Download Cloning of cDNA Encoding NtEPc, a Marker Protein for the

Plant Cell Physiol. 41(2): 129-137 (2000)
JSPP © 2000
Cloning of cDNA Encoding NtEPc, a Marker Protein for the Embryogenic
Dedifferentiation of Immature Tobacco Pollen Grains Cultured in Vitro
Masaharu Kyo1, Hiroaki Miyatake, Kouki Mamezuka and Ken Amagata
Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki, Kagawa, 761-0795 Japan
changes in the process of generation of embryogenic cells
in cultured anthers have been observed in detail in D. innoxia and Nicotiana tabacum using the original method for
inducing pollen embryogenesis (Sunderland and Dunwell
1977). However, the induction mechanism of this phenomenon is poorly understood, probably because anther culture is not suitable for biochemical analysis. The cultured
anthers consist of a heterogeneous cell population including anther wall cells, maturing pollen, dead pollen, and a
small number of embryogenic cells. Unique culture methods for inducing pollen embryogenesis have been developed using N. rustica and N. tabacum immature pollen
(Kyo and Harada 1985, 1986), and Brassica napus microspores (Pechan and Keller 1988), in which the induction
factors, i.e. malnutrition in Nicotiana and heat shock in
Brassica, were evident and the frequencies of embryogenic
pollen cell division were high. Recently, based on the
combination of heat stress and starvation, new culture
methods for embryogenesis from microspores have been
developed in tobacco and wheat (see Touraev et al. 1997).
Several candidate markers for pollen or microspore embryogenesis have been found using these systems (see Reynolds 1997).
Kyo and Harada (1986) cultured the pollen grains of
N. tabacum at the mid-bicellular stage (stage III) which are
characterized by having undeveloped amyloplasts and no
central vacuole. During the culture in the basal medium
without sucrose and glutamine for a few days (the 1st culture), a characteristic form of cytoplasm suspended in the
center of a vegetative cell was observed. Similar morphological changes have also been observed in barley immature
pollen culture (Wei et al. 1986). The characteristic form of
cytoplasm is probably a morphological marker for the
early stage of the androgenic response (Kyo and Harada
1986, Touraev et al. 1997). Cell division was observed 10 d
after the transfer of the cells from the 1st culture to the
medium containing sucrose and glutamine (the 2nd culture). In the 1st culture, stage III pollen grains transform to
cells competent for embryogenesis. Such transformation
was not observed using younger or older pollen grains than
stage III, for example, pollen grains at the early-bicellular
stage (stage II) and the late-bicellular stage (stage IV) which
are characterized by having a large central vacuole and by
having amyloplasts developed little more and no central
vacuole, respectively. However, pollen development is a
naturally continuous process. Therefore, the boundaries
We partially purified three Nicotiana tabacum L. embryogenic pollen-abundant phosphoproteins (NtEPa to c)
which appeared in the cells undergoing a dedifferentiation
process from immature pollen grains to embryogenic cells,
caused by glutamine-deficiency in vitro. All the NtEPs had
a highly conserved N-terminal amino acid sequence. Using
degenerate oligonucleotide probes designed from the amino acid sequences, the cDNA for NtEPc was isolated from
a cDNA library of pollen cultured in glutamine-free medium. The cDNA sequence showed moderate homology with
several type-1 copper-binding glycoproteins and with a
kind of early nodulin though its function could not be
predicted. Expression analysis revealed that the level of
mRNA for NtEPc was high during the dedifferentiation
and also in the very early period of pollen embryogenesis
but it was low in the developmental process of microspores/pollen in anthers, in the in vitro maturation process
and both in the stational and logarithmic growth phases of
tobacco BY-2 cells. Furthermore, an acidic medium pH,
which promoted the induction of dedifferentiation increased the level of mRNA for NtEPc, whereas the presence of 6-benzylaminopurine, which inhibited it, decreased
the level. These results suggest that the expression of
NtEPc gene is correlated with the dedifferentiation but not
with pollen development or cell division.
Key words: cDNA cloning — Dedifferentiation — Nicotiana tabacum L. — Phosphoproteins — Pollen embryogenesis — Protein purification.
Since the first observation in Datura innoxia (Guha
and Maheshwari 1966), embryogenesis from microspores
or pollen has been a remarkable example of the expression
of totipotency and an important tool for the study of plant
breeding (see Maheshwari et al. 1980). Morphological
Abbreviations: BA, 6-benzylaminopurine; Dig, digoxygenin;
NtEPs, Nicotiana tabacum embryogenic pollen-abundant phosphoproteins; RT, reverse transcription; RTase, reverse transcriptase.
