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Isolation and characterization of flower-specific transcripts in Acacia mangium

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Acacia mangium Willd. is a legume tree species native to subtropical and tropical regions of Asia and Australia. Many features of its flower development are common to other legume tree species. To identify genes involved in its floral development, we constructed a subtractive flower cDNA library against vegetative tissues. The 1123 expressed sequence tags (ESTs) represented 576 unique genes. Macroarray analysis further identified 147 of these genes as specific to the early, late or whole flowering process. Eight percent of these flower-specific genes encode MADS-domain-containing transcription factors and MYB proteins. Four percent encode other transcription factors and 10% encode regulatory proteins such as G proteins, kinases and phosphatases. Flower-specific transcripts for gibberellic acid (GA) synthesis and GA-induced proteins, as well as other stress- and pathogenesis-related genes (9%), implicate their involvement in A. mangium flower development. Eighteen percent of the flower-specific genes encode hypothetical proteins and 18% encode proteins of unknown functions. The RNA blot hybridization confirmed and detailed the expression patterns of selected genes. Functions of the A. mangium flower-specific genes are discussed based on comparison with their Arabidopsis homologues, most of which have been implicated in Arabidopsis floral development. Our work suggests general conservation of floral development in A. mangium and Arabidopsis. Further characterization of the conserved and different flower-specific genes will delineate the flowering process of this important legume tree species and facilitate genetic modification of its reproduction
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Summary Acacia mangium Willd. is a legume tree species
native to subtropical and tropical regions of Asia and Australia.
Many features of its flower development are common to other
legume tree species. To identify genes involved in its floral
development, we constructed a subtractive flower cDNA li
-
brary against vegetative tissues. The 1123 expressed sequence
tags (ESTs) represented 576 unique genes. Macroarray analy
-
sis further identified 147 of these genes as specific to the early,
late or whole flowering process. Eight percent of these flower-
specific genes encode MADS-domain-containing transcrip-
tion factors and MYB proteins. Four percent encode other tran-
scription factors and 10% encode regulatory proteins such as
G proteins, kinases and phosphatases. Flower-specific tran-
scripts for gibberellic acid (GA) synthesis and GA-induced
proteins, as well as other stress- and pathogenesis-related
genes (9%), implicate their involvement in A. mangium flower
development. Eighteen percent of the flower-specific genes en-
code hypothetical proteins and 18% encode proteins of un
-
known functions. The RNA blot hybridization confirmed and
detailed the expression patterns of selected genes. Functions of
the A. mangium flower-specific genes are discussed based on
comparison with their Arabidopsis homologues, most of which
have been implicated in Arabidopsis floral development. Our
work suggests general conservation of floral development in
A. mangium and Arabidopsis. Further characterization of the
conserved and different flower-specific genes will delineate
the flowering process of this important legume tree species and
facilitate genetic modification of its reproduction.
Keywords: floral development, flower-specific genes, macroar
-
ray, subtractive cloning.
Introduction
The genus Acacia (family Leguminosae) comprises more than
1300 legume woody shrub and tree species distributed mainly
in the Southern Hemisphere. Acacia mangium Willd., a mem
-
ber of this family, is native to the tropical and subtropical re
-
gions of Asia and Australia. Many aspects of its flower devel
-
opment are common to other Acacia species. The flowers
show weak protogyny and variable degrees of andromonoecy.
Up to 200 small yellow or cream flowers are grouped in an in
-
florescence. Stylar extension is followed by extension of nu
-
merous stamens (as many as 200), and then by anther de
-
hiscence to release a single polyad per spore sac. The polyad is
a composite structure of 16 pollen grains. The flowering pe
-
riod of an adult tree may last 8 months in a year. Cross-pollina
-
tion with other acacias such as A. auricauliformis has been re
-
ported (Sedgley et al. 1992). Because A. mangium has a high
growth rate, good quality fiber, high disease resistance and the
ability to thrive in poor soils, it has been planted widely in In-
donesia, Malaysia and southern China for reforestation and as
a fiber source. Cuttings from adult trees grow readily and
flower at a much younger age than trees derived from seed
(Simmons 1987). Reports on marker-assisted breeding and ge-
netic mapping (Butcher et al. 2002), propagation (Bhaskaran
and Subhash 1996), physiological study (Yu and Ong 2000),
regeneration and Agrobacterium-mediated genetic transfor-
mation (Xie and Hong 2001, 2002) indicate growing interest
in this tropical legume. There are no reports yet, however, on
the molecular or genetic analysis of genes that regulate flower
development in this important tree species.
Our knowledge of flower development at the molecular
level comes mostly from studies of the herbaceous model
plants Arabidopsis thaliana (L.) Heynh. and Antirrhinum.Ac
-
cording to the current model of floral development (see review
by Blazquez 2000), which is based mainly on genetic interac
-
tions between mutants, flowering signals include light (for
long-day plants), gibberellic acid (GA) (for short-day plants)
and a photoperiod-independent pathway that is primarily re
-
lated to temperature changes. The targets for these flower
-
ing signals are the inflorescence and floral meristem identity
genes, whose activities confer floral identity on the newly
emerging primordia. Further downstream are the floral organ
identity genes, whose regionalized expression ensures the cor
-
rect arrangement of floral organs. Floral organ identity genes
comprise at least three classes of homeotic genes, including
the well-known A-, B- and C-function genes (Coen and Mey
-
erowitz 1991, Ma and Claude 2000, Soltis et al. 2002). Most
ABC genes encode transcription factors that carry a MADS-
box DNA-binding domain. Most other genes involved in floral
initiation, floral symmetry and organ polarity encode tran
-
scription factors with domains like the bZIP and the zinc finger
domains (Smyth 2001).
Tree Physiology 25, 167–178
© 2005 Heron Publishing—Victoria, Canada
Isolation and characterization of flower-specific transcripts in Acacia
mangium
XING JUN WANG,
1
XIANG LING CAO
1
and YAN HONG
1,2
1
Temasek Life Sciences Laboratory, 1 Research Link, The National University of Singapore, 117604, Republic of Singapore
2
Corresponding author (hongy@tll.org.sg)
Received February 13, 2004; accepted August 9, 2004; published online December 1, 2004
at University of Portland on May 21, 2011treephys.oxfordjournals.orgDownloaded from
With a better understanding of the genes controlling floral
initiation, floral meristem and floral organ identity, there has
been a growing interest in genes that are targets of these up
-
stream elements. Knowledge of these genes is crucial for un
-
derstanding the molecular mechanisms that lead to organ-spe
-
cific cell and tissue differentiation. In recent years, gene ex
-
pression analysis with DNA microarrays has become a power
-
ful tool for the analysis of developmental processes in animals
and plants. Microarray analysis has implicated 724 genes in
Arabidopsis floral development (Hu et al. 2003). In addi
-
tion, putative downstream target genes for the homeotic genes
APETALA3 and PISTILLATA have been identified by cDNA
microarray (Zik and Irish 2003). A genome-wide microarray
study of the Arabidopsis male gametophytic transcriptome
identified 992 pollen expression mRNAs (Honys and Twell
2003). With the availability of the whole genome sequence of
Arabidopsis, highly specific oligonucleotide probes can be de
-
signed to cover the full genome, thus allowing genome-wide
analysis of spatial and temporal gene expression. With the use
of both cDNA and oligonucleotide arrays on the floral homeo
-
tic mutants apetata1, apetala2, apetala3, pistillata and aga
-
mous, 1453 genes were identified to be specifically or at least
predominantly expressed in one type of floral organ (Wellmer
et al. 2004). Genome-wide oligonucleotide array analysis is
gaining popularity for studying Arabidopsis development and
array data are available from public sources such as the Not-
tingham Arabidopsis Stock Centre’s Affymetrix microarray
database (NASC, website: http://affymetrix.arabidopsis.info/ )
(Craigon et al. 2004). Application of the same gene chips and
protocols allows easy comparison among samples from differ-
ent experiments.
