Download A Histone H3.3-like Gene Specifically Expressed in the Vegetative

Plant Cell Physiol. 46(8): 1299–1308 (2005)
doi:10.1093/pcp/pci139, available online at www.pcp.oupjournals.org
JSPP © 2005
A Histone H3.3-like Gene Specifically Expressed in the Vegetative Cell of
Developing Lily Pollen
Yaeko Sano * and Ichiro Tanaka
Department of Biology, Graduate School of Integrated Science, Yokohama City University, Seto 22-2, Kanazawa-ku, Yokohama, Kanagawa, 2360027 Japan
;
To investigate the progression of the cell cycle during
pollen development, two clones of histone H3 genes, YAH3
and MPH3, were isolated from cDNA libraries of young
anthers and mature pollen of Lilium longiflorum. Northern
blot and reverse transcription–PCR (RT–PCR) analyses
demonstrated that YAH3 transcripts were present in uninucleate microspores and generative cells at the postulated S
phase of the cell cycle as well as in young anthers,
meristematic root tips, and shoot apices that contained proliferating cells. YAH3 therefore appears to be a major type
of histone H3 gene in the lily. In contrast, the expression of
MPH3 was detected only during pollen development, and
expression increased during the development of mid-bicellular pollen to mature pollen. The results of in situ hybridization revealed that the transcripts of MPH3 were
specifically accumulated in the vegetative cell of developing
bicellular pollen. The two histone H3s differed at eight
amino acid positions, and the deduced amino acid sequence
of MPH3 showed identity with histone H3.3, which is a
replacement variant of histone H3. The localization of an
MPH3–green fluorescent protein (GFP) fusion protein differed from that of YAH3–GFP in onion epidermal cells and
tobacco BY-2 cells at stationary phase, which suggests the
preferential ability of MPH3 to be incorporated into chromatin. MPH3 may be expressed replication independently
in vegetative cells at the G1 phase and be incorporated into
highly active chromatin of the vegetative nucleus of bicellular pollen, in a manner similar to histone H3.3 in Drosophila.
Keywords: Cell cycle — Histone H3.3 — Lilium longiflorum
— Pollen development — Vegetative cell.
Abbreviations: CaMV, cauliflower mosaic virus; DAPI, 4′,6diamidino-2-phenylindole; DIG, digoxigenin; GFP, green fluorescent
protein; GN, generative nucleus; ORF, open reading frame; RT–PCR,
reverse transcription–PCR; SN, sperm nucleus; UTR, untranslated
region; VN, vegetative nucleus
The nucleotide sequences reported in this paper have been submitted to the DDBJ/EMBL/GenBank database under the accession
numbers AB195974 (MPH3) and AB195975 (YAH3).
*
Introduction
Drastic changes in gene expression patterns occur during
pollen development in flowering plants (Mascarenhas 1990,
Becker et al. 2003, Honys and Twell 2003, McCormick 2004).
In particular, recent microarray analyses in Arabidopsis have
revealed unique characteristics of the pollen transcriptome
(Becker et al. 2003, Honys and Twell 2003). Although there is
a large overlap between the genes expressed in pollen and
those expressed in sporophytic vegetative tissues, about 10–
40% of genes were detected specifically in pollen. To date,
many pollen-specific genes have been identified. As the proteins encoded by these genes are involved in signal transduction and cell wall biosynthesis and regulation, it is assumed
that these pollen-specific genes are expressed in the vegetative
cell within pollen for pollen germination and tube growth. The
transcripts in sperm cells within pollen have been analyzed in
Nicotiana tabacum and Zea mays (Xu et al. 2002, Engel et al.
2003). This work showed gene expression profiles that differed from those of sporophytic vegetative tissues and pollen
vegetative cells. Thus, the gene expression patterns in larger
gametophytic vegetative cells differ from those in smaller
gametic generative or sperm cells within pollen, as well as
those in developing pollen differing from sporophytic vegetative tissues (Tanaka 1997).
The differential gene expression between vegetative cells
and generative or sperm cells may be due to differences in the
configuration of their chromatin. Chromatin switches between
an open state, which permits transcription, and a more closed
state, which is transcriptionally inactive (reviewed by Fransz
and de Jong 2002). A large-scale manifestation of the open or
closed states is microscopically visible as diffused (open) or
condensed (closed) chromatin. In general, the nucleus of a vegetative cell (referred to hereafter as the vegetative nucleus, VN)
contains diffused chromatin, whereas the nucleus of each generative or sperm cell (referred to hereafter as the generative
nucleus, GN, or sperm nucleus, SN, respectively) contains
highly condensed chromatin (Tanaka 1993). The VN may
therefore be more transcriptionally active than the GN or SN.