The nucleotide sequence reported in this paper has been
submitted to the DDBJ/EMBL/GenBank under accession number AB017533.
1
Corresponding author. Fax: +81-87-891-3021, E-mail: kyo@
ag.kagawa-u.ac.jp.
129
130
NtEPc, a marker for pollen dedifferentiation
between these developmental stages are not very clear,
especially between stages III and IV. In fact one of us
previously found that stage IV pollen also becomes embryogenic in an acidified medium (Kyo 1990).
We designated the transformation process as the
dedifferentiation of immature pollen in TV. tabacum and
N. rustica and previously found biochemical markers for
the process (Kyo and Harada 1990a, b). In both species,
analysis of two-dimensional electrophoretic pattern of
total proteins labeled with [32P]PC>4~ revealed that several
phosphoproteins characteristically appeared in the dedifferentiation process. The intensity of the signals for
those phosphoproteins on X-ray film roughly corresponded to the frequency of the dedifferentiation of immature
pollen under various circumstances, namely, the culture
period, culture conditions and the developmental stage of
pollen. Here we redesignate the phosphoproteins previously referred to as spots a to d in N. tabacum (Kyo and
Harada 1990a) and as spots 1 to 6 in N. rustica (Kyo and
Harada 1990b), as N. tabacum embryogenic pollen-abundant phosphoprotein NtEPa to d and N. rustica NrEPl to
6, respectively. We herein report the purification method
for NtEPa to c, their N-terminal amino acid sequences, the
sequence of cDNA corresponding to NtEPc and the expression pattern of the gene for the cDNA during the
normal development and the dedifferentiation of immature
pollen.
1986), at the final density of 2 to 3 x 104 grains ml l, and cultured
in a petri dish (5 cm in diameter) at 25 °C in the dark without
agitation (the 2nd culture). This culture was necessary to induce
embryogenic cell division for estimating the quality of the harvested cells. The frequency of cell division was observed two
weeks after the onset of the 2nd culture and used to evaluate the
frequency of the dedifferentiation of immature pollen after the 1st
culture. For expression analysis, pollen was prepared from fresh
flower buds and cultured as described in Results and Discussion.
To label NtEPs, [32P]PC>4 (32P;, carrier-free; supplied from
Japan Atomic Energy Research Institute, Toukaimura, Ibaraki)
or a mixture of [35S]methionine and [35S]cysteine (41.4 TBq
mmol" 1 , American Radiolabeled Chemicals, St. Louis, MO,
U.S.A.) was added to the culture medium containing the cells
The BY-2 cell line, used to assess NtEPc gene expression
in cells other than pollen grains, originating from seedlings of
Nicotiana tabacum L. cv. Bright Yellow (Kato et al. 1972), was
supplied from Central Research Institute, Japan Tobacco Co.
(Yokohama, Japan) and was maintained according to a method
reported by Nagata et al. (1981).
Purification of NtEPs—Frozen pellets of approximately 5 x
106 pollen grains were homogenized in 5 ml of ice-cold lysis buffer
which consisted of 100 mM Tris and 2 mM EDTA (pH 7.3 with
HC1), with a glass-Teflon homogenizer on ice. Complete destruction of pollen grains was monitored by observing a few microliter
aliquots of the homogenate under a microscope. The homogenate
was referred to as Fraction I (total fraction). Fraction I was centrifuged (27,000 xg, 1 h, 4°C) and its supernatant was referred to
as Fraction II. The precipitate of Fraction I was resuspended in 10
ml of ice-cold 0.1 xlysis buffer and centrifuged (27,000xg, 1 h,
4°C) again to eliminate proteins soluble to the lysis buffer. The
supernatant was kept in a new tube and referred to as Fraction III.
The precipitate was referred to as NtEP-rich fraction (see Fig. 3).
