Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

Expression proles and genomic organisation of group A

protein phosphatase 2C genes in Brassica oleracea
2,
1, , D. Babula-Skowronska

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

University, Umultowska 89, 61-614, Poznan,

Poland. Email: jsad@amu.edu.pl

These authors contributed equally to the

work.

Received: 29 November 2012; revised version

accepted: 11 May 2013.
doi:10.1111/aab.12039

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

mapped in the Brassica genomes and subsequent analyses

provided evidence of conserved gene structures among
these homologues (Quiros et al., 2001). Despite these
advances we are far from understanding the function of
gene paralogues encoding PP2C group A in Brassicas. It
is well established that Brassica species display complex
genome organisation, reflecting both ancient and more
recent polyploid origins (Helentjaris et al., 1988), one
of the consequences of the triplicated Brassica genome
structure with respect to that of A. thaliana and the
presence of an increased number of duplicated genes
(ONeill & Bancroft, 2000; Babula et al., 2003; Parkin
et al., 2005; Ziolkowski et al., 2006). The genomic
duplications and accumulation of mutations, including
deletions, insertions and inversions, can cause divergence
of gene function. Thus, resultant paralogues may have
become non-functional, contributing to species-specific

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A. Ludwikow

differences and impacting on the development of the

stress response (Liu & Adams, 2007). The occurrence of
numerous Brassica gene homologues makes unravelling
their roles in cellular processes challenging.
The induction of plant stress responses depends on the
perception and subsequent activation of numerous signal
transduction cascades which transmit and amplify stress
signals to the nucleus (Laxalt & Munnik, 2002; Pitzschke
et al., 2009; Pasqualini et al., 2012; Suzuki et al., 2012).
Drought is a major growth limiting factor for crop plants
(Cramer et al., 2011) and the complexity of the drought
stress signalling network was illustrated in model plants
(Xiong et al., 2002; Huang et al., 2012). Recent findings demonstrate that drought conditions activate several
intracellular cascades (Saijo et al., 2000; Hong et al., 2008;
Jubany-Mari et al., 2010). A major player in drought
stress response, the phytohormone ABA, recruits phosphorylation cascades for the generation and transmission
of endogenous signals (Saijo et al., 2000; Fujita et al.,
2009). Protein serine/threonine (ser/thr) phosphorylation/dephosphorylation events lead to essential reversible
changes in the ABA signal transduction network, which
occur through proteinprotein interaction among signalling molecules (Zhu et al., 2007; Park et al., 2009;
Nishimura et al., 2010).
In this study, we investigated the expression profile
and genomic organisation of PP2C group A genes
in B. oleracea to gain an insight into ABA signalling
network conservation in a model plant A. thaliana and
its crop relative. Macroarray analysis of drought-treated
B. oleracea plants identified genes that are differentially
expressed compared to drought stress response genes in
A. thaliana. The most striking change in gene expression
was found for BolC.ABI1 PP2C, which was downregulated
upon drought. This difference encouraged us to search
for functional homologues of group A PP2C in the
B. oleracea genome. Homologous gene sequences have
been identified from available B. oleracea sequence
data and supported by restriction fragment length
polymorphism (RFLP). In addition, simple sequence
repeat (SSR) analysis was used to confirm intergenomic
conservation of selected PP2C in A. thaliana and B. oleracea.
Generally, the presence of some PP2Cs encoding genes
in conserved regions of the A. thaliana and B. oleracea
genomes support their conservation at a functional
level. Expression profiles of putative B. oleracea PP2Cs
were analysed after drought and ABA stimulation to
experimentally verify biological function. We discuss
similarities and differences between transcription profiles
and genomic organisation, noting the importance of
specific members of group A PP2Cs in plant responses to
water deficit.

