Journal of Experimental Botany, Vol. 54, No. 386, pp. 1343±1350, May 2003
DOI: 10.1093/jxb/erg145
RESEARCH PAPER
The molecular characterization of PaHB2, a homeobox
gene of the HD-GL2 family expressed during embryo
development in Norway spruce
Mathieu Ingouff1,3, Isabelle Farbos2, Malgorzata Wiweger1,4 and Sara von Arnold1,5
1
Swedish University of Agricultural Sciences, Department of Forest Genetics, Box 7027, S-750 07 Uppsala
Sweden
2
Swedish University of Agricultural Sciences, Department of Plant Biology, Box 7080, S-750 07 Uppsala,
Sweden
Received 2 October 2002; Accepted 23 January 2003
Abstract
PaHB1 (for Picea abies Homeobox1), an evolutionarily conserved HD-GL2 homeobox gene, speci®cally
expressed in the protoderm during somatic embryogenesis in the gymnosperm Norway spruce has been
reported previously. An additional HD-GL2 gene designated PaHB2 is reported here. During somatic
embryogenesis, the PaHB2 gene is uniformly expressed in proembryogenic masses and in early
somatic embryos, but it is not detectably transcribed
at the beginning of maturation. In mature embryos,
PaHB2 expression was essentially detected in the
outermost layer of the cortex and the root cap. A
similar PaHB2 expression is detected post-embryonically in both the primary root and the hypocotyl.
Phylogenetic reconstructions and intron pattern
analyses revealed that the PAHB proteins fall within
two distinct subclasses comprising highly similar
angiosperm homologues. The PAHB1 subclass consists of protoderm/epiderm-speci®c members. By
contrast, the PAHB2 subclass gathers homologues
with a subepidermal and protodermal/epidermal
activity. This study suggests that at least two distinct
HD-GL2 genes with a layer-speci®c expression
already existed in the last common ancestor of
angiosperms and gymnosperms. The conserved protodermal/epidermal and subepidermal expression of
HD-GL2 genes could be used to study embryo radial
pattern formation across seed plants.
Key words: Embryogenesis, gymnosperm,
homeobox, Picea abies, radial pattern.
HD-GL2,
Introduction
During embryo development in seed plants, the blueprint
of the future adult plant is laid down. This basic body plan
in angiosperms formally consists of a root/shoot axis of
polarity from which embryonic organ systems generate
and a radial organization of primary tissue layers: epidermis (derived from the protoderm), cortex and endodermis
(derived from the ground tissue), pericycle and vascular
tissues (derived from the procambium). Although gymnosperm embryo development generally starts with a freenuclear stage, the mature embryo also features a similar
general body plan: a radial pattern of primary tissues and
two meristematic poles (Romberger et al., 1993; Singh,
1978).
Despite striking histological similarities between embryos of these two groups that diverged approximately 300
million years ago (Stewart and Rothwell, 1993), comparative molecular studies of embryo development between
gymnosperms and angiosperms are rare. Consequently, the
similarities in the developmental processes controlling
embryo development in these two groups are unknown.
Using the gymnosperm Norway spruce somatic embryogenesis as a model (Filonova et al., 2000), the aim was to
characterize the developmental genes conserved across
gymnosperms and angiosperms. This approach might
3
Present address: Unite Mixte de Recherche 5667, Centre National de la Recherche Scienti®que, Institut National de la Recherche Agronomique, Ecole
Normale SupeÂrieure de Lyon, Universite de Lyon I, 46 AlleÂe d'Italie, F-69364 Lyon Cedex 07, France.
4
Permanent address: Plant Breeding and Acclimatization Institute, Radzikow, 05-870 Blonie, Poland.
5
To whom correspondence should be addressed. Fax: +46 18 67 3279. E-mail: [email protected]
Journal of Experimental Botany, Vol. 54, No. 386, ã Society for Experimental Biology 2003; all rights reserved
1344 Ingouff et al.
unravel common developmental processes between the
embryo of the gymnosperms and the angiosperms.