Materials and Methods
This fraction was resuspended in 1 ml of ice-cold, acidic soluPlant and cell materials—Plants of Nicotiana tabacum L. cv. bilization buffer consisting of 100mM acetate-KOH, 1% (w/v)
3 - [ (3 - cholamidopropyl) dimethylammonio] -1 - propane - sulfonate
Samsun were grown under a natural light condition in a room
(CHAPS) and 2 mM EDTA (pH was adjusted to 4.0 with KOH)
regulated at 20° C. Flower buds were collected from plants which
and homogenized again with a glass-Teflon homogenizer on ice.
had been in flower for at least one month (Kyo and Harada
The precipitate obtained after centrifugation (27,000xg, 1 h,
1990a). Surface sterilization of anthers, isolation and culture of
4°C) was referred to as Fraction IV. The supernatant was transpollen were conducted in a manner similar to that described
ferred into a 1.5-ml microcentrifuge tube, filled to 1 ml with icepreviously (Kyo and Harada 1986). However, to obtain sufficient
cold solubilization buffer and mixed with 0.5 ml of ice-cold 60%
material for protein purification we modified the culture method
(w/w) polyethylene glycol (MW 3,350, Sigma). The mixture was
as described below. Flower buds with a corolla length of 21-26
incubated on ice for 1 h and centrifuged (18,500xg, 1 h, 4°C).
mm were collected and kept at 4°C for 0 to 3d. From 50 to 60
The supernatant and precipitate were referred to as Fraction V
flower buds immature pollen at stages III and IV (stage III-IV
and VI, respectively. NtEPs were highly concentrated in Fraction
pollen, hereafter) was aseptically isolated and washed in medium
B"(0.3 M mannitol, 20 mM KC1, 1 mM MgSO4, 1 mM CaCl2; Kyo VI.
and Harada 1990a). Approximately 5 x 106 pollen grains were
Two-dimensional gel electrophoresis and visualization of
isolated, suspended in medium B"in a 300-ml flask at a density of
protein pattern—To prepare the samples for two-dimensional gel
4
1
5 x 10 pollen grains ml" , and cultured with agitation at 100 rpm
electrophoresis, it was necessary to precipitate the soluble proteins
at 25°C in the dark (the 1st culture). To increase the induction
in the liquid fractions, Fractions I, II, III and V. Aliquots corfrequency of the dedifferentiation of stage IV pollen (Kyo 1990),
responding to 106 cells of these fractions were placed into mithe medium pH was decreased to 4.3 or so by adding 100 mM
crocentrifuge tubes and 100% (w/v) trichloroacetic acid (TCA)
EDTA (adjusted to pH 6.8 with KOH, sterilized by autoclaving)
was added at the final concentration of 10%. After mixture, they
to the culture at the final concentration of 1 mM. This medium were placed on ice for 1 h. The precipitated fractions, Fraction IV
acidified by adding EDTA is referred to as medium E, hereafter.
and VI, were resuspended in ice-cold 10% TCA and aliquots
After five days of culture the cells were harvested by centrifugacorresponding to 5 x 105 cells were transferred into microcention (150 xg, 1 min) and stored in 15-ml polypropylene tubes at trifuge tubes. All samples were centrifuged (18,500xg, 30min,
— 80°C. Just before the harvest, 1 ml of the cell suspension was 4°C) and the precipitates were washed with 0.5 ml of acetone and
centrifuged (150 x g, 1 min) and the cells were resuspended in 3 ml then twice with 0.5ml of ether by centrifugation (18,500xg, 5
of medium B" supplemented with 1 mM KH2PO4-K2HPO4 (pH 7), min, 4°C). The ether remaining in the precipitates was allowed to
1 mM sucrose and 3 mM glutamine (medium C, Kyo and Harada evaporate on ice for 1 h. The dried pellet was resuspended in the
NtEPc, a marker for pollen dedifferentiation
sample solution; 9M urea with 5% 2-mercaptoethanol and 2%
Nonidet P-40. After centrifugation (18,500 xg, 10 min, 20°C),
each supernatant was used for two-dimensional gel electrophoresis. The electrophoresis, treatment of gel, and autoradiography
were conducted as described by Kyo and Harada (1990a). To
visualize the protein pattern, silver-staining of the gels were performed according to the method of Davis et al. (1986).
N-terminal amino acid sequencing—The purified sample
(Fraction VI) was solubilized in SDS sample buffer, denatured and
then separated by one dimensional SDS-PAGE (Laemmli 1970).
After the electrophoresis, proteins in the gel were blotted onto
polyvinylidene difiuoride (PVDF) membrane (Immobilon-P, Millipore, MA, U.S.A.) using a semi-dry electroblotter (NA-1512,
Nihon-Eidou, Tokyo, Japan) by the method described by Hirano
(1989). Proteins blotted on the PVDF membrane were stained
with 0.1% Coomassie Brilliant Blue (CBB) R-250 in 50% ethanol.