We previously characterized the GN by the appearance of
specific histone variants during pollen development in Lilium
longiflorum (Ueda and Tanaka 1995a, Ueda and Tanaka
1995b). The expression of genes coding for these histone vari-
Corresponding author: E-mail, [email protected]; Fax, +81-45-787-2217.
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Pollen-specific histone H3 gene in Lilium
Fig. 1 Alignment of nucleotide sequences of YAH3 (upper; accession No. AB195975) and MPH3 (lower; accession No. AB195974). Identical
nucleotides are indicated by asterisks. Open reading frames are shaded.
ants (gH2A, gH2B and gH3) occurs specifically in generative
cells with highly condensed chromatin (Ueda et al. 2000). Xu
et al. (1999) also reported generative cell-specific expression of
similar H2A and H3 histone genes in L. longiflorum.
Thus, histones may be one of the key factors for specific
gene expression during pollen development. However, few
studies have investigated the expression of histone genes during pollen development. Raghavan (1989) and Raghavan et al.
(1992) examined the expression of the histone H3 gene during
pollen development in rice and henbane and found that the
transcripts reached a maximum in mature pollen and were distributed uniformly in the cytoplasm of both the generative and
vegetative cells. This observation is contrary to the common
belief that histone H3 genes are specifically up-regulated during S phase of the cell cycle because neither type of cell within
mature pollen ever synthesizes DNA.
For this reason, we isolated histone H3 genes from young
anthers and mature pollen of Lilium and examined their temporal and spatial expression. The developmental process of lily
pollen is highly synchronized, and successive stages can easily
be estimated from the length of the buds on the plant. As a
result, we isolated a homolog of histone H3.3, which was a variant of histone H3 that was deposited in active chromatin in a
replication-independent manner in Drosophila (Ahmad and
Henikoff 2002a), and it was detected specifically in the vegetative cells. This novel observation may be the first characterization of active VN chromatin.
Results
Two histone H3 genes isolated from young anthers and mature
pollen
Two cDNA clones of histone H3 were obtained by hybridization screening of cDNA libraries of young anthers and
mature pollen and were designated YAH3 and MPH3, respectively. The nucleotide sequences of YAH3 and MPH3 are
shown in Fig. 1. Both sequences have predicted open reading
frames (ORFs) of 408 bp encoding 136 residue proteins including the initial methionine. They showed high homology to each
other in the ORF region (82.8%) and lower homology in the
untranslated region (UTR). Compared with the maize histone
H3 gene (H3C2; accession No. M13378), which was used as
the probe for library screening, YAH3 shows 84.3% identity
and MPH3 shows 79.9% identity in the ORF region (data not
shown).
Temporal expression of YAH3 and MPH3 during pollen development and tissue specificity
Northern blot analysis was performed to examine the temporal expression of the two genes using total RNA samples
extracted from cells at various stages during microspore and
pollen development (Fig. 2). YAH3 was strongly expressed in
anthers from buds 10 mm in length and in microsporocytes
from buds 20 mm in length. After the meiotic division, bicellular pollen from 70 and 80 mm buds after the first microspore
mitosis and uninucleate microspores from 50 and 60 mm buds
before the first microspore mitosis also exhibited YAH3 expres-
Pollen-specific histone H3 gene in Lilium
Fig. 2 Northern blot analysis of Lilium histone H3 transcripts. Total
RNA (10 µg per lane) samples were extracted from anthers from
10 mm buds (10); microsporocytes from 20 mm buds (20); uninucleate microspores from 30 (30), 40 (40), 50 (50) and 60 mm (60) buds;
bicellular pollen from 70 (70), 80 (80), 90 (90), 100 (100), 120 (120),
150 (150) and 170 mm (170) buds; and from mature pollen (MP).
RNA was electrophoresed, stained with ethidium bromide (rRNA),
blotted, and probed with YAH3 or MPH3.
sion. However, the expression was stopped in bicellular pollen
in buds beyond 90 mm in length. On the other hand, a weak
MPH3 signal was detected in bicellular pollen from 120 mm
buds. The signal became stronger during pollen maturation,
although a weak signal was also detected in uninucleate microspores from 50 and 60 mm buds.