Trees differ from the well-studied herbaceous plant species
in that they are adapted to survive for much longer. There is
also wide variation in the age at which trees flower. Therefore,
studies on Arabidopsis will not be entirely adequate for under
-
standing floral development in woody trees. Molecular studies
on flower development in woody species have been limited to
cloning poplar, pine and eucalypt homologues to known floral
meristem genes and ABC genes (Tandre et al. 1995, Mouradov
et al. 1998, Southerton et al. 1998, Brunner et al. 2000). The
generation of transgenic trees with heterologous expression
of Arabidopsis LFY and API can dramatically reduce flower
-
ing time (Egea-Cortines and Weiss 2001), demonstrating that
genes that contribute to flower initiation are conserved; how
-
ever, it is not known if the downstream target genes are also
conserved. These downstream genes may control the defined
positions and numbers of flower organs that are relatively con
-
stant within related species. A greater understanding of floral
development at the molecular level will enable more effective
and specific genetic manipulation of the reproduction process
of the subject plant (Smyth 2001).
To identify genes involved in A. mangium flower develop
-
ment, we started with a subtracted A. mangium flower cDNA li
-
brary. Macroarray analysis was used to profile expression of
the 576 independent genes cloned, leading to the identification
of 147 genes specific to early, late or whole flower develop
-
ment. Besides homologues to ABC genes, genes for transcrip
-
tion factors and other regulatory proteins, 30% of the genes en
-
code for proteins required for basic cellular processes like cell
wall formation. Many of these genes have Arabidopsis homo
-
logues implicated in flower development. This suggests gen
-
eral conservation of flower development in Arabidopsis and
A. mangium. Eighteen percent of the flower-specific genes en
-
code unknown proteins that may be specific to A. mangium
flower development.
Materials and methods
Plant materials
Plant materials were taken from a 10-m-high, 3-year-old A. man
-
gium tree. Flowers at different stages and seedpods were pho
-
tographed. Morphology of flowers at different stages was
examined in detail by scanning electron microscopy (JSM-
5310LV, JEOL, Tokyo, Japan). Based on a previous report on
A. mangium reproductive biology (Sedgley et al. 1992), we di
-
vided the flowering process in A. mangium into four phases.
Phase 1 = floral bud stage 1 (Figures 1A and 1H); the densely
grown buds are small in size (< 0.5 mm) and remain green on
flower spikes less than 5 cm in length. Phase 2 = floral bud
stage 2 (Figures 1B and 1I); flower inflorescences are elon
-
gated up to 10 cm with yellowish buds loosely scattered. Phase
3 = open flower stage 1 (Figures 1C and 1J); the five petals are
open but stamens and carpels are not fully expanded. Phase 4 =
open flower stage 2 (Figures 1D and 1K); flowers are fully
opened with stamens and carpels, and polyads of pollen grains
(Figure 1L) are released during this stage.
Pollination of a few flowers in an inflorescence generates
seedpods each containing 5–10 seeds (Figures 1E and 1F).
Like several other Acacia members, A. mangium has petiole-
derived phyllodes instead of true leaves as the photosynthetic
organ (Figure 1G).
RNA isolation and purification
Total RNA was isolated from young phyllodes (1–2 cm), ma
-
ture phyllodes (about 10 cm), floral buds at stages 1 and 2,
open flowers at stages 1 and 2, young seedpods, young seeds,
stem and roots by a modified hot phenol method. Briefly, 2 g
of sample was ground to a fine powder in liquid nitrogen and
mixed thoroughly with 8 ml of pre-warmed (65 °C) extraction
buffer (50 mM Tris-HCl, pH 6.0, 10 mM EDTA, 2% SDS,
100 mM LiCl), 4 ml of phenol and 4 ml of chloroform. After
incubation at 65 °C for 30 min, the homogenate was centri
-
fuged at 15,000 g for 15 min at 4 °C. The supernatant was ex
-
tracted with equal volumes of phenol and chloroform three
times. Total RNA was precipitated with 4 M LiCl at 20 °C for
2 h followed by centrifugation at 15,000 g for 15 min at 4 °C.
The RNA pellet was washed with 70% ethanol and dissolved
in water containing diethyl pyrocarbonate (DEPC). The sticky
RNA solution was further purified on an RNeasy Plant Mini
Kit column (Qiagen, Valencia, CA).
Flower differential cDNA library construction and sequencing
Total RNA was isolated from phyllode tissue and a mixture of
flowers at Phases 2 and 3. Poly (A) RNA was prepared with a
Qiagen mRNA purification kit. A flower subtracted cDNA li
-
168 WANG, CAO AND HONG
TREE PHYSIOLOGY VOLUME 25, 2005
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brary against phyllode tissue was constructed using the Clon
-
Tech PCR-select cDNA subtraction kit (ClonTech, Palo Alto,
CA). The cDNAs were ligated to pGEM-T easy vector (Pro
-
mega, Madison, WI) and transformed into Escherichia coli
(JM109). Colonies were randomly picked and grown in 96-well
culture plates in 1 ml of LB broth with 100 µg ml
–1
ampicillin.
One µl of overnight culture was used to amplify cDNA inserts
with T7 and SP6 primers by Taq polymerase at an annealing
temperature of 55 °C. The PCR products (10 µl) were sepa
-
rated on 0.9% agarose gel to verify cDNA size, amplification
quantity and quality. Two µl of the derived cDNA inserts were
used for fluorescence cycle sequencing with Big dye termina
-
tor sequencing reagent (Applied Biosystems (ABI), Foster
City, CA) in a volume of 20 µl with T7 or Sp6 primer (96 °C
for 10 s, 50 °C for 5 s and 60 °C for 4 min, 30 cycles). Se
-
quences were determined with an ABI 3700 DNA analyzer. A
homology search against the NCBI protein database was con
-
ducted by BLASTX (http://www.ncbi.nlm.nih.gov/BLAST/).