The stained bands corresponding to NtEPa, b and c were cut out,
destained with 90% ethanol, dried and analyzed by gas-phase
peptide sequencers (model PSQ-1, Shimadzu, Kyoto, Japan;
model 477A, Applied Biosystems, Foster City, CA, U.S.A.;
model 6625, Millipore).
Preparation of total RNA from pollen—Total RNA was
isolated from the cells by the method reported by Chomczynski
and Sacchi (1987) with some modification as described below. The
modification increased the yield of RNA to 1.5 fold higher than
the original method. Just after the harvest, approximately 106
pollen grains were homogenized in 10 ml of an ice-cold denaturing
solution consisting of 4 M guanidine thiocyanate, 25 mM sodium
citrate (pH 7.0), 0.5% (w/v) iV-lauroylsarcosine sodium salt and
0.1 M 2-mercaptoethanol using a glass-Teflon homogenizer. The
homogenate was mixed with 3 volumes of ethanol and one tenth
volume of 3 M sodium acetate (pH4.8) and placed at — 20°C
overnight. After centrifugation (21,000xg, 4°C, 15 min), the
pellet containing RNA was washed in 70% ethanol twice by centrifugation, dried in a vacuum, resuspended in 1 ml of TE buffer
(10 mM Tris, 2 mM EDTA, pH 7.2) containing 1% SDS and incubated at 60°C for 5 min. After adding of 2.5 ml of liquid phenol
supplemented with 0.1 %• (w/v) 8-hydroxyquinoline and saturated
by DEPC-treated distilled water, 0.5 ml of chloroform, and 2 ml
of diethyl pyrocarbonate (DEPC)-treated distilled water, the suspension was mixed well and centrifuged (10,000 x g, 15 min, room
temperature). The aqueous phase was recovered and three volumes of ice-cold ethanol and one-tenth volume of 3 M sodium
acetate were added and it was kept at — 20°C overnight. Total
RNA was precipitated by centrifugation (21,000xg, 30min,
4°C), washed twice with 70% ethanol (21,000xg, 5 min, 4°C),
dried in a vacuum, solubilized in DEPC-treated distilled water
and photometrically quantified. For RT-PCR, total RNA was
prepared from 0.5 to 1.0 xlO 5 pollen grains according to the
method described above.
Construction ofcDNA library—Poly(A)+ RNA was purified
from the total RNA, using OligoTex-dT super (Takara, Kyoto,
Japan) according to the manufacturer's manual. cDNA was constructed from poly(A)+ RNA and cloned into the AZAP II phage
vector, using a ZAP-cDNA synthesis kit (Stratagene, La Jolla,
CA, U.S.A.) according to the manufacturer's manual. The cDNA
library was prepared from the pollen cultured in medium E for 19
h (1.4 x 107 plaque-forming units per microgram DNA of AZAP II
with cDNA).
Screening ofcDNA—Approximately, 103 plaques were plated
on an agarose plate (15 x 7 cm, Eiken, Tokyo, Japan), according
to the manufacturer's manual for a ZAP-cDNA synthesis kit,
lifted to and immobilized on a nylon membrane (Hybond N,
131
Amersham, Buckinghamshire, England). From the result of Nterminal amino acid sequence of NtEPc, a single strand oligonucleotide mixture was designed and synthesized by a DNA synthesizer (Gene Assembler Plus, Pharmacia, Uppsala, Sweden). The
3'-termini of the oligonucleotides were labeled with fiuorescein11-dUTP by terminal deoxyribonucleotide transferase using an
ECL 3-oligolabeling and detection system (Amersham). The
cDNA bound on the membrane was hybridized with the labeled
oligo DNA mixture and visualized according to the manufacturer's manual. Each positive clone on the agarose plate was suspended in SM buffer (0.1 M NaCl, 50 mM Tris, 8 mM MgSO4,
0.01% gelatin, pH 7.5), and reconstructed as pBluescript SKplasmid by an in vivo excision system according to the protocol in
the ZAP-cDNA synthesis kit.
Sequence analysis—The sequence of the cDNA was determined by the dideoxy chain termination method (Sanger et al.