Reverse transcription–PCR (RT–PCR) analysis was performed on the developing bicellular pollen and other tissues
(Fig. 3). YAH3 was expressed in bicellular pollen from both 70
and 80 mm buds and was also expressed in root tips, shoot apices, young leaves and ovules. The MPH3 signal was greater in
mature pollen than in mid-bicellular pollen (120 mm buds), as
shown by Northern blot analysis (Fig. 3). The signal was not
detected in other tissues including stems and bulbs. Thus, the
results of RT–PCR analysis of the expression of both histone
H3 genes during pollen development were similar to those of
Northern blot analysis. The expression of MPH3 was also pollen specific.
Localization of YAH3 and MPH3 transcripts in bicellular pollen
To investigate the location of the expression of YAH3 and
MPH3 within bicellular pollen, in situ hybridization was performed (Fig. 4, 5). In early bicellular pollen, soon after the first
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microspore mitosis, the generative cell was located at one pole
(Fig. 4f). The YAH3 hybridization signal was detected in the
pole (Fig. 4a). When a hybridized section was stained for DNA
with methyl green, the signal surrounded the GN (Fig. 4k). The
generative cell seemed to be pressed against the pole at this
stage. No signal was observed in mid-bicellular pollen whose
generative cell was detached from the pole and was rounded
(Fig. 4b, c). A signal was also undetectable in late bicellular
pollen and mature pollen, whose generative cells were spindle
shaped and yellow (Fig. 4d, e). Thus, the YAH3 signal was only
detected in generative cells from 75 mm buds and was never
detected in vegetative cells during pollen development.
Figure 5 shows the results of in situ hybridization with
MPH3 probes. In the case of the 3′-UTR of MPH3, no hybridization signal was detected in early bicellular pollen (Fig. 5a, b).
A weak signal was first detected in the cytoplasm of the vegetative cell of mid-bicellular pollen from 120 mm buds (Fig. 5c).
It became stronger in the vegetative cells of late bicellular (Fig.
5d) and mature pollen (Fig. 5e). Fig. 5k shows a section of late
bicellular pollen probed with antisense 3′-UTR of MPH3 and
counterstained with methyl green. A yellow area around the
green GN showed no hybridization signal (Fig. 5k). This was
more obvious in broken pollen whose spindle-shaped generative cell showed no hybridization signal (Fig. 5l). In contrast,
the hybridization signal of gH3, which is a GN-specific histone H3 gene (Ueda and Tanaka 1995a, Ueda and Tanaka
1995b), was detected around the GN or in the cytoplasm of the
generative cell within late bicellular pollen (Fig. 5m). Moreover, the hybridization signal in the case of the ORF of MPH3
was detected in the cytoplasm of both vegetative and generative cells (Fig. 5n, o). In contrast to Fig. 5l, the generative cell
of broken pollen showed a strong hybridization signal (Fig.
5o). These results suggest that MPH3 is specifically expressed
in vegetative cells and not in generative cells. Both MPH3 and
gH3 were never expressed in generative cells within early
bicellular pollen from 75 mm buds, where YAH3 was expressed
(data not shown). Thus, the temporal and spatial expression
patterns differed among the three kinds of histone H3 genes.
Fig. 3 RT–PCR analysis of Lilium histone H3 transcripts. cDNAs were synthesized with total RNA samples from bicellular pollen from 70 (70),
80 (80), 90 (90), 100 (100), 120 (120), 150 (150) and 170 mm (170) buds; mature pollen (MP); root tips (Rt); shoot apex (Sa); young leaves (Yl);
old leaves (Ol); stems (St); bulbs (Bu); and ovules (Ov), and used as a template for PCR with gene-specific primers of YAH3, MPH3 or EF1-α.
EF1-α was used as an internal control.
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Pollen-specific histone H3 gene in Lilium
Fig. 4 In situ hybridization analysis of YAH3 expression during pollen development. Bicellular pollen from 75 (a and f), 90 (b and g), 120 (c and h) and 150 mm (d and i) buds and mature
pollen (MP; e and j) were sectioned. Each section was hybridized with an antisense probe (a,
b, c, d and e) or sense probe (f, g, h, i and j) of the 3′-UTR of YAH3. (k) A section of bicellular
pollen from a 75 mm bud that was hybridized with an antisense probe of YAH3 and counterstained with methyl green. G, generative cell; V, vegetative cell. Scale bar: 20 µm.
Fig. 5 In situ hybridization analysis of MPH3 expression during bicellular pollen development. Bicellular pollen from 75 (a and f), 90 (b and g),
120 (c and h) and 150 mm (d and i) buds and mature pollen (MP; e and j) were sectioned. Each section was hybridized with an antisense probe (a,
b, c, d and e) or sense probe (f, g, h, i and j) of the 3′ UTR of MPH3. (k–o) Sections of bicellular pollen from 150 mm buds hybridized with an
antisense probe of the 3′-UTR of MPH3 (k and l), the ORF of gH3 (m) and the ORF of MPH3 (n and o), and counterstained with methyl green. G,
generative cell; V, vegetative cell. Scale bar: 20 µm.