An E value of 0.02 was adopted as the cut-off threshold for ho
-
mology. Independence of the cDNA clones (contigs) was veri
-
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GENE EXPRESSION IN FLOWER DEVELOPMENT OF ACACIA MANGIUM 169
Figure 1. Morphology of
Acacia mangium flower
development. A–D =
Different developmental
stages of Acacia man
-
gium flowers: (A) floral
bud stage 1; (B) floral
bud stage 2; (C) open
flower stage 1; and (D)
open flower stage 2. (E)
Young seedpods. (F)
Mature seedpods. (G)
Phyllodes. H–L = scan
-
ning electron microscopy
of flowers: (H) floral bud
stage 1; (I) floral bud
stage 2; (J) open flower
stage 1; (K) open flower
stage 2; and (L) pollen.
at University of Portland on May 21, 2011treephys.oxfordjournals.orgDownloaded from
fied with Sequencher software (Gene Codes, Ann Arbor, MI)
with parameters of minimum overlap of 20 bp and minimum
match of 85%. Expression profiles of Arabidopsis homologues
were downloaded from the Nottingham Arabidopsis Stock Cen
-
tre’s Affymetrix microarray database (NASC), which contained
all of the oligonucleotide array data from NASC’s Affymetrix
service as of June 2004.
Analysis of gene expression by macroarray
The amplified cDNAs were arrayed on four replicates of
Hybond N membranes (Amersham Biosciences, Piscataway,
NJ) by a Beckman Coulter 2000 laboratory automation work
-
station (Beckman Coulter, Fullerton, CA). Two cDNAs were
arrayed for independent genes with more than two overlapping
cDNAs. Each cDNA was arrayed on two neighboring spots. A
mixture of ribosomal RNAs (18S and 28S) was used as the hy
-
bridization control (with 16 replicates arrayed at the bottom
right of each membrane). The RNAs from flower bud stage 2
(early flower), open flower stage 1 (late flower), phyllode and
young seedpod tissues were reverse transcribed to cDNAs.
The cDNAs were amplified with CDS primer and SMART II
primer provided by Atlas SMART probe amplification kit
(ClonTech) before random prime labeling with
32
P-dATP as
the probe. Arrayed cDNAs were denatured, neutralized and
then cross-linked to Hybond N membrane by UV Stratalinker
(Stratagene, La Jolla, CA). The membranes were prehybrid-
ized in 5 ml of prehybridization solution containing Smart
blocking solution (ClonTech) at 68 °C for 1 h followed by hy-
bridization to denatured probes at 68 °C for 16 –18 h. Mem-
branes were washed twice for 30 min at 68 °C with 2 × SSC
containing 1% SDS, then once for 30 min at 68 °C with 0.1 ×
SSC, containing 0.5% SDS and finally, for 1530 min at room
temperature with 0.2 × SSC. Image and quantitative informa-
tion were recorded with a BioRad GS525 molecular imaging
system (BioRad, Hercules, CA). The mean value for each in
-
dependent gene was normalized against the mean value of ri
-
bosomal cDNAs in the same membrane (value of an independ
-
ent gene/mean value of ribosomal cDNAs in the membrane)
before comparison across membranes.
RNA gel blot hybridization
Purified total RNAs (20 µg) from young phyllode, mature
phyllode, floral bud stages 1 and 2, open flower stages 1 and 2,
stem, young seedpod, young seed and root were separated by
electrophoresis on 1.2% agarose gel containing 6% formalde
-
hyde. Denatured RNA markers and 1-kb DNA markers were
run together with samples as size standards. The RNAs were
transferred to nylon membranes (Hybond N, Amersham Bio
-
sciences) by capillary transfer in 20 × SSC buffer and fixed on
the membrane by UV light (Stratalinker). Before hybridiza
-
tion, a membrane was stained in 0.04% methylene blue to
check the 18S and 28S rRNA bands to evaluate loading unifor
-
mity and integrity of RNAs. The RNA gel blot hybridization
was carried out as described for the arrayed membranes. After
hybridization to
32
P-dATP random prime labeled cDNAs at
68 °C for 16–18 h, membranes were washed twice for 30 min
at 68 °C with 2 × SSC containing 1% SDS, then for 30 min at
68 °C with 0.1 × SSC containing 0.5% SDS followed by 5 min
at room temperature with 0.2 × SSC before exposure to X-ray
films. Membranes were stripped of probes between hybridi
-
zations by adding boiling 1% SDS followed by a 20-min incu
-
bation at room temperature.
Results
Expression profiling of clones from the flower subtracted
cDNA library
The cDNA inserts from the subtracted library ranged in size
from 0.5 to 2 kb. A total of 1123 clones were sequenced, corre
-
sponding to 576 independent genes (contigs) based on contig
analysis by Sequencher.
The macroarray experiment showed different expression
patterns in flower buds, open flowers, phyllodes and young
seedpods (Figures 2A–D), with overall higher expression in
the open flowers. The mixture of rRNAs that was used as the
hybridization control showed similar hybridization intensity
to the four cDNAs probes (square at the bottom right, H12).
Signal intensities recorded by a phosphoimager were normal
-
ized against hybridization controls (normalized expression
level = mean intensity of all spots for each independent gene/
mean intensity of rRNA mixture in each membrane) before
comparison across membranes. An expression level that was
at least three times higher in flower buds or open flowers as in
phyllodes was the criterion used to identify the 147 flower-
specific genes. Within these flower-specific genes, the cut off
for a significant difference in expression between flower bud
and open flower was twofold. Genes that preferentially ex-
pressed in flower buds were defined as early-flower-specific,
whereas genes that preferably expressed in open flower were
defined as late-flower-specific.
Table 1 lists a selection of flower-specific genes with prod
-
uct classification, clone ID, array location, GenBank accession
number, Arabidopsis protein homologue, BLAST score and
gene name for the Arabidopsis homologue with expression
profile as revealed by NASC Affymetrix microarray database
or other experiments. Proteins encoded by the 147 flower-spe
-
cific genes were classified in Figure 3 as follows. (1) Flower
development-related (FD, 1%), including homeotic proteins
and MYB proteins. (2) Other transcription factors (TF, 4%)
containing leucine-rich repeat domains, zinc finger domains
and bZIP domains. (3) Stress-, hormone-, pathogen-related
(SHP, 10%), including one GA synthesis enzyme (CK468636),
GA regulated proteins, auxin-induced proteins, desiccation-
related proteins and senescence-associated proteins. (4) Regu
-
latory proteins (RP, 11%), including G proteins, protein kinases
and phosphatases. (5) Membrane transport (MT, 2%), inclu
-
ding one late-flower-specific major intrinsic family protein
(CK468600), one glucoses-6-phosphate translocator (CK468
-
675) and one transporter-like protein (CK468659). (6) Metab
-
olism (M, 30%), except the three early-flower-specific thioes
-
terase and hydrolyses (CK468549, CK468563 and CK468
-
571) and one early-flower-specific metallothionein-like pro
-
tein (CK468541), the remaining proteins are either late-flow
-
er-specific or expressed uniformly throughout the whole flow
-
ering process. (7) Cytoskeleton (CS, 1%), including one flow
-
170 WANG, CAO AND HONG
TREE PHYSIOLOGY VOLUME 25, 2005
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er-specific actin (CK468668) and one flower-specific tubulin
(CK468656). (8) Unknown function (U, 18%), homologous to
hypothetical proteins from Arabidopsis, alfalfa or castor bean.
