In this study, we identified and characterized cytosine DNA MTase genes that are activated with the onset of reproductive development in rice.. A microarray-based gene expression profile o
Trang 1Sanjay Kapoor1and Meenu Kapoor2
1 Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India
2 University School of Biotechnology, Guru Gobind Singh Indraprastha University, Kashmere Gate, Delhi, India
Introduction
DNA methylation is an epigenetic modification that
regulates key developmental processes, e.g X
chromo-some inactivation, genomic imprinting, gene expression
and protection of genomes from invading transposons, retrotransposons and viruses [1] The methylation of cytosine residues is catalysed by a class of proteins
Keywords
DNA methyltransferase; epigenetics;
methylation; microarray; rice (Oryza sativa)
Correspondence
S Kapoor, Interdisciplinary Centre for Plant
Genomics and Department of Plant
Molecular Biology, University of Delhi South
Campus, Benito Juarez Road, New Delhi
110021, India
Fax: +91 11 24115095
Tel: +91 9811644436
E-mail: kapoors@south.du.ac.in
M Kapoor, University School of
Biotechnology, Guru Gobind Singh
Indraprastha University, Kashmere Gate,
Delhi 110006, India
Fax: +91 11 23900111
Tel: +91 11 23900237
E-mail: kapoorsk@genomeindia.org
*These authors contributed equally to this
work
(Received 21 June 2009, revised 21 August
2009, accepted 1 September 2009)
doi:10.1111/j.1742-4658.2009.07338.x
DNA methylation affects important developmental processes in both plants and animals The process of methylation of cytosines at C-5 is catalysed by DNA methyltransferases (MTases), which are highly conserved, both struc-turally and functionally, in eukaryotes In this study, we identified and characterized cytosine DNA MTase genes that are activated with the onset
of reproductive development in rice The rice genome (Oryza sativa L subsp japonica) encodes a total of 10 genes that contain the highly con-served MTase catalytic domain These genes have been categorized into subfamilies on the basis of phylogenetic relationships A microarray-based gene expression profile of all 10 MTases during 22 stages⁄ tissues that included 14 stages of reproductive development and five vegetative tissues together with three stresses, cold, salt and dehydration stress, revealed spe-cific windows of MTase activity during panicle and seed development The expression of six methylases was specifically⁄ preferentially upregulated with the initiation of floral organs Significantly, one of the MTases was also activated in young seedlings in response to cold and salt stress The molecular studies presented here suggest a greater role for these proteins and the epigenetic process in affecting genome activity during reproductive development and stress than was previously anticipated
Abbreviations
BAH, bromo-adjacent homology; CMT, chromomethyltransferase; Dnmt2, DNA methyltransferase 2; DRM, domains rearranged
methyltransferase; HMM, hidden Markov model; GCRMA, GC robust multi-array average; MTase, methyltransferase; MET1, DNA
methyltransferase 1; NCBI, National Center for Biotechnology Information; SAM, shoot apical meristem; TIGR, The Institute of Genomic Research; QPCR, quantitative PCR.