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PP2C group A in Brassica oleracea

Materials and methods

Plant growth and stress treatments
B. oleracea ssp. alboglabra line A12DH (Warwick Crop
Centre, The University of Warwick, Wellesbourne, UK)
and Arabidopsis thaliana Col-0 (Arabidopsis Biological
Resources Center, The Ohio State University, Columbus,
USA) were sterilised with bleach, chilled for 4 days
then transferred to the growth chamber with a 16 h
photoperiod (300 mol m 2 s 1 photon density, 75%
relative humidity). Three to five plants were grown per
pot for 21 days. For drought stress treatment, plants
were not watered for 5 days until the relative water
content (RWC) of the leaves reached an average of
45 15%. Control plants were watered regularly. RWC
measurements were made from three to five replicates.
For ABA treatment, plants were sprayed with mock or
100 M ABA solution. Plant material (leaf tissue) was
collected at various time points (Fig. 1).
Mapping population
The subset of 50 from 206 dihaploid (DH) lines derived
from a cross between Chinese broccoli (A12DHd; B. oleracea var. alboglabra) and calabrese (GD33DH; B. oleracea
var. italica) by Bohuon et al. (1996) was selected to localise
the PP2C group A gene homologues and additional markers on the previously published map (Sebastian et al.,
2000). The seeds of these lines were kindly provided
by Graham Teakle from the Warwick Crop Centre, The
University of Warwick, Wellesbourne, UK.
Macroarray experiment
Total RNA was isolated from the leaves of five plants
in triplicate using a Qiagen RNA isolation kit. The
corresponding B. oleracea and A. thaliana plant samples
were pooled and 10 g of total RNA was used for 33 P-CTP
labelling with LabelStar Array Kits (Qiagen, Wroclaw,
Poland) according to the manufacturers instructions.
Purified radiolabelled cDNA was used for hybridisation
with custom nylon membrane arrays. To minimise nonspecific binding, labelled DNA in 4SSC, 0.1% SDS was
mixed with 0.1 mg mL 1 poly(dA) DNA, denatured at
95 C for 5 min and hybridised at 65 C for 1 h. The
prehybridisation step was carried out at 65 C for 3 h
in 15 mL of Church buffer (250 mM Na2 HPO4 pH 7.2,
1 mM EDTA, 7% SDS and 1% BSA) with 10 mg mL 1
Salmon sperm DNA. Hybridisation using denatured
labelled cDNA was performed at 65 C for 18 h. After
washing (once in 2SSC, 0.1% SDS, twice in 0.5 SSC,
0.1% SDS and once at 0.1SSC, 0.1% SDS each at 65 C)
membranes were exposed to storage screens for 48 h
125

 et al.
A. Ludwikow

PP2C group A in Brassica oleracea

Log2 Fold change

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

Log2 Fold change

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

Log2 Fold change

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.

and than scanned at 200 m resolution (PhosphorImager

Fuji, Poland) into the supplied Multi Gauge V2.2
Software. The acquired raw data was normalised to the
mean hybridisation signal for ACT2 and then analysed
with ImageQuant TL software. The average expression
values for each condition were calculated from three
independent biological replicates. Control samples were
supplemented by technical replicates, totalling four arrays
per condition. From independent nylon filter arrays only
replicates showing signal intensity at twofold above the
background value were considered present. Background
adjusted signal intensities were further normalised and
analysed to identify significant differences in gene
expression between drought-treated and control samples.
Microsoft Excel was used to manage the macroarray data.
The dataset was submitted in MIAME-compliant format to
the Gene Expression Omnibus (GEO accession number:
GSE39753). The quality of the biological material and
126

spotting as well as the independent confirmation of results

using qPCR are presented in Fig. S1. The performed
analyses showed good quality and reproducibility of the
macroarray experiment.
Nomenclature
The names for newly mapped SSR, RFLP markers and
genes were coded according standard nomenclature
systems for the Brassica markers and genes (stergaard &
King, 2008).
RFLP markers
The source of RFLP probes were B. oleracea genomic
sequences with homology to members of PP2C group A in
A. thaliana: ABI1 (At4g26080), ABI2 (At5g57050), HAB1
(At1g72770), HAB2 (At1g17550), AHG1 (At5g51760)
and PP2CA (At3g11410), developed from the parental

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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).