(9) Novel proteins (N, 18%), without homology to any protein
in NCBI protein databases with E value > 0.02.
Assessing the macroarray results by RNA gel blot hybridization
We used RNA gel blot hybridization to confirm and detail ex
-
pression patterns of thirteen flower-specific genes (clones iden
-
tified by a superscript 2 in Table 1). The RNAs from young
phyllode, phyllode, flower bud stages 1 and 2, open flower
stages 1 and 2, young seedpod, young seed, stem and root were
separated on an agarose gel and transferred to Hybond N
membrane before hybridization to 13 selected cDNA probes
(Figure 4). As expected, all the genes were preferentially ex
-
pressed during flower development. Their expression patterns
obtained by RNA gel blot hybridization correlated well with
those revealed by macroarray analysis, but provided more de
-
tails on transcript size, expression level and pattern. Single
transcripts for all but one probe indicate a high level of speci
-
ficity for both macroarray and RNA blot hybridizations. The
expression patterns of the two MADS-box genes differed. A
PISTILLATA homologue (123-E04, Figure 4H) was expressed
early in flower development (bud stage 1) and its transcription
level was high in open flower stages 1 and 2, whereas the
AGL1 homologue (128-G03, Figure 4M) had highest expres
-
sion level in flower bud stage 2 and open flower stage 1, and its
expression declined to a low level in open flower stage 2. Its
transcript was also present in young seeds but not in seedpods,
suggesting that it is involved in embryo or seed development.
This probe also hybridized with another 1.7-kb transcript,
which is specific to flower bud stage 2 and open flower stage 1,
even after stringent washing. The 1.7-kb transcript could be
from an alternative splicing or from a closely related isoform.
Other transcripts appearing early in flower development in
-
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GENE EXPRESSION IN FLOWER DEVELOPMENT OF ACACIA MANGIUM 171
Figure 2. Macroarray analysis
of gene expression in Acacia
mangium. The four identical
membranes A–D were detected
with cDNA probes generated
from flower bud (A), open
flower (B), phyllode (C) and
young seedpod (D), respec-
tively.
Figure 3. Classification of proteins encoded by the 147 flower-spe
-
cific genes.
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172 WANG, CAO AND HONG
TREE PHYSIOLOGY VOLUME 25, 2005
Table 1. Acacia mangium genes for flower development. Class abbreviations: CS = cytoskeleton; FD = flower development-related; M = metabolism; MT = membrane transport; N = novel; RP = regu-
latory protein; SHP = stress-, hormone-, pathogen-related; TF = other transcription factors; and U = unknown function.
Class Clone Array GenBank Expression during Arabidopsis homologue BLAST Gene name Expression in Arabidopsis
location accession A. mangium E-value by NASC Affymetrix oligo-
no.
1
flowering array unless indicated otherwise
CS 130E07 E08-S7 CK468656 Early and late Tubulin beta-1 chain 2.00E-15 At1g75780 Tricellular and mature pollen
CS 131G02 C06-S8 CK468668 Early and late Actin 7 (ACT7) 4.00E-64 At5g09810 Non-specific
FD 121A06 H02-S2 CK468559 Early MADS2 protein (AGL3) 2.00E-28 At2g03710 Flower and aerial parts
FD 121B06
2
A01-S3 CK468561 Late MYB family transcription factor 5.00E-60 At3g27810 Late flower
(MYB3)
FD 121D08 B05-S3 CK468567 Late Ndr family protein 3.00E-37 At5g56750 Tricellular and mature pollen
FD 121G09 D07-S3 CK468575 Early and late MYB transcription factor 2.00E-25 At3g16350 Tricellular and mature pollen
FD 123E04
2
D09-S4 CK468599 Late Floral homeotic protein 4.00E-10 At5g20240 Early and late flower
PISTILLATA (PI)
FD 124B01 G10-S4 CK468606 Early and late MADS-box protein (AGL9) 5.00E-25 At1g24260 Early and late flower
FD 128F08 D07-S6 CK468630 Early and late Putative MYB family 6.00E-18 At3g16350 Tricellular and mature pollen
transcription factor
FD 128G03
2
E01-S6 CK468631 Early and late MADS-box protein (AGL9) 5.00E-59 At1g24260 Early and late flower
FD 128G05 E02-S6 CK468632 Early and late MYB24 2.00E-36 At5g40350 Late flower
FD 131A05 G12-S7 CK468661 Early and late MADS protein (AGL2) 1.00E-54 At3g02310 Early and late flower
FD 206F09 B12-S7 CK468679 Late Floral homeotic protein 1.00E-08 At3g54340 Early and late flower
APETALA3 (AP3)
FD 129B11 F12-S6 CK468637 Early and late Pollen allergen-like protein 2.00E-46 At1g70850 Root
M 116D12 E10-S2 CK468550 Early and late Phenylalanine ammonia-lyase 1 2.00E-47 At2g37040 Non-specific
(PAL1)
M 123H09 F12-S4 CK468603 Early and late Glucosyltransferase 3.00E-29 At4g09500 Non-specific
M 128A11
2
B01-S6 CK468622 Late Flavonol 3-O-glucosyltransferase 1.00E-19 At2g18570 Seed
M 109D08 B04-S2 CK468540 Early and late Asparagine synthetase I (ASN1) 8.00E-88 At3g47340 Stamen
3
M 109E07 B09-S2 CK468541 Early Metallothionein protein 2B (MT-2B) 6.00E-06 At5g02380 Non-specific
M 116D11 E09-S2 CK468549 Early Acyl-ACP thioesterase 1.00E-64 At1g08510 Early and late flower
M 121C10 A10-S3 CK468563 Early GDSL-motif lipase/hydrolase 6.00E-70 At5g33370 Early and late flower
M 121E01
2
B08-S3 CK468568 Late Bet v I allergen family protein 2.00E-35 At1g70830 Stem
M 121F02
2
C06-S3 CK468571 Early Putative GDSL-motif lipase/hydrolase 2.00E-70 At3g04290 Early and late flower
M 122E01 G10-S3 CK468584 Early and late Xyloglucan:xyloglucosyl transferase 7.00E-85 At5g13870 Seed
M 124A09 G07-S4 CK468605 Early and late GDSL-motif lipase/hydrolase 5.00E-25 At2g03980 Mature pollen
M 124B08 H3-S4 CK468607 Early and late Polygalacturonase 2.00E-59 At3g07840 Tricellular pollen
M 130C01
2
D02-S7 CK468653 Late Short-chain dehydrogenase/ 6.00E-40 At3g01980 Non-specific
reductase (SDR)
M 122A04 E06-S3 CK468579 Early and late GDSL-motif lipase/hydrolase 3.00E-79 At3g16370 Leaf
family protein
M 124A07 G06-S4 CK468604 Early and late Bifunctional nuclease (BFN1) 9.00E-16 At1g11190 Stamen
3
MT 123E08 D12-S4 CK468600 Late Major intrinsic family protein 9.00E-30 At2g36830 Non-specific
MT 130H01 G03-S7 CK468659 Early and late Transporter-like protein 9.00E-06 At5g13740 Early and late flower,
early pollen
Continued on facing page.