1977) modified for an automated sequencer (ALFII, Pharmacia),
using an A.L.F. DNA Sequencing kit (Pharmacia) and double
stranded nested deletion kit (Pharmacia). The GENETYX computer program (Software Development, Tokyo, Japan) was used
for determining the open reading frame, translating and other
editing of the sequence data, BLASTX computer program (Gish
and States 1993, Altschul et al. 1990) for homology search and
MOTIF computer program (Institute Chemical Research, Kyoto
University) for protein sequence motif through GenomeNet
WWW Server (http://www.genome.ad.jp, University of Tokyo
and Kyoto University). The description of functional motifs on
the predicted amino acid sequence were cited from the data base,
PROSITE (http://www.expasy.ch/prosite, University of Geneva).
Northern hybridization—cDNA in pBluescript SK —, was
amplified by PCR using primers designed from the sequences of
T7 and T3 promoters and AmpliTaq DNA polymerase (PerkinElmer, NJ, U.S.A.). The amplified DNA was labeled by Dig-11dUTP using a DIG DNA labeling kit (Boehringer Mannheim,
Mannheim, Germany). Total RNA from cultured pollen, BY-2
cells and the anthers at various developmental stages were prepared by the same method as described above and stored at
— 80°C until use. Four jug of each RNA sample were electrophoretically separated in an agarose gel and transferred onto nylon
membrane (Hybond N + , Amersham) by capillary transfer, according to the standard procedure (Sambrook et al. 1989). RNA
on the nylon membrane was immobilized by UV irradiation using
a Funa-UV-Linker (FS-800, Funakoshi, Tokyo, Japan) and hybridized with a cDNA probe labeled by Dig-11-dUTP as described
above. Hybridization, washing of membrane and detection of
hybridized probes were conducted according to the manufacturer's manual (Boehringer Mannheim). As instructed in the
manual, the use of a hybridization buffer with 7% SDS was
effective for decreasing the background signal.
RT-PCR/Southern hybridization analysis—Reverse transcription was conducted in a 20 jA volume containing 1 fig total
RNA, 1.25 ^M oligo dTi2-i8, 2.5 mM dithiothreitol, 0.5 mM
dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.5 mM dTTP, 1 unit
RNase inhibitor (Gibco BRL, Life Technologies, NY, U.S.A.), 10
units M-MLV RTase (Gibco BRL), and 1 x buffer components for
the RTase, at 37°C for 1 h. The mixture was heated at 94°C for 5
min to destroy RTase and then filled up to 100//I with sterilized
distilled water (sdw). This solution was referred to as 1st strand
cDNA solution. Polymerase chain reaction was conducted in 20
//1-volume containing 2^1 of 1st strand cDNA solution, 0.25 ^M
of each primer, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mM dGTP,
0.2 mM dTTP, 0.5 units Taq DNA polymerase (recombinant
132
NtEPc, a marker for pollen dedifferentiation
type, Gibco BRL), under the following conditions; precycling at
95°C for 5 min, and then 20 cycles of denaturation at 95°C for 30
s, annealing at 62°C for 1 min, and polymerization at 72°C for 2
min. The sequences of NtEPc specific primers, were 5-CTTCATTTCTTTACGGTTTTTGCC and 5-AGCTCCAAATAATGACACCACAAA. The amplified products was separated by electrophoresis using agarose gel, blotted onto nylon membrane and
detected using Dig-labeled cDNA for NtEPc by the same method
as described above.
Results and Discussion
Preparation of pollen materials—-In preliminary experiments, we observed that the intensity of the spots for
phosphorylated NtEPs in the autoradiogram of twodimensional gel electrophoretic pattern was maintained
without a remarked decrease after 24 h of the 1st culture
while the total protein content in the cells decreased with
culture time. This suggested that the relative content of
NtEPs against the total protein was higher in the cells cultured for a longer period. However, as described previously (Kyo and Harada 1990a), the 1st culture period longer than 5 d caused lower frequency of cell division in the
2nd culture probably due to irreversible degradation in
a part of the cells. Therefore, the appropriate length of
the 1st culture period to prepare the cell materials for the
purification of NtEPs was 5 d. For purifying NtEPs, we
used a cell population showing cell division at a higher
frequency than 25% in the 2nd culture.
