
34410a
pW225E1
AC-CTAE01
pO43E1
AC-CACE18
AA-CATE02
C06
0
3
pN21E2
26
27
29
30
31
32
34
37
42
48
49
pO143J1
pN91E1
pO131E1
pO128E1
AC-CATE14
pN47E1
pR54E3NM
pW114E1
pO136E2
AC-CTCE11
AC-CAGJ06
AC-CACJ02
AC-CTCJ02
sN1963a
AC-CAAE16
AA-CATE03
pN148E1
AC-CATE12
AC-CTAJ04
pN215E1
pO153E1
AC-CTCE09
AC-CTCJ04
pN59E2NP
EIN3b
pN91E3
CB10027a
pO123J1
CB10229a
AA-CATE16
AC-CACE02
FITO282b
53
pN113E1
AC-CATE15
pN2J1
pW172E1
56
SN0761a
52
15
16
17
18
19
20
21
22
24
25
27
28
30
40
fBolC.HAB1
CB10211a
45
pO10E2
47
EAT1
0
1
3
4
5
7
8
11
13
14
15
16
17
mBolC.ABI1.b
A4
Ol10H07a
RAB18b
B11amk
SSR_XIC
pO142E1
AC-CACE17
pC2E2
pR36E1NM
AC-CAGJ04
AA-CATE24
pN129E2NM
pN168J1
pR54E4NM
pN87J2
pO52E2
pN53E1NM
pW121E1
pN120E1
AC-CAAE03
AC-CAGE07
AC-CAGE08
pO113E1
pR86E2
pO159J1
pW138J1
pW207E3NM
AC-CAAE06
pW104E2
D10adb
CB10106a
pW188J1
At3g60220a
pW205E1
pR97J1
Ol12D05a
At3g63520a
FITO302a
B8amk
D8dmk
AC-CAAE05
FITO223a
pO143E2
22
A1
CPK6b
D8cmk
mBN72AJ1
pW228E2
pR36E4NM
ACS7
pC2E3
pN107J1
pO118E3
pO29E1
pN97J2
pW104E1
pO59E1
AC-CAGE10
SR0404a
FITO232a
pN216E2
pN101E1
pN64E2
AC-CATE17
24
pN20E2
30
fBolC.PP2CA.b
11
14
15
33
34
36
37
39
40
41
mCa72
51
F7bmk
44
45
49
54
CB10124a
C09
pO43J1
10
20
21
42
49
C08
C07
Ol10F11b
AC-CTCE01
AA-CATE05
AC-CTCE07
AC-CAGJ01
pW101E4NP
AA-CATE04
pR54E1NM
AC-CACE13
pN180E4
pR64E2
pW221E1
CB10234a
AC-CTCJ05
pO124E1
pW103E1
At3g60220b
pW228J1
CB10343a
AC-CATE10
AC-CACE06
pO10E1
CB10010a
AA-CATJ08
pR94E1
pO128E2
pO104E3
AC-CACE12
Ol12E03a
pW197E2
ETR1
mNGA111J1
pS29
pW134J1
Ol10F09a
AC-CATE18
mBNMB4
Na12A02a
AC-CTAJ06
pO104E2
pO169E1
pR113E1
pR3E1
pN86E1
pN184E1
N53E4NP
AC-CAAE12
A5
18
19
20
21
26
27
fBolC.AHG1.b
29
Bras019a
pO131E2
AC-CATE19
pO70E2NP
AC-CTAJ05
pO85J1
AC-CAAE04
AC-CATE01
AC-CAGE11
pR93E1
pO142E2
pw197E3
pO104E1
pW186J1
pO79E1
pW194E1
pW162E1
pR36E2
pO87E2
D8bmk
SSR_WRKY8a
30
34
35
36
38
39
40
41
43
fBolC.HAB2.a
pW123E2
0
2
pN52E2
pN87E3NP
pR116E3
6
7
AC-CTAE04
pO125E1NP
13
14
15
16
21
22
pW137J1
pW167E2
OL11H06a
pN101E2NM
pW114E2
F8amk
RD22a
Ol10A09a
pO119J1
C8cmk
CB10103a
E1bmk
AC-CTAE16
D10bdb
SSR22a
CPK6a
AC-CAGJ05
C8amk
Ol13C03a
pCFH8
pO106E3
AC-CATE21
pO145E1
pN173E2
pW233J1
AC-CTCJ01
FITO247a
pO127E1
pN181E1
pO168J1
pW240E1
pO155E1
pR115E1
pR34E1
pW135E1
AC-CAAE08
AC-CACE04
AA-CATE22
AC-CACE16
AC-CTAJ08
AC-CTAE02
25
26
27
28
30
31
32
33
34
35
A1
36
37
38
39
40
41
45
pN123J1
AC-CATE20
48
49
pN21E1
AA-CATJ03
ACO2c
pO152E2
pN23J1
pO92E2
pN173E1
pN34E1
44
45
48
51
53
CB10139a
70
71
42
50
51
52
56
64
A5
fBolC.ABI2.a
pN180E1
AC-CAAE01
mBN83B1J1
pW212J1
pN105E4NM
pW106E1
pO160E1
pW195J1
pW155E1
pO111E2
pR64E1
LEW6G7E2
pO118J1
pO7E1
D10ddb
pN47E4NM
pN3E1
pW200J1
pW239E1
EIN2
At3g63520b
Figure 2 Genomic localization of the PP2C (group A) genes in B. oleracea. Loci corresponding to six PP2C genes were placed on the B. oleracea genetic
linkage map. These loci were marked in bold. Numbers on the left of each linkage group are Kosambi map distances. The collinearity segments between