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GENE EXPRESSION IN FLOWER DEVELOPMENT OF ACACIA MANGIUM 173
Table 1 continued. Acacia mangium genes for flower development. Class abbreviations: CS = cytoskeleton; FD = flower development-related; M = metabolism; MT = membrane transport; N = novel;
RP = regulatory protein; SHP = stress-, hormone-, pathogen-related; TF = other transcription factors; and U = unknown function.
Class Clone Array GenBank Expression during Arabidopsis homologue BLAST Gene name Expression in Arabidopsis
location accession A. mangium E-value by NASC Affymetrix oligo-
no.
1
flowering array unless indicated otherwise
MT 203F03 E12-S8 CK468675 Early and late Glucose-6-phosphate/ 3.00E-62 At1g77610 Non-specific
phosphate translocator
N 001G09
2
E01-S1 CK468537 Early
N 121B07
2
A02-S3 CK468562 Late
N 122D04
2
C03-S5 CK468582 Early
RP 109D06
2
B02-S2 CK468539 Late Expansin, putative (EXP8) 2.00E-16 At2g40610 Late flower and leaf
RP 122D12 G09-S3 CK468583 Early and late Putative mitogen-activated protein 5.00E-80 At2g18170 Non-specific
kinase (MPK 7)
RP 122G12 A07-S4 CK468586 Late Protease inhibitor/seed storage/lipid 1.00E-23 At1g62510 Seed, high in stomata
transfer protein (LTP) family protein closure mutant gpa-1
RP 122H03 A09-S4 CK468587 Early and late CER1-like protein 1.00E-43 At2g37700 Root
RP 123A10 B08-S4 CK468588 Late Proline-rich family protein (PRP4) 3.00E-30 At4g38770 Flower and leaf, down in plants
defective for SA, JA and
ethylene pathways
RP 123D08 D03-S4 CK468596 Early and late Ras-related GTP-binding protein (RAN3) 5.00E-80 At5g55190 Early pollens
RP 123H08 F11-S4 CK468602 Late Embryo-specific protein-related 4.00E-24 At2g41470 Tricellular pollen
RP 124E10 B10-S5 CK468615 Early and late Late embryogenesis abundant family 1.00E-35 At2g44060 Non-specific
protein
RP 127A11 D08-S5 CK468617 Early and late Expansin, putative (EXP11) 1.00E-59 At1g20190 Leaf
RP 128B02 B03-S6 CK468624 Early and late Protein phosphatase 2C family protein 2.00E-95 At5g66080 Late flower
RP 128C07 C04-S6 CK468628 Early and late Protein kinase family protein 5.00E-76 At3g51550 Flower, leaf and suspension cell
RP 129E12 H11-S6 CK468645 Early and late PRLI-interacting factor 1.00E-40 At5g19900 Early and late flower, mature
pollen
RP 129G04 A06-S7 CK468646 Early and late Leucine-rich repeat transmembrane 2.00E-83 At2g01210 Early and late flower, mature
protein kinase pollen
RP 131C01 H11-S7 CK468663 Early and late DC1 domain-containing protein 3.00E-41 At1g60420 Flower, leaf and suspension cell
RP 203G10 F11-S8 CK468676 Early CBL-interacting protein kinase 9 3.00E-42 At1g01140 Non-specific
SHP 001A01 B01-S1 CK468533 Late Gibberellin-regulated protein 4 3.00E-10 At5g15230 Non-specific
SHP 001C10 C07-S1 CK468536 Early and late Auxin-induced protein IAA9 3.00E-59 At5g65670 Non-specific, high in
suspension cell
SHP 121A01 G12-S2 CK468558 Late Gibberellin-regulated protein 4 7.00E-22 At5g15230 Non-specific
SHP 123B03 B11-S4 CK468591 Early and late Late embryogenesis abundant protein 1.00E-34 At2g46140 Tricellular and mature pollen
SHP 123D11 D05-S4 CK468598 Late Senescence/dehydration-associated 5.00E-66 At2g17840 Aerial parts
protein-related (ERD7)
SHP 124C11 A03-S5 CK468610 Early and late Gibberellin-regulated protein 1 7.00E-26 At1g75750 Sepal
3
SHP 128B07 B08-S6 CK468626 Late Auxin-responsive AUX/IAA family 5.00E-16 At4g14550 Root
protein
SHP 128C02 B11-S6 CK468627 Early Harpin-induced family protein 1.00E-50 At3g11660 Aerial parts
Continued overleaf.
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174 WANG, CAO AND HONG
TREE PHYSIOLOGY VOLUME 25, 2005
Table 1 continued. Acacia mangium genes for flower development. Class abbreviations: CS = cytoskeleton; FD = flower development-related; M = metabolism; MT = membrane transport; N = novel;
RP = regulatory protein; SHP = stress-, hormone-, pathogen-related; TF = other transcription factors; and U = unknown function.
Class Clone Array GenBank Expression during Arabidopsis homologue BLAST Gene name Expression in Arabidopsis
location accession A. mangium E-value by NASC Affymetrix oligo-
no.