Purification of NtEPs—NtEPs, insoluble in a buffer
without detergent (Kyo and Ohkawa 1991) could be separated from soluble proteins and be concentrated in the
pellet. Then NtEPs was solubilized selectively by an acetate
buffer containing CHAPS as described in Materials and
Methods. The two-dimensional gel electrophoretic pattern
of the protein from each fraction prepared by the purifi-
cation method was visualized by silver-staining (Fig. 1A)
and autoradiography (Fig. IB). The silver-stained pattern
of Fraction VI (the final fraction) was clear and almost
identical to the pattern of its autoradiogram. These results
indicated that NtEPs were purified in Fraction VI without
major contamination of other proteins though considerable amounts of NtEPs remained in Fraction IV probably
due to incomplete solubilization from the precipitation. In
Fraction I and IV, the silver stained pattern was dark and
autoradiogram also unclear, due to excess amounts of
loaded protein. However, in Fraction VI the typical pattern
of NtEPs was observed in which NtEPb was separated into
three spots which show similar molecular weights but distinguishable pi values as reported previously (Kyo and
Harada 1990a).
N-terminal amino acid sequences of NtEPs—The partially purified sample, identical to Fraction VI, was prepared from 107 pollen grains, developed by two-dimensional gel electrophoresis, electrically transferred onto a
PVDF membrane and stained with CBB (data not shown).
The faintly stained spots for NtEPa, b and c were cut out
and directly charged to a peptide sequencer to analyze their
N-terminal amino acid sequences. However, a short sequence could be identified as TEFTVGGDKG from the
N-terminus only with NtEPa. The N-termini of NtEPb and
c may have been artificially modified during the first dimensional nonequilibrium pH gradient electrophoresis
(NEpHGE), considering the results described below.
The purified sample prepared from 8xlO 6 pollen
grains was developed by one-dimensional gel electrophoresis of SDS-PAGE, blotted onto a PVDF membrane and
stained with CBB (Fig. 2A). Two aliquots of the sample
corresponding to 106 pollen grains were developed by oneand two-dimensional gel electrophoreses and visualized by
silver staining (Fig. 2B, C). The bands for NtEPa, b and c
NEpHGE
V
VI
Fig. 1 Two-dimensional gel electrophoretic patterns of the protein in the fractions prepared in the purification process. The left panel
in Fraction I corresponds to 3 x 105 pollen grains. The others correspond to 106 pollen grains. (A) Protein patterns visualized by silver
staining. (B) Autoradiograms of the silver stained gel plates.
NtEPc, a marker for pollen dedifferentiation
133
NEpHGE — >
in vitro Maturation
6h
Dedifferentiation
32 h
j£j
U1
NtEPa: TEFTVGGDKGWVVPNtEPb: LEFQVGDTTGWAVPNtEPc: TEFAVGGDKGWAVP-
Fig. 2 One- and two-dimensional electrophoretic patterns of
partially purified NtEPs and the blot for sequencing their N-terminal regions. (A) CBB-stained PVDF blot prepared from 8 x
106 pollen grains. The arrowheads on the edge indicate the bands
corresponding to NtEPa, b and c, which were cut out for N-terminal sequence analysis. Corresponding N-terminal amino acid
sequences were shown on the left side. (B) and (C) Silver-stained
protein patterns of the purified sample developed by one- and
two-dimensional gel electrophoresis, respectively. Both panels
correspond to 106 pollen grains. The three samples, (A) to (C)
were run in the same SDS-PAGE plate.
on PVDF membrane could be identified by comparison
with the one and two-dimensional gel electrophoretic patterns visualized by silver staining. Fortunately, there were
no serious contaminant proteins in the area from 30 to 14
kDa where NtEPs were localized. Each band for NtEPa, b
and c visualized by CBB was cut out and directly charged
to peptide sequencers and at least fourteen amino acid
residues could be identified in each sample (Fig. 2). The 3
different spots of NtEPb described above were treated
together as a single sample because it was impossible to
separate them by one-dimensional SDS-PAGE. However,
in the amino acid sequence analysis, there was no position
at which two or more amino acids were indicated. This
suggests that the three proteins in NtEPb possess the same
sequence in the examined region. Among the identified Nterminal sequences of NtEPa, b and c, 55% of the amino
acid residues were identical.
De novo synthesis of NtEPs—Stage III-IV pollen was
cultured in medium E or medium B"with 3 mM glutamine.
The mixture of [35S]methionine and [35S]cysteine was added
just after or 26 h after the start of the culture and was fed
by the cells for 6 h. From the harvested cells NtEP-rich
fractions (see Materials and Methods) were prepared, developed by two-dimensional gel electrophoresis and visualized by autoradiography (Fig. 3). In the early period of
culture, faint spots for NtEPa and c were observed. After
32 h dense spots for NtEPb and c were observed but no
spot for NtEPa. Such appearance of dense spots for NtEPs
was not observed on the culture in medium B" with glutamine. These results suggest that NtEPb and c were actively
synthesized in the cells cultured in medium E and that
NtEPa were not synthesized as much as NtEPb or c were.