Homeobox genes encode homeodomain (HD) transcription regulators found in all kinds of eukaryotic organisms
including yeast, animals and plants. The homeodomain
proteins are typically involved in cell differentiation and
patterning (Gehring et al., 1994; Chan et al., 1998;
Kappen, 2000). The AtML1 (for Arabidopsis thaliana
meristem L1 layer) gene (Lu et al., 1996; Sessions et al.,
1999), related counterparts in maize ZmOCL (for Zea mays
outer cell layer) (Ingram et al., 1999, 2000) and rice
(ROC1) (Ito et al., 2002) are essentially protodermal/
epidermal-speci®c in angiosperm embryos. They belong to
the Homeodomain-Glabra2 (HD-GL2) plant-speci®c family (Lu et al., 1996) and their members are characterized by
an amino-terminal homeodomain linked to a putative
leucine zipper (HD-ZIP) followed by a sterol/lipid-binding
START domain (Ponting and Aravind, 1999; Tsujishita
and Hurley, 2000). The recently cloned Norway spruce
homeobox gene PaHB1 (Picea abies Homeobox1) belongs
to the HD-GL2 family (Ingouff et al., 2001). Similarly to
its angiosperm homologues, PaHB1 expression switches
from a ubiquitous expression to a protoderm-speci®c
localization during somatic embryo development (Ingouff
et al., 2001). The conserved protoderm-speci®c pattern of
HD-GL2 genes among highly divergent plants raises the
possibility of conserved function and similar developmental pathways during embryo development in seed plants.
Recently another HD-GL2 gene, ANTHOCYANINLESS2
(ANL2) was isolated in Arabidopsis (Kubo et al., 1999).
The anl2 mutant has an aberrant radial root patterning
(Kubo et al., 1999). The anl2 roots produce extra cells
called intervening cells, located between the cortical and
epidermal layers. ANL2 might be involved in maintaining
subepidermal-layer identity during plant development.
A new member of the HD-GL2 family from Norway
spruce called PaHB2 is reported here. The PaHB2 gene
structure and the corresponding predicted PAHB2 protein,
are highly similar to those of the ANL2 gene. PaHB2
transcripts are uniformly expressed in early somatic
embryos to become preferentially localized in the outer
cortical layer and the root cap in mature embryos. A
similar expression pattern is perpetuated post-embryonically in the primary root and the hypocotyl. Phylogenetic
reconstructions and intron pattern analyses revealed that
the PAHB proteins belong to two distinct subclasses with
highly similar angiosperm homologues. The PAHB1
subclass gathers protoderm/epiderm-speci®c angiosperm
members whereas the PAHB2 subclass is more heterogeneous and consists of homologues with a subepidermal
or epidermal activity. This study suggests that at least two
distinct HD-GL2 genes with a layer-speci®c expression
already existed in the last common ancestor of angiosperms and gymnosperms.
Materials and methods
Plant material
Embryogenic cell lines of Norway spruce (Picea abies (L.) Karst)
were initiated from mature zygotic embryos and established as
described by Egertsdotter and von Arnold (1993). The cell line
95:88:22 was stored in liquid nitrogen and then thawed 2 months
before being used in these experiments. Embryogenic cultures were
maintained under proliferation conditions and induced to mature
according to Filonova et al. (2000).
Surface-sterilized seeds of Norway spruce were germinated in
Magenta jars containing sterile vermiculite and water. The seedlings
were grown at 22 °C under continuous light (200±300 mmol cm±2 s±1).
After 6 weeks, each part of the seedlings (needles, hypocotyl,
epicotyl, and root) was collected and frozen at ±80 °C until use.
Immature female and male strobili cones were harvested from a
40-year-old Norway spruce tree, 2 weeks prior to pollen release.
RNA extraction, cDNA synthesis
Total RNA was extracted according to Chang et al. (1993). Five
micrograms of DNase I-treated total RNA were subjected to a
reverse-transcription using SuperScript II (Gibco-BRL) and an
adapter-dT17 primer (5¢-GACTCGAGTCGACATCGAT17(G/A/C)-3¢)
according to the manufacturer's instructions. The absence of
genomic DNA contamination in single-strand cDNA (ss-cDNA)
samples was monitored by PCR (35 cycles: 94 °C/20 s, 55 °C/20 s,
72 °C/1 min) using P9 and P11 oligonucleotides. These two primers
are speci®c for a p34cdc2 protein kinase from Norway spruce
(cdc2Pa) and span two introns (Kvarnheden et al., 1995).