1
flowering array unless indicated otherwise
SHP 129A10 F03-S6 CK468636 Early and late Gibberellin 20-Oxidase, putative 2.00E-40 At5g51810 Carpel
3
SHP 130G02 F09-S7 CK468657 Early and late Cold-acclimation protein, putative 3.00E-48 At2g15970 Leaf
(FL3-5A3)
SHP 131B05 H07-S7 CK468662 Early and late Auxin-responsive protein/indoleacetic 2.00E-23 At1g04250 Carpel
3
acid-induced protein 17 (IAA17)
SHP 131G06
2
C08-S8 CK468670 Late Gibberellin-regulated family protein 1.00E-25 At5g59845 Stamen
3
TF 116F02 F05-S2 CK468551 Early and late Basic helix-loop-helix (bHLH) family 6.00E-54 At5g62610 Non-specific
protein
TF 124D09 A12-S5 CK468613 Late Leucine-rich repeat protein 5.00E-16 At3g20820 Non-specific
TF 129C03 G02-S6 CK468638 Early and late Dof-type zinc finger domain-containing 2.00E-19 At3g21270 High in stem
protein (ADOF2)
TF 129H09 B04-S7 CK468650 Early and late bHLH family protein 2.00E-19 At1g74500 Root
TF 130D04 E01-S7 CK468655 Early and late bZIP transcription factor 2.00E-11 At1g75390 Early and late flower
TF 130H05 G05-S7 CK468660 Early and late Zinc finger (GATA type) family protein 3.00E-09 At5g25830 Biocellular and tricellular
pollen
U 121E09 C01-S3 CK468570 Early and late Expressed protein 5.00E-37 At3g62730 Stem
U 121D07
2
B04-S3 CK468566 Late Hypothetical protein 4.00E-86 At4g32460 Late flower and seed
1
All 147 flower-specific sequences were submitted to GenBank with accession numbers CK468533CK468679. A complete list can be obtained on request.
2
Expression patterns were confirmed with RNA blot hybridization.
3
Identified by cDNA and oligonucleotide array of Wellmer et al. (2004).
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cluded those for two novel genes (122-D04, Figure 4K, and
001-G09, Figure 4F) and a GDSL-motif lipase gene (121-F02,
Figure 4B). The first novel gene transcript peaked at flower
bud stage 2 and open flower stage 1, degraded in open flower
stage 2 and remained in young seed. However, the lipase tran
-
script and the second novel gene transcript were specific to
flower bud stage 2. Transcripts for a DC1 domain-containing
protein gene (130- C01, Figure 4D), a novel gene (121-B07,
Figure 4G), a hypothetical protein gene (121-D07, Figure 4L)
and an expansin gene (109-D06, Figure 4E) were specific to
open flower stage 1. A Myb gene transcript (121-B06, Figure
4A) was present at open flower stages 1 and 2. Similarly, one
flavonol 3-O-glucosyltransferase gene (128-A11, Figure 4J)
and a Bet v I allergen family protein gene (121-E01, Figure
4C) expressed strongly in open flower stages 1 and 2.
Discussion
Subtractive hybridization allows comparison of two popula
-
tions of mRNAs in order to obtain clones of genes that are
preferentially expressed in one population (Duguid and Di
-
nauer 1990). Based on this technique, mRNA populations are
first converted to cDNAs. The cDNA population with differen
-
tially expressed transcripts (tester) and the reference cDNA
(driver) are hybridized and the hybrid sequences are then re-
moved. The remaining unhybridized cDNA represent genes
that are preferentially expressed or tester-specific. In our ex-
periment, the cDNA from the flower was used as the tester and
the cDNA from the vegetative and photosynthetic phyllode
was used as the reference (driver). Differential cDNA was se-
lectively amplified, cloned and sequenced. Of the 1123 ESTs,
only eight (0.7%) encoded proteins involved in photosynthe
-
sis, indicating satisfactory efficiency of subtraction.
Array analysis is a powerful experimental approach for dis
-
covering and characterizing genes with differential expression
levels and patterns. Newly discovered genes can be assigned
putative functions by sequence analysis against known genes
from other species and comparison of expression patterns.
Microarray was used to monitor the expression of 1800 genes
during strawberry fruit development and identified 200 genes
whose expression varied with developmental stage. A novel
strawberry alcohol acyltransferase gene that plays a crucial
role in flavor biogenesis in ripening fruit was identified (Ahar
-
oni et al. 2000). Reymond et al. (2000) used microarrays to an
-
alyze the expression of 150 genes in mechanically woun
-
ded leaves of Arabidopsis and identified one COI1-dependent
gene. The expression of this gene was induced by insect feed
-
ing but not by wounding. We found 147 genes preferentially
expressed in the early, late or entire flowering process. Be
-
cause Arabidopsis flower development has been best studied,
we compared our genes with the Arabiodopsis homologues to
determine their particular roles in A. mangium flowering.
Among the many public sources of microarray data for Arabi
-
dopsis genes, we chose Arabidopsis full genome oligoarray
data provided by Nottingham Arabidopsis Stock Centre’s Affy-
metrix microarray database. The NASC Affymetrix oligo Gene-
Chip (ATH1-121501) covers the full Arabidopsis genome.
Specificity of hybridization was ensured by using 11 probe
pairs per gene. Importantly, the same chip and protocol were
used for all experiments to allow comparisons among samples
from different experiments. By June 2004, the database had
data for more than 1000 chips, including experiments on
TREE PHYSIOLOGY ONLINE at http://heronpublishing.com
GENE EXPRESSION IN FLOWER DEVELOPMENT OF ACACIA MANGIUM 175
Figure 4. An RNA blot
hybridization of selec
-
ted genes. The cDNA
probes were hybridized
to RNAs from young
phyllode (YP), phyllode
(P), floral bud stage 1
(B1), floral bud stage 2
(B2), open flower stage
1 (O1), open flower
stage 2 (O2), young
seedpod (YSP), young
seed (YS), stem (St)
and root (Ro). (A) 121-
B06 (CK468561), a
Myb gene; (B) 121-F02
(CK468571), a lipase
gene; (C) 121-E01
(CK468568), a Bet v I
allergen family protein
gene; (D) 130-C01
(CK468653), a DC1 do
-
main containing protein
gene; (E) 109-D06
(CK468539), an expan-
sin gene; (F) 001-G09 (CK468537), a novel gene; (G) 121-B07 (CK468562), a novel gene; (H) 123-E04 (CK468599), a PISTILLATA homolo
-
gue; (I) 131-G06 (CK468670), a GA-stimulated transcript; (J) 128-A11 (CK468622), an anthocyanin synthesis gene; (K) 122-D04 (CK468
-
582), a novel gene; (L) 121-D07 (CK468566), a gene for a hypothetical protein; (M) 128-G03 (CK468631), an AGL1 homologue; and (28S)
staining of 28S ribosomal RNA.
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flower development (Craigon et al. 2004). Expression profiles
from this database were verified and supplemented with data
from recent microarray studies on Arabidopsis floral develop
-
ment (Honys et al. 2003, Hu et al. 2003, Zik et al. 2003,
Wellmer et al. 2004).
The MADS-domain-containing proteins are a diverse class
of transcription factors that are involved in regulating develop
-
mental processes. In plants, they were first discovered to be re
-
quired for floral meristem and organ identity. We identified six
flower-specific MADS-box genes (4% of total number of
flower-specific genes). Macroarray analysis, as well as RNA
blot hybridization, indicated their varied expression levels and
patterns during A. mangium flower development. The AGL2
and AGL9 homologues (131-A05, 128-G03, 124-B01)ex
-
pressed throughout the flowering process, just like AGL2 and
AGL9 in Arabidopsis. It has been found that almost all of the
plant MADS-box genes that are involved in the floral transi
-
tion belong to the AP1/AGL9 subfamily (Theissen et al. 1996)
and their expression extends to later stages (Moon et al. 1999).