PP2C group A in Brassica oleracea

detection system (Applied Biosystems, Foster City, CA,

USA). A 15 L PCR reaction contained 2 L of diluted
cDNA, 200 nM of each gene-specific primer designed
with PrimerExpress software and 7.5 L of SYBR Green
master mix. The qRT-PCR amplifications were carried
out using the following thermal profile: 50 C for 2 min,
95 C for 10 min, followed by 40 cycles of 95 C for 15 s
and 60 C for 1 min. Data analysis was performed using
SDS 2.2.1 software (Applied Biosystems). As a reference,
18S rRNA and ACT2 were used. Table S5 includes the
primer sequences used for the qPCR in this study. The
serial dilutions of genomic DNA (from 100 to 10 000
copies) were used to set up the calibration curve. The
results presented here are the mean of the relative
gene expression ratios [log2 fold change scale; FC = log2
(ratio/ratio)] of two independent experiments (n = 6).
After the PCR, a melting curve was generated to test the
amplicon specificity.
Phylogenetic analysis
Assignment of the putative B. oleracea PP2Cs was
performed using the NCBI database by a BLAST search
for A. thaliana PP2Cs homologues. Reciprocal BlastX
searches were performed to confirm the best homologue
match against the A. thaliana sequences (TAIR database).
The retrieved nucleic acid sequences were manually
adjusted to identify protein coding regions and converted
to the corresponding amino acid sequences using ORF
Finder (NCBI). A rooted neighbour-joining phylogenetic
tree was constructed using the ClustalX version 2.0
and displayed using the MEGA 5 version program. The
resulting tree was verified by bootstrapping using 1000
bootstrap replicates. Only sequences with a significant
alignment (an amino acid alignment of 224 residues
with at least 70% coverage, Appendix S1) and including
phosphatase catalytic domain sequences were included in
the analysis.

Quantitative real-time PCR

Quantitative real-time PCR (qPCR) was performed as
et al., 2009). The leaves
described previously (Ludwikow
of three biological replicates from five plants each were
pooled for the isolation of total RNA using TRIZOL reagent
(Invitrogen, Carlsbad, CA, USA); 3 g of total RNA was
then digested with RQ DNase (Promega, Madison, WI,
USA) and half the volume of DNase-treated aliquot was
used for first-strand cDNA synthesis using a First Strand
cDNA Synthesis Kit (Fermentas, Gdansk, Poland). The
second half of DNase-treated aliquot was used as the
DNAse treatment control. After 10-fold dilution of cDNA
samples, qPCR reactions for each sample were set up in
triplicate and run twice on separate plates. PCR reactions
were conducted in an ABI PRISM 7900 HT sequence

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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,
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PP2C group A in Brassica oleracea

31 transcripts increased and eight transcripts decreased in

abundance upon drought stress (Table S1). In A. thaliana
seven genes were induced by drought and 19 were
downregulated (Table S2). Nine gene transcripts (WRKY4
and WRKY11 members of WRKY transcription factors
family, FAB2 a stearoyl-ACP desaturase, SUS1 a protein with sucrose synthase activity, CSD2 copper/zinc
superoxide dismutase 2, GPX1 glutathione peroxidase 1,
GBF1 G-box binding factor 1, ABI1 ABA insensitive 1,
LSD1 lesion simulating disease 1) showed significantly
different expression in B. oleracea and A. thaliana. Among
these genes, FAB2, GPX, ABI1 and LSD1 genes were oppositely regulated in the two species. Interestingly, while
FAB2, GPX1 and LSD1 gene expression was induced, the
ABI1 (PP2C from group A) gene was downregulated in
B. oleracea. This result seems to indicate that ABI1 was
not preferentially expressed in response to drought stress
in B. oleracea. This in turn suggests that ABI1 may have
a limited or different role in the regulation of drought
stress response in B. oleracea. Because ABA-induced genes
and associated gene networks (ABI1 is the key negative
regulator of ABA signalling) are particularly important for
crop improvement, we focused our further attention on
an analysis of the functional divergence of PP2Cs group
A encoding genes in B. oleracea and A. thaliana.
Identification of the PP2C group A gene homologues
in B. oleracea
To date, the complete genome sequence of B. oleracea
is not known. To identify the homologous gene copies
of a particular group A member from B. oleracea, we
performed a BLASTN search against available databases
using full coding sequences from Arabidopsis. Among the
retrieved sequences we identified a considerable group of
genomic survey sequences (GSS), sequences that shared
significant sequence homology with particular PP2Cs in
Arabidopsis. In addition, the extracted EST sequences
provided evidence that at least seven corresponding
genes are expressed in B. oleracea. These are BolC.ABI1,
BolC.ABI2, BolC.HAB2, BolC.HAI3, BolC.HAI1, BolC.PP2CA
and BolC.AHG1; the gene names follow the gene
nomenclature system proposed for Brassica genus by
stergaard and King (2008). A reciprocal analysis was
performed to confirm that identified entries indeed have
the best match to the Arabidopsis sequence. Afterwards,
redundant sequences were removed from the initial
dataset. Gene names and the corresponding accession
numbers are listed in Table S3.
To verify computational gene number predictions
and to identify gene specific copies of particular PP2C,
we aligned the identified B. oleracea hits along with
corresponding Arabidopsis genes and coding sequences.
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et al.
A. Ludwikow