Trang 2known as DNA methyltransferases (MTases) These
proteins in prokaryotes and eukaryotes possess a
catalytic domain with conserved motifs that are
arranged in a specific order, thus reflecting their origin
from a common ancestor In eukaryotes, this domain
is associated with N-terminal extensions having a
vari-able number of domains conserved across different
members of MTase subclasses [2] This structural
arrangement facilitates the interaction of MTases with
a variety of cellular proteins [3] The diverse types of
MTase that exist in eukaryotes are known to be
involved in two basic kinds of methylation activities:
maintenance methylation and de novo methylation
When a methyl group is added to cytosines of a
hemimethylated DNA after DNA replication, the
pro-cess is referred to as maintenance methylation
Methyl-ation of cytosines in nonmethylated DNA is referred
to as de novo methylation This process is responsible
for establishing new methyl patterns that are then
maintained by maintenance MTases
In plants, four main subfamilies of MTase have been
identified: domains rearranged methyltransferase
(DRM), DNA methyltransferase 1 (MET1), DNA
methyltransferase 2 (Dnmt2) and
chromomethyltrans-ferase (CMT) [4–7] DRMs are similar to human
Dnmt3, which is required for establishing methylation
patterns during development In Arabidopsis, three
DRM genes are known, AtDRM1, AtDRM2 and
At-DRM3 AtDRM1 and AtDRM2 are required for
de novo methylation of cytosines in all sequence
con-texts, CpG, CpNpG and at asymmetric positions [8]
The MET1 class encodes MTases resembling the
ani-mal Dnmt1 In Arabidopsis, the MET1 proteins are
encoded by a small multigene family and these
pro-teins function predominantly as maintenance
methylas-es, methylating CpG residues [4] MET1-like genes
have been identified in a variety of plant species, such
as pea, maize, tobacco and Brassica [9–12] Dnmt2 has
been reported in yeast, Drosophila, animals and plants,
suggesting an ancestral origin and its involvement in
essential cellular functions Unlike other MTases,
Dnmt2s have broader substrate specificity The human
Dnmt2 also acts as an RNA MTase and has been
shown to specifically methylate cytosine 38 in the
anti-codon loop of tRNA(Asp) [13] CMTs are
plant-spe-cific MTases that are characterized by the presence of
a chromo (chromatin organization modifier) domain
and a bromo-adjacent homology (BAH) domain in the
N-terminal region In Arabidopsis, three CMTs have
been reported and AtCMT3 is known to control
meth-ylation at CNG and CNN sites in Arabidopsis [3,5]
There is significant variability in the types and
numbers of MTases utilized by various organisms
Although a single methylase has been identified in many protozoa, insects and fungi, more than 10 genes are known in higher plants [2] The availability of complete genome sequences of numerous eukaryotes provides an unprecedented opportunity to explore the diversity among this class of protein in various organ-isms and to study their roles in regulating critical developmental processes at the whole genome level
In the present study, an attempt was made to gain insight into the expression pattern of rice DNA MTases during panicle and seed development Utilizing an in-house generated microarray-based gene expression dataset, expression profiles of all rice MTases were compiled from stages⁄ tissues of vegetative, panicle and seed development, together with seedlings subjected to desiccation, cold and salt stress conditions Wherever possible, expression profiles of rice genes were compared with expression patterns of Arabidopsis MTases avail-able in public databases
Results
Identification of rice cytosine-specific DNA MTases
Putative DNA MTases were searched in the Rice Genome Annotation Project (http://rice.plantbiology msu.edu), formerly at The Institute of Genomic Research (TIGR) using the keyword ‘cytosine methyl-transferase’ and the hidden Markov model (HMM) corresponding to the DNA MTase domain as described
in Pfam (http://www.pfam.sanger.ac.uk/) The domains were searched using hmmer, version 2.3.2 (http:// hmmer.janelia.org/) All proteins with an E-value above
e)10were considered as putative MTases The values for putative DRMs were lower than the other MTases due
to rearrangement of conserved motifs in their catalytic domains (Fig S1) These MTases were further verified
by other search tools (see below) In total, 10 nonredun-dant loci were identified from HMM analysis (Table 1) Eight of these loci have previously been reported [2] We identified two additional loci corresponding to putative CMTs and de novo MTase The length of the open read-ing frames encoded by all putative MTases varied from
1152 to 4584 bp and the derived proteins ranged from
383 to 1527 amino acids (Table 1) The presence of conserved domains was also verified using the Simple Modular Architecture Research Tool (http://smar-t.embl-heidelberg.de/) and the National Center for Bio-technology Information (NCBI) conserved domain database (http://www.ncbi.nlm.nih.gov/Structure/cdd/ cdd.shtml) All 10 MTases possessed the MTase domain with conserved motifs at the carboxyl terminus Most of
Trang 3Serial no.