Cloning of PaHB2 cDNA
The PaHB2 cDNA was isolated using two previously described
degenerate oligonucleotides d-HOX2 and d-HOX3 (Ingouff et al.,
2001). The corresponding amino acids in the HD-GL2 homeodomain are HPDDEKQR and QVKFWFQ, respectively.
A 3¢-RACE PCR reaction was carried out for 35 cycles (94 °C/20
s, 47 °C/20 s, 72 °C/1 min) using ss-cDNA from proliferating
cultures and the oligonucleotides d-HOX2 and adapter (5¢-GACTCGAGTCGACATCGA-3¢). One tenth of the ®rst PCR reaction
was then ampli®ed with the nested d-HOX3 and the adapter
oligonucleotides.
The ampli®cation products were visualized by agarose gel
blotting, probed with the full-length PaHB1 cDNA and washed at
low stringency.
Two major signals were obtained in the d-HOX3-adapter PCR
reaction: one at 2.1 kb and another at 2.5 kb. The two bands were
puri®ed from an agarose gel and cloned in pGEM-T-Easy vector
(Promega) to be sequenced. The partial 2.1 kb-insert sequence was
identical to PaHB1 whereas the partial 2.5 kb-insert sequence was
similar but not identical to PaHB1. Speci®c primers derived from the
sequenced 2.5 kb-fragment were used to carry out 5¢-RACE-PCR
according to Zhang and Frohman (1998).
The integrity of the PaHB2 sequence was checked by sequencing
three independent PaHB2 PCR products with the dye-terminator
PRISM ready reaction kit (Amersham-Pharmacia) and an ABI
PRISM 377 automated sequencer (Applied Biosciences).
Analysis of exon/intron organization
Total genomic DNA was extracted according to Bernatzky and
Tanksley (1986). Seventy nanograms of DNA were used as a
substrate for the PCR reaction (94 °C/15 s, 59 °C/15 s, 68 °C/5 min,
35 cycles) with the oligonucleotides 5HS0 (5¢-GAGGTATTAGGCTACAAGGGTTTG-3¢) and 3HR6 (5¢-GAAGCTTCAGTTTCTGTCTGTC-3¢). Alignments of sequences from the PCR-ampli®ed
Molecular characterization of PaHB2 1345
genomic fragment with the full-length PaHB2 cDNA were carried
out to determine the exon±intron boundaries.
Phylogenetic analysis
Amino acid sequences deduced from 20 HD-GL2 genes were
aligned using CLUSTAL W (Thompson et al., 1994) and further
re®ned manually (complete alignment available from the authors on
request). The phylogenetic analyses were based on the amino acid
region between the homeodomain and the carboxy-terminal end of
the protein. Regions with gaps in the alignment were excluded from
the analyses. Phylogenetic trees were constructed with Neighbor±
Joining (NJ) and maximum parsimony (MP) methods. NJ analyses
were carried out in MEGA2 (Kumar et al., 2000) by using the
Poisson correction distance. The MP searches were conducted in
PAUP version 4.0b (Swofford, 1999). Heuristic search strategies,
consisting of ASIS addition sequences followed by TBR branch
swapping, were performed. Nodal support was estimated using
bootstrap analyses based on 1000 and 500 replicates in NJ and MP
analyses respectively.
Expression pattern analysis by RT-PCR
One-tenth of each ss-cDNA sample was ampli®ed by PCR (94 °C/15
s, 55 °C/15 s, 72 °C/1 min, 35 cycles) with the oligonucleotides
3HS7 (5¢-CTGGCTTTGCCATTCTT-3¢) and 3HR6 (5¢-GAAGCTTCAGTTTCTGTCTGTC-3¢) located in the 3¢-untranslated region
(UTR) of PaHB2. The PCR products were visualized by agarose gel
blotting and probed with the full-length PaHB2 cDNA under
stringent conditions.