Fig. 3 Two-dimensional gel electrophoretic patterns of the protein contained in NtEP-rich fractions prepared from the pollen
grains cultured in medium B'with 3 mM glutamine or in medium
E for 6 h and 32 h. The pollen grains were fed with [35S]methionine and [35S]cysteine for 6 h before harvest.
Isolation of cDNA for NtEPc—Based on a part of
the identified N-terminal amino acid sequence of NtEPc,
EFAVGGDKGWA, the 32-mer oligonucleotide mixture,
5'-GA(A/G) TT(C/T) GCI GTI GGI GA(C/T) GA(C/T)
AA(A/G) GGI TGG GC, was synthesized. Their 3-termini
were labeled by fluorescein-11-dUTP as the probe for
screening cDNA for NtEPc. A cDNA library was prepared
from stage III-IV pollen cultured in medium E for 19 h.
From 1.4 x 104 clones, 17 positive clones were isolated and
were sequenced in a part of their cDNA. The 6 clones encoded the amino acid sequence of NtEPc, EFAVGGDKGWA with complete identity in the region examined. The
other 11 clones possessed no sequences homologous with
the probes and no sequences encoding the N-terminal
amino acid sequences of NtEPs. The 32-mer oligonucleotide mixture may hybridize to cDNA for NtEPa because
NtEPa had only 2 amino acid residues different from
NtEPc in the N-terminal region (Fig. 2). However, the
cDNA for NtEPa could not be obtained probably because
the level for mRNA for NtEPa was much lower than that
for NtEPc considering the results described above (Fig. 3).
As shown in Figure 4, the whole sequence of the
cDNA for NtEPc is 702 bp in length. The longest open
reading frame consists of 501 bp, and encodes a protein of
166 amino acids with a calculated molecular weight of
18,503. The deduced amino acid sequence represents a
probable signal peptide sequence in the hydrophobic
region from Met1 to Ala24, which is followed by the sequence of the identified N-terminal sequence of NtEPc
(Fig. 4), suggesting that the NtEPc precursor is transported
into a certain organelle through the membrane system.
Furthermore, the hydrophobic regions, Val70 to Thr74,
Leu104 to He108, Leu119 to Leu126 and Thr155 to Tyr166 may
contribute to the formation of the transmembrane region,
suggesting the localization of the mature NtEPc on the
membrane. These speculations are consistent with our
previous observation of the insolubility of NtEPc in the
absence of detergent. The putative molecular weight and pi
of NtEPc without the probable signal peptide region are
134
NtEPc, a marker for pollen dedifferentiation
M
70
80
90
D
S
S
K
A
100
F
L
Y
110
120
130
1 4 0
150
1 6 0
170
1 8 0
TGGCGGTGACAAAGGATGGGCTGTTCCTAAAGTAAAAGATGATCAAGTGTATGACCAGTG
G
G D K G W A
V P K V K D D Q V V D Q W
190
200
210
220
230
240
GGCTGGAAAAAATAGGTTTAAGATTGGCGACACCTTAAGTTTTGAGTATAAGAAAGATTC
A G K N R F K I G D T L S F E Y K K D S
250
260
270
280
290
300
GGTTCTGGTGGTGACCAAAGAAGAGTACGAGAAATGCAAGTCGAGTCATCCAATTTTCTT
310
S
N
N
320
G
K
T
I
330
Y
K
L
E
340
Q
P
350
G
L
Y
Y
360
F
I
S
370
380
390
400
410
420
TGGAGTATCAGGACATTGCGAGAGGGGTTTGAAAATGATAATCAAAGTTTTAGAACCAGA
G V S G H C E R G L K M I
I K V L E P E
430
440
450
460
470
480
ATCCCCACCTCAATCTGCTAACCAGACTTCCCCAACTAGTAGTGGTACTACTGCAGCTGC
490
T
P
T
T
500
M
A
F
510
V
V
S
L
520
F
G
A
530
L
L
Y
540
*
550
560
570
580
590
600
610
620
630
640
650
660
670
680
690
700
Fig. 4 Nucleotide sequence of cDNA for NtEPc and its putative
amino acid sequence. Underlined sequence coincides with the
identified N-terminal amino acid sequence of NtEPc. Double-underlined region carries multicopper oxidase 1 signature.
calculated to be 15,541 and 8.1, respectively. These values
are not consistent with those evaluated from the results of
the two-dimensional gel electrophoresis probably due to
post-translational modification by glycosylation and phosphorylation.