Gene name Chromatin database identification
Accession numbers of
Locus identification
UniProtKB accession number
ength (bp)
KOME clone accession number Location on pseudomolecules
Annotation Project (Release
Length (amino acids)
Molecular mass
12001.m10527, 12001.m150650, 12001.m150651
12005.m04966, 12005.m64178
12003.m05744, 12003.m34976, 12003.m34977, 12003.m34978
a Genes
Trang 4the proteins also had amino terminal extensions with
dif-ferent types of domain, such as ubiquitin-associated
domain (UBA), BAH or chromo domains depending on
the type of MTase Rice MTases were classified into
known subfamilies and the unnamed loci were assigned
names on the basis of their phylogenetic relatedness to
corresponding members of the MTase subfamilies in
humans and other plants (see below) In total, three
CMT(OsCMT1–3), one OsDnmt2 showing similarity to
vertebrate Dnmt2, four DRM (OsDRM1a, OsDRM1b,
OsDRM2and OsDRM3) having similarity with human
Dnmt3and two MET1 genes (1 and
OsMET1-2) were identified on the basis of these searches
(Table 1)
Phylogeny and genome distribution
In order to analyse evolutionary relatedness of rice
proteins, complete amino acid sequences of all MTases
were aligned with corresponding proteins from maize,
Arabidopsis, Chlamydomonas, mouse, human,
Drosoph-ilaand yeast using clustalx 1.83 [14] A bootstrapped
phylogenetic tree was generated using the
neighbour-joining method This revealed the existence of rice
proteins that belonged to all four subclasses of MTase
known in plants (Fig 1) Rice contains a single
puta-tive OsDnmt2 gene whose product shows 80%
simi-larity to maize Dnmt2, 67% to Arabidopsis AtDnmt2
and 38% to yeast PMT1 proteins at the amino acid
level These genes code for small proteins similar to
prokaryotic MTases that possess only the MTase
domain with conserved motifs (Fig 2)
Four loci having highly similar amino acid
sequences with corresponding Arabidopsis AtDRMs
were identified in rice The products of LOC_
Os12g01800 and Loc_Os11g01810, respectively, were
found to be >90 and 60% identical to Arabidopsis
AtDRM1 Hence, these genes have been named
OsDRM1a and OsDRM1b (Table 1) All rice DRMs
exhibited characteristic altered arrangement of
con-served motifs in the MTase domain with motifs I and
X juxtaposed, similar to DRMs of Arabidopsis, Zea
maysand Nicotiana (Figs 2 and S1) OsDRM3 showed
greater similarity to Arabidopsis AtDRM3 in the
rearranged MTase domain than to AtDRM2 and
AtDRM1 in this subclass Phylogenetic analysis
showed that DRM3 genes of rice and Arabidopsis
sepa-rated from the lineage that gave rise to DRM2 and
DRM1earlier in evolution and evolved independently
Among the CMTs, three genes were identified in rice
that showed significant similarity to the corresponding
Arabidopsisgenes at both the nucleotide and the protein
level (more than 60%) (Fig 1) Rice CMTs have been
named OsCMT1–3 on the basis of their similarity to corresponding proteins in Arabidopsis and similarity in the expression profiles in both the plant species (see below) OsCMT1–3 are characterized by the presence of
a BAH domain in the N-terminal half and a conserved chromo domain inserted between motifs I and IV in the MTase domain (Figs 2 and S2A) OsCMT2 differs from the other two proteins in encoding an additional stretch
of 193 amino acids ahead of the BAH domain in the amino terminal region (Fig S2A) The chromo domain
of rice CMTs possesses conserved aromatic amino acids, tyrosine at position 412 (Y412) and tryptophan at position 409 (W409) However, a phenylalanine was observed to be present in all plant proteins at position
382 in place of a tyrosine at the same position in animal proteins, with Arabidopsis LHP1 being an exception (Fig S2B) We have identified two closely related puta-tive MET1-type genes, OsMET1-1 and OsMET1-2, in rice, confirming the two previous reports [17,18] Both genes encode a conserved BAH domain and a C-termi-nal catalytic domain similar to the animal Dnmt1 proteins (Figs 1 and 2) The putative DNA MTase from the green alga, Chlamydomonas reinhardtii, did not possess any of the conserved regulatory domains identi-fied in other MTases At least three of the MTases in this alga, CrDMT3407, CrDMT3403 and CrDMT3408, appear to have evolved independently from the com-mon ancestor that gave rise to the higher plants and humans de novo MTases Localization of all rice MTase genes on pseudomolecules showed unbiased genomic distribution Except for OsDRM1a and OsDRM1b, which were observed to be located in segmentally dupli-cated regions of chromosomes 11 and 12, all the other genes were observed to be located in unique regions specific to each chromosome (Table 1)