In situ hybridization
The proembryogenic masses of proliferating cultures, maturing and
mature somatic embryos placed, respectively, 1 week and 3 weeks
on ABA, and germinated seeds were ®xed for 5 h under vacuum in
4% paraformaldehyde, 5% acetic acid and 50% ethanol. The tissues
were dehydrated through a graded series of ethanol (50±100% [v/v])
and embedded in Paraplast Plus (Sigma).
A 253 bp PCR fragment located in the 5¢-UTR of PaHB2 was
ligated into pGEM-T Easy (Promega). The sense and antisense
riboprobes were labelled with digoxigenin using T7 and SP6
polymerases (DIG RNA Labeling Kit, Roche Biochemicals). The
pre-hybridization, hybridization and washes were performed according to Langdale (1992) with the modi®cations described by CantoÂn
et al. (1999).
Results
Molecular cloning of PaHB2 cDNA
To identify additional HD-GL2 genes expressed during
somatic embryogenesis in Norway spruce, two previously
described degenerate oligonucleotides binding in the
homeobox and RT-PCR were used (Ingouff et al., 2001).
Two main PCR products of approximately 2100 bp and
2500 bp were ampli®ed from Norway spruce proliferating
embryogenic cell cDNA. The sequences obtained from
the 2100 bp PCR product were identical to PaHB1. The
sequenced 2500 bp PCR fragment was different from
PaHB1 and designated PaHB2. Speci®c primers were
designed and used to isolate the full-length PaHB2 cDNA
by RACE experiments (Zhang and Frohman, 1998).
The longest PaHB2 cDNA was 3110 bp long (Genbank
accession number AF328842), with a putative ORF of
2126 bp. The PaHB2 gene encodes a predicted protein of
708 amino acids, which is highly similar to HD-GL2
proteins. PAHB2 is 60% and 56% identical to the
Arabidopsis proteins ANL2 and GL2-1, respectively, and
60% identical to OCL1 from maize. The N-terminal part of
PAHB2 (amino acids 1±25) is shorter than in its close
relatives. When this region is omitted in the sequence
comparisons, PAHB2 shares 68% identity with ANL2 and
63% identity with GL2-1 and OCL1. PAHB2 shows 56%
identity with PAHB1 (62% with the N-terminal region
excluded).
The PAHB2 protein presents a typical HD-GL2 protein
domain organization: an N-terminal homeodomain linked
to a non-canonical leucine zipper domain, and a putative
StAR-related lipid transfer (START) domain. The
N-terminal end of PAHB2 comprises a short stretch rich
in aspartate and glycine, which is reminiscent of the
N-terminal regions of HD-GL2 proteins.
The longest 5¢- and 3¢-untranslated regions (UTR) of
PaHB2 examined are 397 bp and 587 bp, respectively. A
BLAST search in the UTR database (Pesole et al., 2000)
with the 5¢-UTR of PaHB2 showed no signi®cant sequence
similarity. A similar sequence analysis of 3¢-UTR revealed
a highly conserved 17-nt long motif in the PaHB2 gene and
close homologues (ANL2, OCL1, OCL2, OCL3). This
motif was also present in PaHB1 and most of the
angiosperm HD-GL2 genes used in this study (Table 1).
It is noteworthy that this motif was not detected in FWA,
GL2 and PDF2 from Arabidopsis, HAHR1 from sun¯ower
and GhGL2 from cotton.
PaHB2 encodes a HD-GL2 protein homologous to
ANL2
To visualize the evolutionary relationships between members of the HD-GL2 family, an unrooted phylogenetic tree
was generated by the Neighbor±Joining distance method,
presented in Fig. 1A, using an amino acid alignment
comprising the HD-ZIP motif, the START domain and the
downstream region of the protein until the carboxyterminal end. The phylogenetic analysis revealed that the
HD-GL2 family consists of at least three distinct
subgroups comprising proteins from monocot and dicot
plants. In the maximum parsimony analysis, the general
tree topology was unchanged but the de®ned subgroups
were supported with lower bootstrap values (data not
shown).