In our analysis of B. oleracea, 20 sequences demonstrate

informative similarity to members of the PP2C group
A. The key player of ABA signalling, i.e. BolC.HAB1
was represented by a single hit per accession. In
contrast, BolC.HAB2, BolC.HAI3, BolC.HAI1, BolC.PP2CA
and BolC.AHG1 were found to hit multiple sequences. The
nucleotide sequence alignments of putative BolC.ABI1,
BolC.HAB1, BolC.HAB2, BolC.HAI3, BolC.HAI2, BolC.HAI1
and BolC.AHG1 sequences revealed significant divergence,
which suggests that multiple copies of these genes exist
in the B. oleracea genome.
Gene expression profile of PP2C group A members in
B. oleracea
To explore the relationships between sequence homology
and expression pattern for B. oleracea and A. thaliana,
PP2C genes transcription levels were analysed at the
early stage of drought stress. Because 12 out of the 20
putative BolC.PP2Cs revealed a significantly high sequence
divergence, specific primers were designed and individual
gene expression patterns were verified using qPCR.
To confirm the specificity of each primer pair for the
corresponding BolC.PP2C gene, the amplified product was
verified by sequencing. Primer pairs designed to amplify
single loci AtABI1 and AtABI2 were used to examine
the cumulative expression of putative BolC.ABI1 and
BolC.ABI2 genes.
As expected, PP2C group A genes exhibited a
preferential expression in drought-stressed A. thaliana
(Fig. 1). In contrast, only 6 of the 20 BolC.PP2C
genes under scrutiny were significantly upregulated by
drought stress. Indeed, AtABI2 and putative BolC.ABI2
as well as AtHAB2 and BolC.HAB2.a showed similar
induction patterns, while BolC.ABI1 gene expression was
suppressed by drought stress confirming macroarray
results in independent experiments. The expression
level of BolC.HAB1 and BolC.PP2CA.a was significantly
higher under drought conditions than that reported
in Arabidopsis. Furthermore, all identified BolC.HAB2
isoforms were transcriptionally active in drought stress
conditions. These results suggest that B. oleracea has
advanced signalling pathways to upregulate stress
response genes. The transcription level of BolC.AHG1.a,
BolC.HAI3.a-c, BolC.HAI2.b and BolC.HAI1a was hardly
detectable under drought conditions or in the non-treated
control.
Finally, we tested if the drought-induced expression
of BolC.PP2Cs is ABA-regulated. The results showed that
only three of the PP2C genes studied (BolC.ABI1, putative
BolC.ABI2 and BolC.PP2CA.a) were upregulated by ABA at
1.5 h and/or 2.5 h time points. These results are consistent
with a previous report (Xue et al., 2008) that showed

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that the expression of PP2C genes involved in the ABA