Gene expression analysis
To gain insight into the developmental windows during which rice MTase genes are expressed, spatial and tem-poral expression patterns of these genes were analysed during different developmental stages⁄ tissues For this purpose, an in-house generated microarray expression dataset prepared by using 57 k Affymetrix GeneChip rice genome arrays was utilized, as mentioned previously (Affymetrix Inc., Santa Clara, CA, USA) The develop-mental stages⁄ tissues of rice plants used in the present investigation are summarized in Table S1 The rice gen-ome array contains a total of 57 381 probe sets, of which
55 515 correspond to 51 279 rice transcripts and the rest, 1866, are hybridization, poly A and housekeeping controls In our dataset, the average intensity values of the nonrice control probe sets were found to be < 10
Trang 5Therefore, the value ‘10’ was taken as the cut-off to
dis-tinguish between expressed and nonexpressed genes in a
particular tissue⁄ developmental stage
On the basis of signal intensities obtained for rice
transcripts, three genes, OsCMT1, OsDRM1a and
OsDRM1b, did not express in any of the 22 stages⁄
tissues analysed (Table S2) The remaining six genes
showed specific⁄ preferential enhancement in transcript
abundance at the onset of reproductive development
as compared to their counterparts in Arabidopsis (Table S3) These genes also showed moderate to low level expression in vegetative tissues (Fig 3)
OsMET1-1 transcripts accumulated at low level (£ 5; amounting
to no expression) in most of the stages analysed, except P1-I to P1-III, where the average expression value was 11 in all three stages The OsMET1-2
Fig 1 Phylogeny of rice cytosine DNA MTases An unrooted, neighbour-joining tree was constructed by alignment of total protein sequences from rice (Os), Arabidopsis (At), Zea mays (Zm), Nicotiana (Nt), yeast (Sp), Chlamydomonas reinhardtii (Cr), Drosophila (Dm), human (Hs) and mouse were downloaded from the chromatin database The names of proteins mentioned for each organism indicate the chromatin database identification Putative maintenance MTase from rice OsMET1-1 and OsMET1-2 cluster with other functionally character-ized maintenance MTases from Arabidopsis and other organisms and are shown connected by blue lines Putative rice CMTs and similar methylases from other plants are connected by green lines Evolutionarily conserved DNMT2 proteins from animals and plants, including rice, are shown with purple lines De novo MTases from animals and plants, including, rice are shown connected by brown lines The scale bar shows 0.1 amino acid substitutions per site.
Trang 6transcripts accumulated at higher than the cut-off
value of 10 in all the samples analysed, with the
high-est transcript accumulation observed in the SAM
(shoot apical meristem), the early stages of panicle
ini-tiation (P1-I and P1-II) and the early seed development
stages (Tables S2 and S4) In comparison with leaf
tissues (mature leaf and Y leaf), there was an 87-,
77-and 81-fold increase (P£ 0.005) observed in the
tran-script accumulation of OsMET1-2 in the SAM, early
panicle and early seed stages, respectively In response
to abiotic stress conditions, more than a two-fold
reduction (P£ 0.05) in the transcript levels of this gene
was observed in both salt and dehydration stress,
whereas, 3 h cold stress did not show any significant
effect OsDnmt2, the only representative of the Dnmt2
subclass, was found to express at moderate levels in all
the stages⁄ tissues analysed Similar to OsMET1-2, the
OsDnmt2 transcripts also accumulated at higher levels
in the SAM and early stages (P1-I–III) of panicle
development, but the enhancement in transcript levels
was only about two-fold The OsDnmt2 transcript
levels were significantly reduced in seed stages
(Tables S1–S4; more than two-fold downregulation at
P£ 0.05) The two DRM genes, OsDRM2 and -3,
exhibited similar expression profiles and significant
expression levels throughout rice development
Differ-ential expression analysis revealed that both the genes