Pollen-specific histone H3 gene in Lilium
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Fig. 6 (A) Alignment of deduced amino acid sequences
of MPH3 and YAH3 of Lilium longiflorum and histone
H3.3s and histone H3.1s of Arabidopsis thaliana and
Drosophila melanogaster. Residues varying between the
two sequences from the same species are indicated with
black backgrounds. (B) Phylogenetic analysis of amino
acid sequences of histone H3 variants in plants (Lilium,
Arabidopsis and Medicago) and Drosophila. The accession numbers were as follows: Drosophila H3.1
(AB019400), Drosophila H3.3 (X82257), Arabidopsis
H3.1 (M35387), Medicago H3.1 (X13674), Arabidopsis
H3.3 (X60429) and Medicago H3.2 (U09465). Numbers
above branches are the bootstrap values.
MPH3 is similar to plant histone H3.3
The deduced amino acid sequences of the two lily H3s
were 93.4% similar over their entire length, differing at eight
residues (Fig. 6A). A homology search showed that the
deduced amino acid sequence of MPH3 showed homology
with Arabidopsis histone H3.3-like protein or histone H3.III
(referred to hereafter as Arabidopsis histone H3.3), a replacement histone H3 variant of Arabidopsis thaliana, while YAH3
showed homology with the major histone type of Arabidopsis
H3.1 or H3.I (referred to hereafter as Arabidopsis histone
H3.1). The eight residues that differed between MPH3 and
YAH3 include the four residues (position 32, 42, 88 and 91)
that differ between histone H3.3 and H3.1 of Arabidopsis.
Moreover, among these four residues, three (position 32, 88
and 91) correspond to three of the four positions (32, 88, 90
and 91) that differ between H3.3 and H3.1 of Drosophila (Fig.
6A). Phylogenetic analysis of amino acid sequences indicated
that MPH3 is grouped with Arabidopsis histone H3.3 and
Medicago histone H3.2 (a kind of histone H3.3), while YAH3
is grouped with major histone H3.1 of plant species (Fig. 6B).
MPH3–green fluorescent protein (GFP) fusion proteins preferentially localized in chromatin of onion epidermal cells and
tobacco BY-2 cells
Among the eight residues that differed between MPH3
and YAH3, seven (except for position 32) were in the histone
fold domain, which is necessary for the formation of histone
octamers (Fig. 6A). To investigate the ability of the histones to
become incorporated into chromatin, we constructed fusion
genes encoding each H3 histone and GFP under the control of
cauliflower mosaic virus (CaMV) 35S promoters. These constructs were introduced into onion epidermal cells and tobacco
BY-2 cells using a particle gun, and the localization of transiently expressed fusion proteins was investigated (Fig. 7).
When a GFP gene alone was introduced into onion epidermal
cells, GFP-derived fluorescence was observed in the cyto-
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Pollen-specific histone H3 gene in Lilium
Fig. 7 Expression of histone H3–GFP fusion proteins in onion epidermal cells (a–c) and tobacco BY-2 cells (d–l). GFP (a, d, g and j), MPH3–
GFP (b, e, h and k) and YAH3–GFP (c, f, i and l) were introduced into cells by particle bombardment. BY-2 cells were counterstained with DAPI.
GFP signal (d, c and f), fluorescence from DAPI (g, h and i), and of the GFP signal viewed under a light microscope (a, b, c, j, k and l). Arrows
indicate nucleoli. Scale bars: 20 µm.
plasm as well as in the nucleus (Fig. 7a). In contrast, the
MPH3–GFP signal was only observed at the nucleus (Fig. 7b).
On the other hand, YAH3–GFP showed nuclear localization
with a distinct preference for nucleolus (Fig. 7c). Thus, the
nuclear localization of MPH3 and YAH3 was different in the
introduced cells. This was ascertained further in tobacco BY-2
cells at the stationary phase whose nucleoli were distinct by
4′,6-diamidino-2-phenylindole (DAPI) staining (Fig. 7d–l).
Similarly to onion epidermal cells, BY-2 cells showed the GFP
fluorescence both in the cytoplasm and in the nucleus when a
GFP gene alone was introduced (Fig. 7d, g). In contrast, the
MPH3–GFP signal corresponds to DAPI fluorescence in BY-2
cells (Fig. 7e, h). On the other hand, the strong YAH3–GFP
signal localized at a part of the nucleus where DAPI did not
stain (Fig. 7f, i). Thus, while YAH3–GFP mainly localized at
the nucleolus, MPH3–GFP localized in the chromatin domain
in BY-2 cells at the stationary phase.