signalling network is strongly correlated. The other genes
were not significantly affected by ABA treatment. This
indicates the temporal differences in the expression of
BolC.PP2Cs. Note that AtAHG1 and AtHAI3 genes were
also not regulated by ABA under these experimental
conditions. It seems that BolC.HAB1, BolC.HAB2.ac,
BolC.HAI1.a and BolC.HAI2ab isoforms have lost their
function in early ABA signalling.
Genomic organisation of the PP2C group A gene
homologues in B. oleracea
On the basis of the gene expression profile obtained for B.
oleracea, six of nine PP2C group A members were selected
to determine their gene chromosomal organisation in
this species. First, gene-specific primer pairs within the
identified Brassica GSSs with the sequence homology to
AtABI1, AtABI2, AtHAB1, AtHAB2, AtAHG1 and AtPP2CA
were designed. The amplified genomic fragments ranging
in size from 300 bp to 500 bp were isolated, cloned and
then used as RFLP probes in the B. oleracea mapping
population developed by Sebastian et al. (2000). The
DH lines of the mapping population were screened for
polymorphisms (RFLP) to establish their usefulness in the
mapping process. Due to the high nucleotide sequence
homology (8392%) between analysed members of the
group, the specificity of individual genomic fragments was
verified by sequencing and later confirmed by mapping to
regions of the sequence collinearity in the A. thaliana and
B. oleracea genomes. Additionally, the gene-specific loci
were detected with a marker system known as SSR. The
SSR patterns were found in the A. thaliana ABI1, HAB1 and
HAB2 genes as well as in the corresponding Brassica GSSs.
Altogether, the 10 PP2C loci were detected by
mapping analysis based on Southern hybridisation using
gene-specific probes (Fig. 2). In addition, single SSR
corresponding to ABI1 was polymorphic in the analysed
mapping population and hence was assigned to the B.
oleracea map as well (see Fig. 2, locus mBolC.ABI1.b on the
top of the C07 linkage group; m = microsatellite). Because
of the high sequence homology among the members
of the PP2C gene family, hybridisation patterns were
compared for individual genes to exclude the possibility of
their cross-hybridisation in the Brassica genetic mapping.
The detected loci were distributed among seven out of
nine linkage groups including two loci of ABI1 on the
C01 and C07 linkage groups, two loci of ABI2 on the
C03 and C09 linkage groups, one locus of HAB1 on the
C06 linkage group, two loci of HAB2 on the C05 and
C08 linkage groups, two loci of AHG1 on the C03 and
C07 linkage groups and two loci of PP2CA on the C01
and C07 linkage groups (Fig. 2). The data suggested the

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PP2C group A in Brassica oleracea

presence of three to four homologues of these genes

in the B. oleracea genome contrary to the single copy
genes in the A. thaliana genome. An exception is HAB1,
which appeared as a single-copy gene in both genomes.
However, due to the limited level of polymorphism, not
all loci of the remaining genes could be identified and
placed on the genetic map.
Comparative analysis of loci order along the linkage
groups of B. oleracea and the chromosomes of A. thaliana
revealed the presence of 8 out of the 10 loci in collinear
regions of both species (Fig. 2). The results of the analysis
also showed that both mapped copies for the ABI1 gene
located on the C01 and C07 linkage groups of B. oleracea
were detected in the collinear regions of the A. thaliana
A4 chromosome containing five and four loci, each. Both
collinear regions include the conserved organisation of
two loci, ABI1 and pN107, which proves their common
evolutionary origin. Furthermore, one of two copies of
the ABI2 gene located on the B. oleracea C09 linkage
group was also detected in the collinear region of the
A. thaliana A5 chromosome, including nine loci with the
length of 5 cM. A unique copy of the HAB1 gene mapped
in the C06 linkage group of B. oleracea was found in the
collinear region of the A. thaliana A1 chromosome with
six loci covering a length of 26 cM.
Overall, the linkage pattern obtained from the
B. oleracea genetic map indicates that PP2Cs under
study are encoded by duplicated genes (homologues)
dispersed in the genome. Most of them were proved in
qPCR expression profiling to be transcriptionally active
(BolC.ABI1.a from the linkage group C01; BolC.HAB2.a
from the linkage group C08; BolC.HAB2.b from the linkage
group C05; BolC.HAB1 and from the linkage group C06).
This observation was in agreement with our earlier report
on the expression of B. oleracea duplicated genes involved
in the ethylene signalling (Babula et al., 2006).
Phylogenetic analysis
Identification of multiple copies of group A PP2C
members raise questions about their phylogeny. In view
of the fact that we have identified the partial sequences
of PP2Cs, limited to the PP2C domain, we were interested
in whether the identified PP2Cs are truly orthologous.
To answer this a phylogenetic tree was inferred by the
Neighbour-Joining method from the predicted amino
acid sequences. The phylogenetic analysis was limited
to the overlapping fragments of the phosphatase domain.
This part of the analysis provided a reasonable bootstrap
estimation of the tree topology. The results presented
here show that the B. oleracea PP2Cs are grouped with
corresponding Arabidopsis sequences to form distinct
clades with specific bootstrap support (Fig. 3). This
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A. Ludwikow

PP2C group A in Brassica oleracea

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).