We suggest, therefore, that the AGL9 homologue may play an
important role in floral transition in A. mangium like its homo
-
logues in other plants. Acacia mangium AGL2 may also serve
the same fundamental roles in the development of all floral or
-
gans (Flanagan and Ma 1994). The AGL3 homologue (121-
A06) expresses early in flowering, like Arabidopsis AGL3,but
not in aerial parts of the plant, suggesting that it has a more
specific role in A. mangium flower development than does
AGL3 in Arabidopsis (Huang et al. 1995). The PI and AP3
homologues (123-E04 and 206-F09) express during early flo-
wer development but with higher expression during late flower
development, consistent with their possible function of main-
taining organ identity (function B). Myb genes are well known
for controlling anthocyanin synthesis (Sablowski et al. 1994,
Moyano et al. 1996, Uimari and Strommer 1997). Recent stud
-
ies also implicated Myb genes in regulating anther and pollen
development (Higginson et al. 2003, Murray et al. 2003, Stei
-
ner-Lange et al. 2003). We identified four Myb genes (128-
G05, 121-B06, 121-G09, 128-F08) that express during the
flowering process in Acacia. The Arabidopsis homologues for
two Myb genes (121-G09, 128-F08) express strongly and spe
-
cifically in tricellular and mature pollen, suggesting that they
have important roles in pollen development. Our approach
also identified several genes for other transcription factors, in
-
cluding one zinc finger (GATA-type) family protein (131-
H05), a protein with a leucine-rich repeat domain (124-D09)
and another with bZIP domain (130-D04). The Arabidopsis
homologue for the zinc finger family protein is specific to
biocelluar and tricellular pollen, strongly suggesting similar
involvement in pollen development for its A. mangium homo
-
logue. The A. mangium and Arabidopsis bZIP transcription
factor homologues all express throughout flowering, in line
with possible roles in floral symmetry and organ polarity
(Smyth 2001).
Besides transcription factors, many other flower-specific
genes for putative regulatory proteins were identified. They in
-
cluded genes for two receptor protein kinases (128-C07, 129-
G04), one GTP-binding protein (123-D08) and a protein phos
-
phatase 2C (128-B02). Their Arabidopsis homologues also ex
-
press in flower development, suggesting their similar involve
-
ment. Two flower-specific expansins were identified (109-D06
and 127-A11). Expansins function in cell wall extension and
increase wall stress relaxation (Cosgrove 1998). The two A. man
-
gium expansin genes that we identified were expressed in the
later flowering stages (see Table 1 and Figure 4E) when rapid
growth and fertilization occur. Similarly, the Arabidopsis homo
-
logue to one of these genes (109-D06) is expressed during late
flower development (Wellmer et al. 2004).
About 30% of the flower-specific genes were involved in
metabolism, including genes involved in amino acid metabo
-
lism such as asparagine synthetase (129-D08), genes for thio
-
esterase, hydrolases and lipases (116-D11, 121-C10, 121-F02,
124-A09), genes for cell wall-associated products like endo-
xyloglucan transferase (122-E01) and polygalacturonase (124-
B08). Arabidopsis homologues to a GDSL-motif lipase and
asparagine synthetase were identified as target genes regulated
by APETALA3 and PISTILLATA in Arabidopsis (Zik and Irish
2003). These genes are involved in basic cellular processes
like cell wall formation and cell metabolism and are highly ex
-
pressed in order to meet the demand for rapid growth of floral
organs. The highly specific expression of Arabodopsis homo
-
logues in seed (xyloglucan transferase), mature pollen and
tricellular pollen (GDSL-motif lipase) suggests similar in-
volvement of A. mangium homologues in seed and pollen de-
velopment.
A high percentage of A. mangium flower-specific genes
(8%) encode proteins involved in phytohormone and stress re-
sponses. The genes for a gibberellin oxidase (129-A10) and
four GA-regulated proteins (001-A01, 121-A01, 124-C11 and
131-G06) implicate GA in A. mangium flower development.
This is in line with the finding that the Arabidopsis gibberellin
20-oxidase homologue is located in the carpel and two Arabi-
dopsis GA-regulated protein homologues are localized in the
stamen and sepal (Wellmer et al. 2004). The Arabidopsis
homologue for one of the three auxin-induced proteins (131-
B05) is expressed in the stamen, whereas those for the other
two are nonspecific. Arabidopsis homologues to 15 A. man
-
gium flower-specific genes are not specific to floral develop
-
ment; this may be because of divergent functions of similar
types of proteins in different plants. These 15 genes encode
regulatory proteins and proteins involved in metabolism.
We found a high percentage of homologues to hypothe
-
tical proteins (18%, predicted open reading frames mostly
from Arabidopsis whole genome sequence) and flower-spe
-
cific genes encoding unknown proteins (18%). This is in
agreement with the finding of Sterky et al. (1998) that as much
as 37% of independent ESTs from the cambium region of pop
-
lar encode either novel proteins or hypothetical proteins. Be
-
cause the mean length of sequenced regions was only 600 bp,
it is possible that sequenced regions were too short for a homo
-
logy search in the database.
There has been increasing interest in introducing beneficial
traits into tree species. Successes in genetic modifications to
lignin synthesis have been reported for poplar (O’Connell et
al. 2002, Pilate et al. 2002). A barrier to the exploitation of ge
-
netic modifications in trees is the concern that transgenes in
genetically modified trees might be passed to natural forests
176 WANG, CAO AND HONG
TREE PHYSIOLOGY VOLUME 25, 2005
at University of Portland on May 21, 2011treephys.oxfordjournals.orgDownloaded from
because trees live much longer than herbaceous species and
birds can carry seeds over long distances. Effective gene con
-
tainment is therefore necessary before genetically modified
trees can be used in commercial plantations. The requirement
for gene containment poses technical challenges for those tree
species relying on seeds for propagation. It is, however, less of
an issue for tree species like A. mangium that can be readily
propagated vegetatively. A genetically modified A. mangium
plant with its reproduction controlled can be propagated and
its clones planted on a large scale, because the sterile clonal
trees will pose no threat to the natural environment. However,
A. mangium is characterized by early flowering and a long an
-
nual flowering period, features that are not beneficial to com
-
mercial wood production because reproduction drains energy
at the expense of vegetative growth. Thus, control of reproduc
-
tion in a genetically modified A. mangium not only elimi
-
nates uncontrolled gene flow, but may increase its vegetative
growth. The large number of flower-specific genes in A. man
-
gium provides many possibilities for genetic modification.