Discussion
Cell cycle progression during pollen development
During microspore and pollen development in angiosperms, DNA synthesis occurs twice: once in the uninucleate
microspore after meiotic division and once in the generative
cell within bicellular pollen. In L. longiflorum, the two DNA
synthesis events occur just before and just after the first microspore mitosis (Taylor and McMaster 1954, Tanaka et al. 1979).
The occurrence of DNA synthesis during pollen development
has also been reported in other plant species (Zarsky et al.
1992, Binarova et al. 1993). On the other hand, histones are
generally synthesized during DNA synthesis (S phase). In this
study, the expression of YAH3 intimately depended on the presence of proliferating cells or S-phase cells (Fig. 3). YAH3 was
highly expressed in meristematic root tips, shoot apices, young
leaves and ovules. We presumed that young leaves also contained proliferating cells, while old leaves stopped cell proliferation. Ovule also contained proliferating somatic cells
surrounding an embryo sac. Therefore, we assume that YAH3 is
the usual somatic type of histone H3 gene, which is expressed
replication dependently. The fact that YAH3 showed homology with Arabidopsis histone H3.1 (Fig. 6), the major type of
histone H3, also supports the conclusion that YAH3 is a major
type of histone H3 of L. longiflorum. The samples from 10 and
20 mm buds also showed high levels of expression of YAH3
(Fig. 2). The pre-meiotic anthers from 10 mm buds probably
contained proliferating endothecium, and microsporocytes
from 20 mm buds were contaminated with proliferating tapetum cells.
Pollen-specific histone H3 gene in Lilium
Under the conditions in our greenhouse, the first microspore mitosis occurred in uninucleate microspores from 65 mm
buds. YAH3 was expressed in uninucleate microspores in 50–
60 mm buds and in bicellular pollen in 70–80 mm buds (Fig.
2), suggesting that they were in S phase. Furthermore, the
result of in situ hybridization of bicellular pollen from 75 mm
buds with the YAH3 signal in the cytoplasm of the poleattached generative cell (Fig. 4a, k) strongly suggests that the
generative cells at this stage are in S phase, because the GN
begins DNA synthesis while attached to the plasma membrane
of the generative pole just after microspore mitosis (Tanaka and
Ito 1984, Tanaka 1997). From the results of this study, we conclude that the uninucleate microspores in 50–60 mm buds and
the generative cells within bicellular pollen in 70–80 mm buds
were in S phase. Accordingly, the generative cells within bicellular pollen in buds >90 mm are in G2 phase.
MPH3 is expressed in the vegetative cells at G1 phase
In contrast to YAH3, MPH3 was specifically expressed
during pollen development (Fig. 3). Neither somatic tissue nor
ovules, the female reproductive organs, expressed MPH3. The
expression of MPH3 increased during the development of midbicellular pollen (buds 120 mm in length) to mature pollen.
The vegetative cell is in G1 (or G0) phase for its entire life.
During the interval, the generative cell is at G2 phase, as
described above. Therefore, it is apparent that MPH3, unlike
YAH3, is a replication-independent histone H3 gene.
Ueda et al. (2000) found another histone H3 variant gene,
gH3, which is also pollen specific. The temporal gene expression pattern of gH3 during pollen development is similar to that
of MPH3. Although both are expressed in a replication-independent manner, the spatial expression patterns of the two histone H3 genes are quite distinct. The transcripts of MPH3 were
accumulated in the vegetative cells of developing bicellular
pollen (Fig. 5c, d, e, k, l), while the transcripts of gH3 were
specifically accumulated in the generative cell of late bicellular pollen (Fig. 5m). In rice and henbane, the transcripts of the
histone H3 gene reach a maximum in mature pollen and are
distributed uniformly in the cytoplasm of both generative and
vegetative cells (Raghavan 1989, Raghavan et al. 1992). The
probe used in these studies is a 1.3 kb genomic clone of rice
histone H3 (accession No. M15664), which showed 85.5%
identity with YAH3, and 80.4% identity with MPH3 in the ORF
region. This may allow for the detection of histone H3 gene
expression, such as MPH3 and gH3, together. Indeed, when an
ORF sequence of MPH3 with lower specificity was used as a
probe, both cell types showed hybridization signals (Fig. 5n,
o). Therefore, MPH3 is the first histone H3 gene to be identified that is specifically expressed in vegetative cells in G1 phase.