indicates that the identified sequences are closely related

to the members of the PP2C group A in Arabidopsis. The
generated tree recovered two major clades of sequences.
The first clade groups together with the sister clades of
PP2CA, AHG1 and HAI2 sequences with a very high
bootstrap (95%), suggesting that all of the corresponding
loci are orthologous to each other and descended from
a common ancestral locus. The second clade corresponds
to the ABI1-ABI2-HAB1-HAB2 branch that receives
relatively strong support at 99%. The topology relations
speak in favour of the orthologous origin of AtHAB1
and BolC.HAB1.a (98% bootstrap). The same can be
concluded for AtHAB2 and BolC.HAB2.a. The ABI1/2
subgroup with 99% bootstrap values includes AtABI1,
AtABI2 and BolC.ABI1.ac sequences. BolC.ABI1.ac
are more closely related to AtABI1 (89% bootstrap) but
130

non-orthologous. Finally, the remaining AtABI2 should

be considered as apparently an independent locus, which
was not expected.

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

Ann Appl Biol 163 (2013) 124134

2013 Association of Applied Biologists

 et al.
A. Ludwikow

Figure 3 Phylogenetic relationships of Arabidopsis thaliana and Brassica

oleracea based on group A PP2C proteins. A neighbour-joining
phylogenetic tree was created from the phosphatase catalytic domains
of A. thaliana and B. oleracea PP2Cs rooted with Physcomitrella
patens XP 001765872.1. The optimal tree with the sum of branch
length = 1.98599134 is shown. The percentage of replicate trees in
which the associated taxa clustered together in the bootstrap test (1000
replicates) is shown. Branches corresponding to partitions reproduced
in less than 50% bootstrap replicates are collapsed. The evolutionary
distances were computed using the Poisson correction method and are in
the units of the number of amino acid substitutions per site. The analysis
involved 18 amino acid sequences. All ambiguous positions were removed
for each sequence pair. There were a total of 224 positions in the nal
dataset. Evolutionary analyses were conducted in MEGA5. All predicted
Brassica PP2C genes are listed in Table S3.

sequences and disclosed polymorphisms, we were able

to determine the chromosomal organisation and confirm
the duplication status of selected members of the PP2C
family in the Brassica C genome.
Several gene expression studies, using different
approaches, have recently been conducted to the search
for specific gene expression patterns under drought
conditions in A. thaliana and B. oleracea (reviewed in

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

Ann Appl Biol 163 (2013) 124134

2013 Association of Applied Biologists

PP2C group A in Brassica oleracea

ABA signalling and leads to enhanced drought tolerance.

As members of the PP2C group A family are important
regulators of ABA signalling, we identified homologous
gene copies in available databases and examined their
transcription profile in drought conditions and ABA
response using a qPCR approach. Relevant differences
were identified in B. oleracea both in the transcript
abundance and gene expression patterns, which implies
a divergence in stress response between A. thaliana and
B. oleracea. Comparison of ABI1, ABI2 and PP2CA gene
expression following ABA treatment showed that there is
a substantial overlap between A. thaliana and B. oleracea.
Therefore, the results imply that BolC.ABI1, BolC.ABI2 and
BolC.PP2CA.a function as early ABA signalling elements,
which may be involved in the regulation of drought stress
tolerance in Brassica. Moreover, although AtHAB1 and
AtHAB2 were significantly induced upon ABA treatment,
B. oleracea homologous genes BolC.HAB1, BolC.HAB2.a,
BolC.HAB2.b and BolC.HAB2.c had a hardly detectable or
constitutive level of transcription. This suggests that these
proteins are not downstream elements of the early ABA
signalling pathway. Another interesting result was the
transcription profile of BolC.AHG1.a, BolC.HAI2.ac and
BolC.HAI1.a which, in comparison to the homologous A.
thaliana genes, were not induced either by drought or
by ABA. All this strongly implies that AtHAI2 and AtHAI1
gain the function as early regulators of ABA signalling and
drought stress response. In summary, this work showed
the different reactions of members of the PP2C family
to water stress treatment and ABA treatment signifying
the very low conservation of differential gene expression
between these two species.
The authors of previous comparative studies of Brassica
species and A. thaliana postulated a duplicated genome
structure of Brassica species (Kowalski et al., 1994;
Sadowski et al., 1996; Parkin et al., 2005; Ziolkowski
et al., 2006; Kaczmarek et al., 2009). To understand
the possible mechanisms of PP2C gene family expansion
in the B. oleracea genome, chromosomal localisation of
the PP2C genes and the phylogenetic analysis of the
PP2C family were performed. The phylogenetic analyses
revealed that the majority of the BolC.PP2Cs and AtPP2Cs
clustered together, thus we considered that they were of
an orthologous origin and, as such, these members of
the group might play similar functional roles in the same
signalling pathways. Nevertheless, some B. oleracea copies
of PP2C genes form distinct clades suggest the speciesexpansion of PP2C members in Brassica. The genetic
analysis has corroborated the presence of three copies
for ABI2, HAB2, PP2CA and AHG1 genes and at least four
copies for the ABI1 gene in the B. oleracea C genome,
each corresponding to unique genes in A. thaliana. The
only exception was the HAB1 gene, which is probably a
131