A. thaliana and B. oleracea genomes are shown on right. The linkage group and loci were coded according to the nomenclature system recommended
by the Steering Committee for the Multinational Brassica Genome project; f and m letter in front of the locus name designate RFLP and SSR marker assay
types, respectively (stergaard & King, 2008).
Discussion
To address questions with respect to the evolution and
the function of the gene members of PP2Cs group A,
we investigated the genes and their expression profiles
in closely related Brassicaceae species: B. oleracea and A.
thaliana. The most important and unexpected finding
was the discovery of expression divergence among the
group A members of the PP2C gene family in B. oleracea
in relation to well established Arabidopsis orthologues.
In addition, we identified and characterised multiple
gene copies of PP2Cs group A. On the basis of specific
et al.
A. Ludwikow
Ludwikow
et al., 2011). Genome expression data for
B. oleracea is highly limited, notably that there is still
not completed whole-genome sequencing and there is no
et al., 2011).
Brassica transcriptomics database (Ludwikow
However, as the Brassica and Arabidopsis genera diverged
only ca. 17 Mya, exon sequences display a high level of
similarity (>83% at the nucleotide level). Therefore, most
coding sequences available for Arabidopsis in the databases
can be used for initial gene analysis in related crop
species. On the basis of this approach, we observed a most
striking feature of the B. oleracea transcriptional profile:
downregulation of the ABI1 homologue under drought
conditions. This result suggested that BolC.ABI1 is not
essential for the development of drought stress response
or, on the contrary, may prevent the negative feedback of
et al.
A. Ludwikow
Acknowledgements
This work was supported by the Polish Committee for Scientific Research Grants PBZ-MNiSW-2/3/2006/19/IGR/4,
MNiSW-2/3/2006/19/IGR/3 and 682/N-COST/2010/0.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Fig. S1. Technical and biological quality assessment
of macroarray experiment. (A,B) Correlation between
technical replicates. Scater plots comparing normalized
signal intensities of the technical replicates for A.
thaliana (A) and B. oleracea (B) control sample pairs.
Pearson correlation coeficient are presented on the
graph; (C) Pearson correlation between the biological
replicates. Mean Pearson correlation (R) was calculated
for normalized signal intensities of all three pairs of
biological replicates; (D) Confirmation rate of the cDNA
nylon array ratios with qPCR. Scatter plot of the log2 of
the relative change for ABI1 and ERF1 genes in A. thaliana
and B. oleracea as determined by qPCR and the cDNA
nylon array analysis. The RAB18 gene expression was
134
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