The gymnosperm protein PAHB1 belongs to the highly
supported subclass including ATML1, PDF2, GL2-2,
GL2-3, ZMOCL5, and O39. However, the spruce protein
PAHB2 together with these angiosperm proteins, ANL2
and GL2-1 from Arabidopsis and OCL1, OCL2 and OCL3
from maize represent a second subclass. The third subclass
only consists of angiosperm proteins: GL2, GL2-4 and
GL2-5 from Arabidopsis, OCL4 from maize, HAHR1
from sun¯ower, and GHGL2 from cotton.
1346 Ingouff et al.
Table 1. Conservation of a 17-nt long sequence element in the 3¢-untranslated region (UTR) of PaHB2 mRNA and other mRNA
from HD-GL2 genes
This motif was not detected in the following genes: FWA, GL2 and PDF2 from Arabidopsis, HAHR1 from sun¯ower and GhGL2 from cotton.
Nucleotides that are not conserved in the motif are indicated in small letters. Accession numbers of the genes used in this analysis: PaHB1
(AF172931), PaHB2 (AF328842), this paper, ANL2 (AF077335), AtML1 (U37589, At4g21750), GL2-1 (AJ224338, At3g61150), GL2-2
(AC000098, At1g05230), GL2-3 (AC005700, At2g32370), GL2-4 (Z97344, At4g17710), GL2-5 (AB013394, At5g46880), O39 (U34743), OCL1
(Y17898), OCL2 (AJ250984), OCL3 (AJ250985), OCL4 (AJ250986), OCL5 (AJ250987).
Species
Gene name
Motif
Position
Picea abies
PaHB1
PaHB2
ANL2
AtML1
GL2-1
GL2-2
GL2-3
GL2-4
GL2-5
O39
OCL1
OCL2
OCL3
OCL4
OCL5
UGGUUCGGGUAUU_GACU
UGGUUCGGGUAUU_GACc
UGGUUCGGGUAUU_GAaU
aGGUUCGGGUAUU_GACU
UGGUUCGGGUAUU_GACU
aGGUUCGGG aAUUuGACU
aGGUUCGGG a uUU_GACU
UGGUUCGGGUAUU_GACU
UGGUUCGGGUAUU_GACU
aGGUUCGGGUAUU_GACU
UGGUUCGGGUAUU_GACU
UGGUUCGGGUAUU_GACU
UGGUUCGGGUAUU_GACU
UGGUUCGGGaAUU_GACU
UGGUUCGGGUAUU_GACU
3022±3038
2962±2978
6699±6715
10525821±10525838
22642839±22642856
1513103±1513086
13694388±13694405
8821132±8821115
18643928±18643945
2781±2797
2760±2776
2323±2339
3453±3469
3907±3923
2649±2665
Arabidopsis thaliana
Phalaenospsis
Zea mays
The HD-GL2 genes present a very similar overall exon±
intron organization with nine possible conserved intron
positions numbered from 1 to 9 (Tavares et al., 2000;
Ingouff et al., 2001). The intron positions 1, 2, 4, 5, 7, and
9 are found in all the members whereas intron positions 3,
6 and 8 can be present/absent in different combinations.
Figure 1B shows the position of these introns (when
available) in relation to the deduced amino acid sequence
of the gene. It has previously been shown that the
phylogenetic classi®cation of HD-GL2 proteins is supported by the exon±intron structures of the corresponding
genes (Ingouff et al., 2001). For example, in the PaHB1
subclass the corresponding genes all feature the intron
positions from 1 to 9, except for AtML1, which lacks
position 3.
The PaHB2 exon±intron boundaries are located at
positions 1, 2, 3, 4, 5, 6, 7, and 9 (Fig. 1B). It is noteworthy
that the genes belonging to the PaHB2 subclass (ANL2,
FWA, GL2-1, and OCL1) closely resemble PaHB2 gene
structure except for intron position 3 that seems to have
been lost in genes of all subclasses (for example AtML1,
ANL2 and GL2) (Fig. 1A, B). Even if PaHB2 features a
unique intron position (position 3) among this subclass, a
speci®c intron pattern can be de®ned for the PaHB2
subclass, presence of position 1, 2, 4, 5, 6, 7, and 9 and
absence of position 8. In the third subclass, the exon±intron
boundaries (see GL2, GL2-4 and GL2-5) correspond to a
unique combination in the HD-GL2 family. They are
characterized by the presence of the positions 1, 2, 4, 5, 7,
8, and 9 and the absence of the intron position 6.