were upregulated by approximately two-fold in the
SAM and early stages of panicle development in
com-parison with any of the vegetative stages (mature leaf,
Y leaf, root and seedling) used in this study A signifi-cant downregulation (up to 4.8- and 2.5-fold for OsDRM2 and -3, respectively) in their accumulation was also observed during late seed development stages (Table S4) Two Arabidopsis counterparts of the rice DRM genes also expressed throughout development, but, interestingly, in contrast to that in rice, AtDRM2 and -3 showed up to four-fold enhancement in tran-script accumulation during seed development stages and showed higher expression in vegetative tissues as compared with rice genes (Fig 3) For the two CMT genes, OsCMT2 and -3, highly differential and con-trasting profiles were observed OsCMT2 showed mod-erate expression with specific downregulation (up to two-fold at P£ 0.001) in the SAM and early panicle stages (P1-I, II, III, P1 and P2) (Tables S2 and S4) However, OsCMT3, which expressed at very low levels (average normalized expression value of 24) during late panicle and seed development stages, showed up to 140-fold enhancement in the SAM and stages of early panicle development The Arabidopsis AtCMT3 gene also exhibited a similar reduction in transcript accumu-lation during seed development, but unlike OsCMT3,
it expressed at significantly higher levels in vegetative tissues Interestingly, transcripts of OsCMT2 accumu-lated in seedlings in response to cold and salt stress, whereas the transcriptional activity of this gene was unaffected under dehydration conditions (Table S3) In contrast, OsCMT3 showed approximately a six- and four-fold reduction in transcript accumulation in rice
Fig 2 Schematic representation of conserved domains and motifs identified among proteins of each subclass of rice MTases Blue rectan-gles represent the conserved motifs in the catalytic MTase domains, whereas the conserved domains in the N-terminal regulatory region are represented by red, yellow and purple rectangles The numbers on the right denote the length of each protein.
Trang 7seedlings subjected to salt and dehydration stress,
respectively (Table S2)
Statistically significant and differential expression
profiles of three genes, OsCMT3, OsDRM3 and
OsMET1-2, were further validated by quantitative PCR
(QPCR) The transcript accumulation patterns for all
three genes observed by QPCR were similar to those
observed from the microarray analysis (Fig 4)
Tran-scripts for OsCMT3 and OsDRM3 showed maximum
accumulation in the SAM and early stages of panicle
initiation (P1-I, P1-II and P1-III) and thereafter their
levels declined in mature organs and during seed
forma-tion Transcripts for OsMET1-2, on the other hand,
showed the highest accumulation during the S2 stage of
seed development, which coincides with the onset of
differentiation of embryonic organs such as the SAM
and the radicle 3–4 days after pollination (Table S1)
Discussion
In this investigation, putative cytosine DNA MTase genes encoded in the rice genome were identified and characterized Transcript profiling of these genes dur-ing the transition of plants from the vegetative to the reproductive phase has provided weighted insight into the developmental timing of the activity of these genes The specific activation of OsCMT2 in seedlings sub-jected to cold and salt stress and the downregulation
of OsCMT3 expression under salt and drought stress
is interesting and provides the foundation for further
in depth analysis to study the possible involvement of these proteins in affecting overall genomic activity under abiotic stress
The first part of our analysis identified 10 putative cytosine DNA MTases that have sequence similarities
Fig 3 Microarray-based expression analysis of DNA MTase genes of rice and Arabidopsis The expression profiles of rice genes were analy-sed in vegetative tissues (Y leaf, mature leaf and roots), SAM, six stages of panicle development (P1–P6), three substages of P1 (P1-I, P1-II and P1-III), five stages of seed development (S1–S5) and under three abiotic stress conditions (cold, salt and dehydration) For Arabidopsis, the expression profiles of two vegetative stages (leaf and root), five stages of flower development (F1–F5), five stages of silique
dendrogram is shown on the left-hand side of the expression maps.