 et al.
A. Ludwikow

PP2C group A in Brassica oleracea

unique copy in both B. oleracea and A. thaliana genome.

These results are not compatible with previous reports
(Xue et al., 2008) showing a similar number of group A
members in Arabidopsis and rice despite the difference in
gene number and genome size.
Because of the limited polymorphism level in the
mapping population applied for genetic analysis, not
all duplicated loci of the above genes were located
on the B. oleracea map (Fig. 2). However, our genetic
mapping results are supported by the incomplete genomic
data collected from whole genome shotgun-sequencing
(Ayele et al., 2005) and phylogenetic analysis conducted
in this study. Furthermore, a similar degree of these
gene duplications and their genomic organisation are
observed in B. rapa. One exception, the AHG1 gene exists
as a unique copy in B. rapa. The uniqueness of some
genes in the duplicated B. oleracea genome was observed
also in other crucial genes of stress signalling, such as
RPS2 and EIN2 (Quiros et al., 2001; Babula et al., 2006).
Absence of these genes from the B. oleracea genome
reflects gene loss events, which are often an observed
phenomenon in the duplicated chromosomal segments
of Brassica species (ONeill & Bancroft, 2000; Park et al.,
2005). Eight out of the eleven detected loci homologous
to the analysed PP2CA genes were located within the
collinearity regions in B. oleracea and A. thaliana (Fig.
2). Accurate identification of orthologues/homologues in
paleopolyploids such as A. thaliana and Brassica species
deduced on the basis of synteny and collinearity may
provide a valuable framework for functional analysis.
Genes localised within the conserved chromosomal
location in Brassica and A. thaliana genomes may also
expose functional conservation based on orthologous
origin (Simillion et al., 2002; Babula et al., 2006).
However, the detailed analysis of the expression patterns
of duplicated gene copies has shown that half of them
exhibit a functional divergence.
In conclusion, this study substantially increases our
knowledge of type 2C protein phosphatases with an
emphasis on the function and their chromosomal
organisation in B. oleracea. We demonstrate herein the
functional divergence among the group A members
in B. oleracea. We have shown that the particular
genes of group A in B. oleracea function in ABA or
drought signalling. Phylogenetic analyses revealed sets
of orthologous and species-specific gene duplications in
Brassica. Our observations are a starting point in studies
on the genetic plasticity of the ABA signalling pathway in
Brassica based on the presence of duplicated genes with
diverged functions. Finally, this study provides novel
findings with potential implications for strategies in plant
selection and engineering of drought-tolerant crop plants.
132

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

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et al.
A. Ludwikow

validated to confirm quality of biological samples used in

the macroarray experiment. In general, the qPCR analysis
confirms the reliability of the nylon array results. Relative
change was calculated as fold change ratio to the nontreated control to indicate genes up- or downregulated,
respectively. A gene was considered validated by qPCR
analysis if revealed consistent trend with the nylon array
data. Ath A. thaliana, Bol B. oleracea var. alboglabra
Appendix S1. Protein alignment of PP2Cs homologues
Table S1. Drought stress response gene homologues
in B. oleracea identified in the macroarray experiment
Table S2. Drought stress responsive genes in A. thaliana
identified in the macroarray experiment
Table S3. Gene names and accession numbers
Table S4. Primers used for RLFP and SSR analysis
Table S5. Primers used for qRT-PCR analysis

Ann Appl Biol 163 (2013) 124134

2013 Association of Applied Biologists