Some histone H3 variants are expressed outside of the S
phase, including H3(P) of the ciliated protozoan Euplotes (Jahn
et al. 1997, Ghosh and Klobutcher 2000), which is not
expressed in proliferating cells but only in cells during sexual
phase. Other histone H3 variants, Cid and H3.3 in Drosophila,
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are also expressed outside of S phase (Ahmad and Henikoff
2002b). While the S phase-specific major type of histone H3
provides the bulk of nucleosome assembly when the genome is
duplicated, the replication-independent histone H3 variants
may also have the ability to be incorporated into non-replicating chromatin. To investigate the chromatin incorporation of
MPH3, GFP expression vectors were introduced into onion
epidermal cells in G1 phase and tobacco BY-2 cells at stationary phase. MPH3–GFP localized at the whole chromatin
domain in the nucleus. In contrast, there was clear preferential
staining of nucleoli with YAH3–GFP (Fig. 7). Such a tendency
has been observed in other transiently expressed replicationdependent histones (unpublished data). Alternatively, the result
suggests that MPH3 may easily be incorporated into the chromatin of G1 cells.
MPH3 may play a role in the transcriptional activity of VN
The deduced amino acid sequence of MPH3 showed identity with histone H3.3, which is a replacement variant of histone H3 (Fig. 6). The variant histone H3.3 is nearly identical to
major histone H3.1, differing at only a few amino acid positions (Hendzel and Davie 1990, Ahmad and Henikoff 2002b,
Tagami et al. 2004). In recent years, plant H3.3-like genes have
been found in A. thaliana (Chaubet et al. 1992, Chaubet-Gigot
et al. 2001), Medicago sativa (Kapros et al. 1992, Robertson et
al. 1996) and in some other plants. Among the eight residues
which differ between MPH3 and YAH3, positions 32, 88 and
91 differ between histone H3 and histone H3.3-like protein in
many other species in addition to Arabidopsis and Drosophila
(Wells et al. 1986). The difference in position 42 was also
observed in Arabidopsis histone H3s, and that in position 79
was observed in Tetrahymena histone H3s (Thatcher et al.
1994). The remaining three residues only showed difference
among species but not between histone H3s in the same
species. The expression pattern of MPH3, however, is distinct
from those of histone H3.3 genes of other plants. For example,
Arabidopsis H3.3 genes do not show preferential expression in
reproductive tissues such as developing pollen (Chaubet et al.
1992). Although Drosophila H3.3A shows strong expression in
the testes (Akhmanova et al. 1995), histone H3.3 genes in other
species show very little specific expression in reproductive
tissues.
In Drosophila, histone H3.3 is deposited in active chromatin, while the major type of histone H3.1 is incorporated strictly
into newly formed nucleosomes during DNA replication
(Ahmad and Henikoff 2002a). Such a difference in chromatin
incorporation may be due to differences in amino acid residues. Three of four amino acid residues that differ between
H3.3 and H3.1 in Drosophila correspond to three of eight that
differ between MPH3 and YAH3 (Fig. 6A). The result of the
introduction of a GFP fusion protein (Fig. 7) suggests the
incorporation of MPH3 into non-replicating chromatin.
The vegetative cell in which MPH3 transcripts accumulate is a terminal cell that does not undergo further mitotic divi-
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Pollen-specific histone H3 gene in Lilium
sion in vivo. The vegetative cell is transcriptionally active in
order to produce a large amount of materials that are involved
in pollen maturation, germination and tube growth. For this
reason, the chromatin of the VN is largely diffused. Histone H1
gradually decreases in the VN after microspore mitosis, and the
VN in mature pollen contains little histone H1 in the lily
(Tanaka et al. 1998). Such a low level of the linker histone may
place the chromatin of VN into a diffuse and transcriptionally
active state such that MPH3 transcripts are accumulated in the
vegetative cell. Consequently, MPH3 may be incorporated into
the active VN chromatin, as in Drosophila H3.3, and play a
role in the maintenance of the transcriptional activity. To investigate this possibility, we are currently generating an antibody
against the specific amino acid sequence of MPH3 and are
examining the localization in bicellular pollen.