The phylogenetic relationships between HD-GL2
proteins are supported by the intron-exon structures of
the corresponding genes.
Expression pattern of PaHB2
Expression of PaHB2 in different organs and in embryogenic cultures was analysed by RT-PCR (Fig. 2) using
PaHB2 speci®c oligonucleotides. An ampli®cation product was obtained in all analysed parts of the seedling
(needles, epicotyl, hypocotyl, and root), in reproductive
organs (immature male and female strobili) and during
somatic embryogenesis in proliferating cells and in mature
embryos (Fig. 2).
The spatial expression pattern of PaHB2 was further
investigated by RNA in situ hybridization during somatic
embryogenesis (Fig. 3) and in the primary root (Fig. 4).
Control experiments with the DIG-labelled PaHB2 sense
riboprobe were performed on sections of tissues from
proliferating embryogenic cultures consisting of proembryogenic masses and early somatic embryos (data not
shown) and maturing somatic embryos (Figs 3D, F, I, K,
4B) and con®rmed that hybridization of the antisense
probe was speci®c (Figs 3A, C, E, H, J, 4A). In
proembryogenic masses of proliferating embryogenic
cultures, PaHB2 transcripts were detected in all embryonic
cells (data not shown). In early somatic embryos which
still do not feature any sign of tissue differentiation,
PaHB2 was mainly expressed in the embryonal mass and
in the embryonal tube cells that are derived from periclinal
cell divisions of the embryonal mass (Fig. 3A). These
results suggest that PaHB2 is expressed in the apical
region of the somatic embryo during early somatic embryo
development. However, since the vacuolated cells are
damaged during the sample preparation, it cannot be
excluded that PaHB2 is expressed in suspensor cells.
At the beginning of late embryogeny, a distinct
protoderm is de®ned and a root meristem is visible at the
Molecular characterization of PaHB2 1347
proximal part of the embryo (Fig. 3B). At this stage, in situ
hybridization experiments showed no hybridization signal
speci®c to PaHB2 transcripts in any tissue of the maturing
embryo after 1 week on ABA (compare Fig. 3C, D, E, F)
suggesting that PaHB2 was not expressed. Figure 3G
shows a mature somatic embryo after 3 weeks on ABA
where all the primary tissues are de®ned (protoderm,
cortex, vascular tissues) and the cotyledons have expanded. In sections hybridized with the antisense riboprobe a speci®c signal was detected in the outermost layer
of the embryonal cortex (Fig. 3H). The PaHB2 transcripts
were also detected in the root cap (Fig. 3J). Expression was
neither detected in the cotyledons and in the shoot apical
meristem (data not shown).
After germination, the PaHB2 gene is expressed in the
cortex of the root and the hypocotyl, and in the root cap
(Fig. 4A). No PaHB2 mRNA was detected in the vascular
tissue. PaHB2 transcripts were also clearly excluded from
the root meristem (Fig. 4A). Control experiments on
comparable sections with the sense probe did not show any
signals (Fig. 4B).
Discussion
PaHB2 is a member of the HD-GL2 family
A highly conserved gymnosperm HD-GL2 gene PaHB1
has recently been characterized using degenerate oligonucleotides (Ingouff et al., 2001). In this work, an
additional gene PaHB2 coding for a typical predicted
HD-GL2 protein is described. PAHB2 has an N-terminal
homeodomain linked to a truncated leucine zipper (a
dimerization domain) followed by the START domain (a
predicted lipid/sterol binding domain). PAHB2 shares
consistent sequence identity with the HD-GL2 proteins
Fig. 2. Expression pattern of PaHB2 during somatic embryogenesis
and in different organs. RT-PCR reactions were carried out on
reverse-transcribed mRNA samples from (1) needles, (2) epicotyl, (3)
hypocotyl, (4) root, (5) immature male strobile, (6) immature female
strobile, (7) proliferating cells, and (8) mature somatic embryos.