Populus has become the model woody plant to complement
the genetic resource being developed in Arabidopsis (Taylor
2002). Some of the reasons for this choice include its high
growth rate, commercial value, amenability to genetic modifi
-
cation, small genome, commitment to sequencing the whole
genome and the availability of many ESTs. The limitations of
poplar as a model woody plant are that it is dioecious, that it
takes a long time to flower (6 years) and that Populus species
are representative of trees in the Northern Hemisphere only.
We argue that a representative tree in the Southern Hemisphere
should also be developed as a model woody plant. Acacia spe-
cies are representative of tree species in the Southern Hemi-
sphere and there is wide genetic diversity among more than
1300 species within the genus; they are among the fastest
growing trees on earth and are leguminous. Species like A. man
-
gium have great commercial value, are amenable to genetic
transformation (Xie and Hong 2002), are monoecious and
flower at an early age (within 2 years in vegetatively propa
-
gated clones). Acacia is a diploid tree species (2n =2x = 26)
with 14–15 linkage groups and the integrated map spans 950
to 1045 cM (Butcher et al. 2002) (Populus has a diploid ge
-
nome with 2n = 38 and a genetic map spanning 2927 cM for a
genome size of about 550 Mb (Wu et al. 2000)). The finding
that most of the genes we identified were single copy genes as
revealed by genomic Southern blot hybridization (data not
shown) also suggests a simple genome for A. mangium. All
these features indicate that A. mangium has the potential to
serve as another model tree species.
In conclusion, we identified 147 genes that were specifically
expressed during flower development in A. mangium, a repre
-
sentative legume tree species in the Southern Hemisphere. Many
of the genes have Arabidopsis homologues that are implicated
in flower development, suggesting general conservation in flo
-
ral development. The remainder of the genes either have Ara
-
bidopsis homologues that are nonspecific to flower develop
-
ment or code for unknown proteins. Further characterization
of these conserved and different genes will increase our under
-
standing of flower development in general, and facilitate ma
-
nipulation of reproduction in tree species.
Acknowledgments
We thank Miss Joanne Leong, Mr. Lin Qingwen, Ms. Chan Yangsun
and all members of the sequencing laboratory for technical help. We
also thank Dr. Megan Griffith for critical review of the manuscript.
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178 WANG, CAO AND HONG
TREE PHYSIOLOGY VOLUME 25, 2005
at University of Portland on May 21, 2011treephys.oxfordjournals.orgDownloaded from
... To the best of our knowledge, no descriptive palynological studies have been reported yet. However, Wang et al. [58] documented only the isolation and characterization of flower-specific transcripts in Acacia mangium. ...
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... The QTL FL_06-07(2) in chr. III was associated with Mtr1g116330, a PRLI-interacting factor specifically expressed during flower development in Acacia mangium 54 . In rice, genetic analysis and fine mapping of a set of complementary genes controlling late heading (flowering time) also identified a PRLI-interacting factor as a putative candidate for LH1 55 . ...
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... For example, using SSH, bractspecific genes have been successfully identified in the ornamental tree Davidia involucrata (Li et al., 2002), and genes responsive to benzothiadiazole (BTH; used to induce systemic acquired resistance) in the tropical fruit tree papaya (Qiu et al., 2004). Genes involved in flowering have also been isolated from carnation (Dianthus caryophyllus; Ok et al., 2003) and black wattle (Acacia mangium; Wang, Cao and Hong, 2005) using this method. ...
... A partir de la descripción de las rutas relacionadas con la floración en A. thaliana, Wang et al. (2005) identificaron 147 genes expresados durante la floración en A. mangium. De estos, el 26% están relacionados con el metabolismo (asparaginasintetasa, tioesterasa, hidrolasas y lipasas), el 18% son nuevos, 18% son genes específicos con función desconocida, 10% de ellos tienen funciones relacionadas con el desarrollo floral y 11% son reguladores de proteínas. ...
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Controlling gene expression levels is one of the key regulatory mechanisms used by living cells to sustain and execute their operations. Monitoring gene expression has been for more than a decade an important molecular tool for providing clues to gene function and for new perspectives on spatial and temporal cellular activities. Genome sequencing and the availability of large sets of expressed sequence tags (ESTs) from numerous organisms urged the development of efficient and accurate methods for large-scale and genome-wide analyses of genetic variation and expression patterns. Novel methods based on either gel separation (Bruce et al. 2000), counting of tags and signatures of DNA fragments (Brenner et al. 2000) or on specific hybridizations of nucleic acids to macro — and microarrays (Schena et al. 1995; Lockhart et al. 1996; Desprez et al. 1998) are currently available.
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The MADS-box encodes a novel type of DNA-binding domain found so far in a diverse group of transcription factors from yeast, animals, and seed plants. Here, our first aim was to evaluate the primary structure of the MADS-box. Compilation of the 107 currently available MADS-domain sequences resulted in a signature which can strictly discriminate between genes possessing or lacking a MADS-domain and allowed a classification of MADS-domain proteins into several distinct subfamilies. A comprehensive phylogenetic analysis of known eukaryotic MADS-box genes, which is the first comprising animal as well as fungal and plant homologs, showed that the vast majority of subfamily members appear on distinct subtrees of phylogenetic trees, suggesting that subfamilies represent monophyletic gene clades and providing the proposed classification scheme with a sound evolutionary basis. A reconstruction of the history of the MADS-box gene subfamilies based on the taxonomic distribution of contemporary subfamily members revealed that each subfamily comprises highly conserved putative orthologs and recent paralogs. Some subfamilies must be very old (1,000 MY or more), while others are more recent. In general, subfamily members tend to share highly similar sequences, expression patterns, and related functions. The defined species distribution, specific function, and strong evolutionary conservation of the members of most subfamilies suggest that the establishment of different subfamilies was followed by rapid fixation and was thus highly advantageous during eukaryotic evolution. These gene subfamilies may have been essential prerequisites for the establishment of several complex eukaryotic body structures, such as muscles in animals and certain reproductive structures in higher plants, and of some signal transduction pathways. Phylogenetic trees indicate that after establishment of different subfamilies, additional gene duplications led to a further increase in the number of MADS-box genes. However, several molecular mechanisms of MADS-box gene diversification were used to a quite different extent during animal and plant evolution. Known plant MADS-domain sequences diverged much faster than those of animals, and gene duplication and sequence diversification were extensively used for the creation of new genes during plant evolution, resulting in a relatively large number of interacting genes. In contrast, the available data on animal genes suggest that increase in gene number was only moderate in the lineage leading to mammals, but in the case of MEF2-like gene products, heterodimerization between different splice variants may have increased the combinatorial possibilities of interactions considerably. These observations demonstrate that in metazoan and plant evolution, increased combinatorial possibilities of MADS-box gene product interactions correlated with the evolution of increasingly complex body plans.