Trang 8with known de novo and maintenance MTases of
eukaryotes encoded in the completely sequenced rice
genome Phylogenetic analysis with genes in other
organisms revealed that the DNA methylation system
in rice utilizes the same basic set of MTases functional
in eukaryotes However, duplications and multiplicity
in gene numbers was observed in DRM and OsMET1
subfamilies in rice
In the case of duplicated genes, it is usually observed
that either both the genes remain active and function
redundantly in an organism or the regulatory role of
these genes is divided or is shared between the two
part-ners equally or one of the gene member functions
prominently while the other either weakly complements
the function of the main gene or becomes a pseudogene
In the case of OsDRM1, it was observed that the
conse-quence of duplication of this gene has probably resulted
in inactivity of both OsDRM1a and OsDRM1b, as no
transcripts could be detected for either of these genes
during the stages of development analysed in this study
It is also plausible that OsDRM1a and OsDRM1b
could be expressed during a much narrower
develop-mental window and in specific tissues of floral organs,
as we analysed the expression of these genes in whole
panicles that were developmentally segregated on the
basis of their total length The expression of
Arabidop-sis AtDRM1 was also very weak during floral organ
ini-tiation and in mature seeds In the case of OsMET1,
two previous reports have shown expression of both
OsMET1-1 and OsMET1-2 genes by RT-PCR and
ribonuclease protection assay in actively dividing tissues
from various vegetative and floral organs studied in
japonica variety cv Nipponbare and Taipei 309, although transcripts of OsMET1-2 were observed to accumulate between 7- and 12-fold higher than those of OsMET1-1 in both the cultivars [17,18] In our micro-array experimental set-up, expression signals for OsMET1-1 transcripts were below the cut-off limit of
10 in most of the stages⁄ tissues analysed, except in early stages of panicle development, whereas the expression
of OsMET1-2 varied from moderate to high levels spe-cifically during early stages of floral organ initiation and in mature seeds Interestingly, however, the pattern
of transcript accumulation as seen from the values of signal intensities for both OsMET1-1 and OsMET1-2 overlapped in vegetative organs and at all stages of panicle and seed development (Table S2) It can there-fore be speculated that OsMET1-2 possibly functions
as the major maintenance MTase
The putative functions of genes can be inferred from
a comparison of their gene expression profiles with that of known genes in other organisms The expres-sion pattern of OsCMT3 at different developmental stages showed similarity with the expression of AtCMT3 in young actively dividing tissues of roots and during the formation of inflorescence and floral meristems and the initiation of floral organs (P1-I and P1-II in rice and F1 and F2 in Arabidopsis) AtCMT3
is known to regulate DNA methylation by interacting with the N-terminal tail of histone H3, which is simultaneously methylated at lysine 9 and lysine 27 by kryptonite, a histone H3 lysine 9 MTase and an unknown protein [3,19] All the rice CMTs share simi-larity in their N-terminal regulatory domains (BAH
Fig 4 QPCR results for three selected genes, OsCMT3, OsDRM3 and OsMET1-2, and their correlation with microarray data The y-axis represents raw expression values obtained from the microarray analysis in the form of relative transcript abundance The x-axis depicts developmental stages of panicle and seeds as described in Table S1 Standard error bars are shown for data obtained using both techniques.