Materials and Methods
Plant materials
Lily (L. longiflorum cv. Hinomoto) plants were grown in a greenhouse. The various stages of pollen development in the lily can easily
be estimated from the length of the buds. Under our greenhouse conditions, meiotic division and the first microspore mitosis occur in buds
that are about 13–27 and 65 mm in length, respectively. Therefore,
anthers at pre-meiotic interphase were obtained from 10 mm buds,
while microsporocytes at meiotic prophase I were collected from 15
and 20 mm buds. The uninucleate microspores were obtained from 30,
40, 50 and 60 mm buds, and the bicellular pollen was collected from
70, 75, 80, 90, 100, 120, 150 and 170 mm buds. Mature pollen and
ovules were collected from the dehiscent anthers and ovaries of flowers 3 d after anthesis. Root tips, shoot apices, young and old leaves,
stems and bulbs were collected from the lily plants. Onion bulbs were
purchased in a store. The tobacco BY-2 (N. tabacum L. cv. Bright Yellow 2) suspension cells were maintained by weekly subculturing in
Murashige and Skoog’s medium at 27°C in the dark. The BY-2 cells at
stationary phase were obtained 10 d after subculture.
Isolation of RNA
Cells and tissues were frozen, ground to a powder in liquid nitrogen, and then lysed in a buffer containing 100 mM Tris–HCl (pH 8.0),
10 mM EDTA (pH 8.0), 100 mM LiCl and 1% SDS. The subsequent
isolation procedure was carried out using the SDS–phenol method
(Shirzadegan et al. 1991), and the materials were precipitated with lithium chloride. Total RNA samples were treated with DNase I (Roche,
Mannheim, Germany) to remove contaminating genomic DNA.
Poly(A)+ RNA samples for cDNA libraries were purified using the
Oliotex™-dT30<Super> mRNA Purification kit (TAKARA BIO INC.,
Otsu, Japan).
Hybridization screening
A cDNA library was constructed using poly(A)+ RNA of either
young anthers obtained from 15 mm buds or mature pollen with a
cDNA Library Construction System (Novagen, Madison, WI, USA).
Hybridization screening of both the young anther and mature pollen
cDNA libraries was performed using a digoxigenin (DIG)-labeled
probe of the histone H3 sequence, which was obtained by PCR from
cDNA of lily root tips with primers 5′-CGCAAGCAGCTGGCCACCAA-3′ and 5′-GCGAGCTGGATGTCCTTGGG-3′ corresponding to
the ORF region of maize histone H3 (H3C2; accession No. M13378).
Positive plaques were isolated, and the nucleotide sequences were
determined using a BigDye Terminator cycle sequencing kit (Applied
Biosystems, Foster City, CA, USA) with a DNA sequencer (ABI
PRISM 310 Genetic Analyzer; Applied Biosystems). Database
searches were performed using the BLAST and FASTA algorithms
(http://www.ddbj.nig.ac.jp). The amino acid sequences of histone H3
were aligned using a program in GENETYX software. Then a phylogenetic tree was constructed by the neighbor-joining (NJ) method.
Bootstrap analysis with 1,000 replications was performed.
Northern blot analysis
A 10 µg aliquot of total RNA was separated in a 1% agarose gel
in the presence of 2.2 M formaldehyde and visualized by ethidium bromide staining. The RNA was then transferred to a nylon membrane
(Hybond-N; Amersham Bioscience, Piscataway, NJ, USA), and the
membrane was baked at 80°C for 2 h. DIG-labeled DNA probes were
prepared by PCR with a DIG-labeling (Roche) kit using the 3′-UTR
sequences of YAH3 and MPH3 as templates. The RNA was hybridized
overnight at 50°C in a buffer containing the DNA probes. The membrane was washed once in 2× SSC, 0.1% SDS at room temperature for
10 min and twice in 0.5× SSC, 0.1% SDS at 55°C for 15 min. The
hybridization signals were detected using CSPD (chemiluminescent
substrates for alkaline phosphatase; Roche) as instructed by the
manufacturer.
RT–PCR analysis
First-strand cDNA synthesis was performed in 20 µl of reaction
mixture containing 5 µg of total RNA, 50 pmol of oligo(dT) (18mer)
primer, 10 nmol of dNTP mixture and 4 U of AMV reverse transcriptase (TAKARA BIO INC.). The reaction mixture was incubated
at 42°C for 50 min and stopped by heating at 70°C for 15 min. After
the reaction, the solution was diluted 1 : 10 with distilled water, and
aliquots of 1 µl were added to 50 µl of PCR solution containing the
gene-specific primers.