Fig. 1. Molecular analysis of PaHB2. (A) Phylogenetic tree showing
the relationships between Homeodomain-Glabra2 (HD-GL2) proteins.
The tree presented here is a Neighbor±Joining tree based on an amino
acid sequence alignment. The phylogeny was estimated by Poisson
corrected distances using MEGA2. Branch lengths are proportional to
the number of amino acid substitutions. Numbers at nodes are
bootstrap support values. Angiosperm and Picea proteins are indicated
in regular and bold cases, respectively. Alignments were derived from
the following sources: Norway spruce PAHB1 (AF172931) and
PAHB2 (AF328842), this paper; Arabidopsis ANL2 (AF077335),
ATML1 (U37589), FWA (AAG09302), GL2 (Z54356), GL2-1
(AJ224338), GL2-2 (AC000098), GL2-3 (AC005700), GL2-4
(Z97344), GL2-5 (AB013394), and PDF2 (AB056455); cotton
GHGL2 (AAK19610); sun¯ower HAHR1 (L76588); Phalaenopsis
O39 (U34743); maize OCL1 (Y17898), OCL2 (AJ250984), OCL3
(AJ250985), OCL4 (AJ250986), and OCL5 (AJ250987). (B) Exon±
intron organization of the HD-GL2 family. Exons are represented by
light boxes and intron positions are indicated by vertical bars.
Conserved intron positions are numbered as de®ned by Tavares et al.
(2000). Unique intron positions, mostly lying in the N-terminal region
upstream of the homeobox, are presented. In this analysis, the ®rst
conserved intron position is located in the homeobox (shaded in grey).
1348 Ingouff et al.
Fig. 3. Localization of PaHB2 mRNA by in situ hybridization during somatic embryo development. Longitudinal sections of somatic embryos
were hybridized with PaHB2 DIG-labelled antisense (A, C, E, H, J) and sense (D, F, I, K) riboprobes. Control section (B, G) was stained with
toluidine blue. (A) Early somatic embryo. Bar 75 mm. Note the signal in all cells in the embryonal mass and embryonal tube cells. No signals
were detected when the sense probe was used for hybridizations (data not shown). (B) Maturing somatic embryo at the beginning of late
embryogeny after one week on ABA. Bar 200 mm. (C, D) Higher magni®cation of sections from a maturing somatic embryo hybridized with
antisense (C, E) and sense (D, F) riboprobes. Note the absence of any speci®c signal. (G) Mature somatic embryo after 3 weeks on ABA. Bar
300 mm. Higher magni®cation of sections from a mature embryo hybridized with antisense (H, J) and sense (I, K) riboprobes. A speci®c signal
was detected in the outer embryonal cortex and in the root cap. ec, embryonal cortex; em, embryonal mass; et, embryonal tube cells;
pd, protoderm; rc, root cap; rm, root meristem; vt, vascular tissues.
Cell-speci®c expression of PaHB2 during embryo
development and post-embryonic growth
Fig. 4. Localization of PaHB2 mRNA by in situ hybridization in the
primary root. Longitudinal sections of primary roots were hybridized
with PaHB2 DIG-labelled antisense (A) and sense (B) riboprobes. Bar
250 mm. co, cortex; rc, root cap; rm, root meristem; vt, vascular
tissues. A speci®c signal was detected in the cortex and in the root
cap.
downstream of the START domain. However, this Cterminal end of the HD-GL2 protein does not match any
known functional domain.
A highly conserved 17 nt-long motif is present in the
3¢-UTR of the two gymnosperm and most of the
angiosperm HD-GL2 genes used in this study (Table 1).
This motif was not detected in genes outside the plantspeci®c HD-GL2 family. Homeobox genes expressed
during embryogenesis in metazoans sometimes feature
regulatory elements located in their 3¢-UTR that in¯uence
mRNA subcellular localization (Bashirullah et al., 1998),
mRNA translation (Dubnau and Struhl, 1996; RiveraPomar et al., 1996) and mRNA stability (Fontes et al.,
1999). Further molecular analyses are required to test if
this motif has any regulatory function.