Trang 9conserved aromatic amino acids that are also present
in Arabidopsis and maize CMT and HP1 and the
poly-comb group proteins from humans and Drosophila
(Fig S2B) [20,21] These amino acids are known to
form a methyl ammonium recognition cage in the
complex that interacts with the methylated histone
peptides Amino acids on either side of these aromatic
amino acids were also observed to be highly conserved,
suggesting that the rice proteins can also adopt higher
order structures similar to other proteins and possibly
interact with methylated lysines in a manner similar to
AtCMT3
Although the observations presented in this study
strongly suggest that the subsets of cytosine DNA
MTases for performing putative de novo and
mainte-nance methylation activities during reproductive
devel-opment are largely conserved across monocot and
dicot species, finer differences in terms of specific genes
targeted by these proteins probably occur in a
species-specific manner Further investigation into the roles of
these proteins in development and the biochemical
mechanisms underlying these activities could extend our
knowledge and understanding of the role of DNA
methyl-ation in programming reproductive development in rice
Materials and methods
Identification of genes, chromosomal localization
and phylogenetic analysis
Cytosine DNA MTases encoded in the rice genome were
identified using search tools as described previously [15,16]
Briefly, a name search and the HMM analysis were used to
identify cytosine DNA MTases encoded in the rice genome
The specific sequences were downloaded from the Rice
Genome Annotation Project (http://rice.plantbiology.msu
edu) and an HMM profile was generated using hmmer
2.1.1 software (http://hmmer.wust.edu) This resulting
pro-file was then used to search the proteome database of rice
available at TIGR using the Basic Local Alignment Search
Tool with the filter off option The details of encoded
pro-teins [length, isoelectric point (pI), molecular mass] were
obtained from the Rice Genome Annotation Project
(http://rice.plantbiology.msu.edu) and chromatin database
(http://www.chromdb.org) Conserved domains in encoded
proteins were identified by using the Simple Modular
Architecture Research Tool version 3.4 or the NCBI
con-served domain database The newly identified genes were
named on lines of nomenclature used for the genes
identified previously and on the basis of their phylogenetic
constructing the tree using the neighbour-joining method followed by bootstrap analysis using 1000 replicates [14]
Plant material
tis-sues were collected from field-grown rice plants (Oryza
drought and cold, were given to rice seedlings as described previously [15] Briefly, salt stress was given to 7-day-old seedlings by transferring them to NaCl solution (200 mm) for
drought stress was induced by drying the plants on tissue paper and spreading them on a Whatmann 3 mm sheet for
3 h Seven-day-old seedlings with roots submerged in water for 3 h were used as controls for all stress treatments
Microarray hybridization and analysis of data
49 824 rice transcripts were used to prepare a compendium
of transcriptome profiles of 22 stages of vegetative and repro-ductive development and stress response in rice [15] The microarray analysis of 17 stages has been described previ-ously and the data were deposited in the Gene Expression Omnibus database at the NCBI under the series accession numbers GSE6893 and GSE6901 In the present investiga-tion, five more stages, namely Y leaf, SAM, P1-I, P1-II and P1-III were included to analyse gene expression patterns dur-ing early stages of panicle differentiation Sixty-six cell
further analysed [14,15] Expression data for cytosine MTase genes were extracted using the gene locus indentifications listed in Table 1 Wherever more than one probe set was available for one gene, the probe set designed from the 3¢ end was given preference A differential expression analysis was performed by taking a mature leaf as the reference to identify genes expressing at more than the two-fold level in various stages of reproductive development (panicle and seed), with
differ-ential expression analysis was performed with no correction
Expression analysis of Arabidopsis MTases
Expression data for Arabidopsis, derived using Affymetrix
with those used for rice, were downloaded from the Gene Expression Omnibus database at the NCBI under the series accession numbers GSE5620, GSE5621, GSE5623,
Trang 10GSE5624, GSE5629, GSE5630, GSE5631, GSE5632 and
GSE5634 The downloaded files were imported using the
microarray analysis software, array assist, and normalized
further analysis leading to the generation of heat maps was
performed in a manner identical to that for rice [15]
QPCR analysis
Real time PCR reactions were carried out using RNA
the microarray analysis The primers for amplification were
designed using primer express, version 2.0 (PE Applied
Biosystems, Foster City, CA, USA), preferentially from the
3¢ end of the gene The specificity of each primer was
checked using the Basic Local Alignment Search Tool of
the NCBI Two biological replicates were taken and for
each three technical replicates were carried out Actin was
used as the endogenous control RT-PCR was performed
using the ABI Prism 7000 Sequence Detection System and
software (PE Applied Biosystems) [14,15] The data were
normalized to facilitate the profile matching that obtained
from the microarrays and bar charts were plotted using
Microsoft excel
Acknowledgements
A senior research fellowship awarded by the Council
for Scientific and Industrial Research (CSIR) to R.S.,
P.D., M.S and a junior research fellowship to G.M
are acknowledged This work has been funded by the
Department of Science and Technology and the
Department of Biotechnology, Government of India
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