The conditions for PCR were as follows: 94°C for 5 min, followed by cycles of denaturation at 94°C for 30 s, annealing for 30 s,
and extension at 72°C for 1 min, with an additional extension step at
72°C for 5 min. The number of cycles and the annealing temperature
were changed for each gene as follows: 35 cycles and 60°C for MPH3,
32 cycles and 56°C for YAH3, and 28 cycles and 57°C for EF1-α. The
gene-specific primers 5′-CTAATTCGTCTGTTATTGTG-3′ and 5′AAGCTTTTGTTTCGCGAGAA-3′ for the 3′-UTR region of MPH3,
5′-CGAGAGGGCTTGAGCATG-3′ and 5′-AACCCTACATATTCGCAAG-3′ for the 3′-UTR region of YAH3, and 5′-GAGGCAGACTGTTGCTGTCGG-3′ and 5′-AGCAGACTGAAATGAAGATGC-3′ for EF1α were used. PCR products were resolved by agarose gel electrophoresis or polyacrylamide gel electrophoresis and visualized by
ethidium bromide staining.
In situ hybridization
Cells were fixed in phosphate-buffered saline (PBS) containing
4% paraformaldehyde for 2–6 h and were then dehydrated in a graded
ethanol series, placed in xylene and embedded in paraffin. Serial sections 7–15 µm thick were placed on a slide coated with 3- aminopropyltriethoxysilane (Sigma, St Louis, MO, USA). The sections were dewaxed in xylene, hydrated in a graded ethanol series, treated with proteinase K, hydrolyzed in 0.2 M HCl, 0.25% acetic anhydride, and
dehydrated in a graded ethanol series. DIG-UTP-labeled sense and
antisense RNA probes were prepared with a DIG-RNA labeling kit
(Roche) using the 3′-UTR sequences of YAH3 and MPH3 and the ORF
sequence of MPH3 and gH3 as templates. The sections were hybridized overnight at 50°C in a buffer containing the RNA probes. The
slides were washed in 50% formamide in 2× SSC at 50°C, treated with
RNase A at 30°C, and washed in 2× SSC and then 0.2× SSC at 50°C.
Pollen-specific histone H3 gene in Lilium
Hybridization signals were detected using an NBT–BCIP mixture as
described by the manufacturer (Roche). For staining of DNA, the
slides were counterstained with 0.3% methyl green solution for 5 min.
The sections were observed using bright field microscopy.
Construction of GFP expression vectors
The CaMV 35S-sGFP(S65T)-NOS plasmid (Chiu et al. 1996)
was kindly provided by Dr. Y. Niwa of the Prefectural University of
Shizuoka. The plasmid was digested with SalI and NcoI. The MPH3
gene was obtained by PCR amplification of cDNA using primers that
introduce SalI and NcoI sites (5′-GGGTCGACATGGCCCGTACGAAGCAGACT-3′ and 5′-CATGCCATGGAGGCACGCTCGCCACGGAT-3′). The YAH3 gene was obtained by PCR amplification of
cDNA using primers that introduce SalI and NcoI sites (5′-GGGTCGACATGGCCCGCACCAAGCAGAC-3′ and 5′-CATGCCATGGAAGCCCTCTCGCCGCGGAT-3′). The conditions for PCR were as
follows: 94°C for 5 min, followed by 30 cycles of denaturation at 94°C
for 30 s, annealing at 68°C for 30 s, and extension at 72°C for 1 min,
with an additional extension step at 72°C for 5 min. Both PCR products were digested with SalI and NcoI and subcloned into the
CaMV35S-sGFP(S65T)-NOS plasmid.
Particle bombardment
Onion epidermal cells and tobacco BY-2 cells were placed on an
agar gel containing Murashige and Skoog’s medium. Plasmid DNA
was introduced into the cells using a particle gun (PDS-1000/He; BIORAD, Hercules, CA, USA). The conditions for bombardment were a
vacuum of 28 inches Hg, helium pressure of 1,100–1,350 p.s.i., and a
target distance of 6–9 cm using 1.6 µm of gold microcarrier. After
bombardment, cells were incubated for 4 h to overnight at 27°C in the
dark. Then, cells were fixed in 3% paraformaldehyde and viewed
under blue-light irradiation to monitor fluorescence from GFP. They
were also counterstained with DAPI.
Acknowledgments
The authors thank Dr. K. Ueda (Akita Prefectural University)
for kindly providing the gH3 clone, Dr. Y. Niwa (Shizuoka Prefectural University) for kindly providing the GFP plasmid, Ms. K.
Yanagihashi for technical support, and Dr. H. Shiota and Dr. N.
Mogami (Yokohama City University) for helpful advice. This work
was supported in part by a Grant-in-Aid for research from the
Yokohama Academic Foundation to Y.S and a Grant-in-Aid for
Scientific Research (C) (project number 16570057 to I.T).
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(Received January 23, 2005; Accepted May 23, 2005)