Unlike the case for model angiosperm plants (Arabidopsis
and maize), the relationships between the initial cells and
the primary tissues are not clearly established in
gymnosperm embryos. However, the PaHB2 transcripts
are detected before any signs of tissue differentiation in the
young embryo, but it is not detectably transcribed at the
beginning of the maturation when cortex speci®cation is
thought to take place (Romberger et al., 1993; Filonova
et al., 2000). Later PaHB2 expression becomes restricted
to the outer cortex cell layer and the root cap in the mature
embryo. Therefore, it is at present unclear if PAHB2 is
involved in the speci®cation and/or maintenance of the
cortex identity.
After germination PaHB2 speci®c expression pattern is
maintained in the cortex of both roots and hypocotyls and
in the root cap, demonstrating that the mechanism
responsible for the post-embryonic expression of PaHB2
is already in place during embryo maturation.
Correlation between structure and function of PaHB
genes and angiosperm homologues
Despite a high degree of similarity, the two-gymnosperm
PAHB proteins, PAHB1 (Ingouff et al., 2001) and PAHB2
(this report), fall within two distinct monophyletic
subclasses. These observed phylogenetic relationships
were further supported by comparative analyses of
HD-GL2 gene structure. The fact that each PAHB protein
is phylogenetically more closely related to angiosperm
counterparts than to the other spruce protein suggests that
at least two ancestor proteins belonging to two distinct
Molecular characterization of PaHB2 1349
subgroups were already present before angiosperms and
gymnosperms separated, about 300 million years ago
(Stewart and Rothwell, 1993).
By contrast to PAHB1, which groups with members from
dicot and monocot plants having an outer cell layer-speci®c
activity, PAHB2 clearly belongs to the well-supported
subgroup comprising two Arabidopsis proteins (ANL2,
GL2-1) and three maize proteins (OCL1, OCL2 and OCL3).
The anl2 mutant is affected in the radial pattern of the
primary root; roots produce intervening cells, located
between the cortical and epidermal layers (Kubo et al.,
1999). Thus ANL2 activity is probably taking place in the
subepidermal layers of the roots. The spruce PaHB2 gene
exhibits a spatial expression localized in the cortical layers
of both the primary root and the hypocotyl and in the root
cap. Similarly to ANL2 and PaHB2, a subepidermal
expression was described for OCL2 in the meristem
(Ingram et al., 2000). The other two maize genes have an
expression restricted to the protoderm layer (OCL1) and to
the suspensor of the embryo (OCL3) (Ingram et al., 1999,
2000). Although the PAHB2 subclass includes genes with
distinct tissue-speci®c expression, these data show that the
PAHB2 subclass comprises members which have a subepidermal activity in both angiosperms and gymnosperms.
During plant embryogenesis, the ®rst histologically
detectable tissue is the protoderm. Subsequent steps of
radial patterning are con®ned to the inner cells where the
ground and conductive tissues will be de®ned. Both PaHB
genes switch from a uniform expression in early somatic
embryos to a distinct tissue-speci®c expression in nearby
cells at different stages of embryo maturation. The
protoderm-speci®c expression of PaHB1 clearly precedes
the outer cortex-speci®c expression of PaHB2. At present
it is not known if this differential layer-speci®c expression
of HD-GL2 genes in gymnosperms is mimicked by the
corresponding PaHB homologues in angiosperm embryos.
The protoderm/epiderm-speci®c and the subepidermspeci®c pattern of HD-GL2 genes are present among
HD-GL2 genes of highly divergent seed plants. The HDGL2 genes could therefore be used as molecular markers to
study radial pattern formation across seed plants.
Acknowledgements
We thank Ulf Lagercrantz for helping with the phylogenetic analyses
and critical reading of the manuscript and Lada Filonova and Peter
Bozhkov for providing the histological sections. This work was
supported by the Swedish Council for Agricultural and Forestry
Research (MI), the Swedish International Development Cooperation
Agency (MW) and a Marie Curie post-doctoral fellowship (IF).
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