Expression analysis of NtEPc gene—Northern hybridization was conducted to survey the level of mRNA for
NtEPc in the developmental process of microspores/pollen
in the anther (Fig. 5A), in the in vitro maturation and the
dedifferentiation processes of stage III-IV pollen (Fig. 5B),
Developmental Stage
in the induction process of cell division in the 2nd culture
and in BY-2 cells at the stational and logarithmic growth
phases (Fig. 5C).
The total RNA samples were obtained from the anthers mainly containing pollen-mother cells (PM), tetrad
(T), early-unicellular (U), late-unicellular (I), early-bicellular (II), mid-bicellular (III), late-bicellular (IV) and premature pollen (V), which were taken out from fresh flower
buds with corolla lengths of 4-5, 6-8, 9-10, 11-13, 15-17,
20-22, 25-26, 30-40 mm, respectively (Fig. 5A). At stages
U to V, the hybridization signals were detectable but the
intensity was much lower than that in the dedifferentiation
process described below. For inducing the in vitro maturation and the dedifferentiation, we used stage III-IV
pollen and cultured in flasks with agitation. Approximately
90% of viable pollen developed to show mature form in the
medium B" supplemented with 3 mM glutamine while approximately 70% of viable pollen showed a characteristic
form of cytoplasm suspended in the center of a vegetative
cell in medium E, which seemed to be a morphological
marker for the dedifferentiation as described above. In the
dedifferentiation process the mRNA level remarkably increased with the culture time, however, in the in vitro
maturation it remained (Fig. 5B). After the 1st culture in
medium E for 3 d, we selected cells floated against medium
B" containing 50% Percoll as described by Kyo and Harada
(1987). By this fractionation it was possible to obtain a
highly homogeneous cell population of dedifferentiated
pollen. The fractionated cells were resuspended in medium
C and cultured in a flask with agitation. In the 2nd culture,
the cells began to divide asynchronously within 10 d at the
frequency of approximately 30%. As shown in Figure 5C
the level of mRNA for NtEPc was very high during the first
5 d but it decreased after the 9th d and finally was undetectable on the 15th d. These results suggest that NtEPc
plays a certain role in the dedifferentiation process in the
1st culture and in the very early state of embryogenesis in
the 2nd culture, but not in the processes of the microspore/pollen development and the in vitro maturation.
Maturation (h) Dedifferentiation (h)
2nd culture (d)
0
2
5
9
15
BY-2 (h)
~0
25
W
Fig. 5 Northern hybridization analysis. (A) Total RNA samples were prepared from anthers containing pollen-mother cells (PM),
tetrad (T), early-unicellular (U), late-unicellular (I), early-bicellular (II), mid-bicellular (III), late-bicellular (IV) and pre-mature (V)
pollen grains. (B) Total RNA samples were prepared from stage III-IV pollen cultured for indicated periods in medium B"supplemented
with 3 mM glutamine or medium E for inducing the in vitro maturation and the dedifferentiation, respectively. (C) Total RNA samples
were prepared from the cells cultured for the indicated days after the beginning of the 2nd culture in medium C. BY-2 cells in the
stationary phase were harvested from the 2-week-old culture and resuspended in a fresh medium. Cells were harvested at the indicated
time after the inoculation. Ethidium bromide-stained patterns of total RNA are shown at the bottom.
NtEPc, a marker for pollen dedifferentiation
A well-established cell line, BY-2, shows high proliferation activity and the percentage of mitotic cells in the
population 25 h after the inoculation to a fresh medium
was approximately 8% in our experiments. To investigate
whether the expression of NtEPc gene is correlated with
cell cycle progression or proliferation activity, we examined the level of mRNA for NtEPc in BY-2 cells at the
stationary phase (2-week-old culture) and at the logarithmic growth phase (25 h and 48 h after the transfer to fresh
culture medium). As shown in Figure 5C, no mRNA was
detected in any of the three samples. These findings suggest
that NtEPc is not related to starting or driving of the cell
cycle.
In medium E the dedifferentiation could be promoted
even in stage IV pollen, and at the same time the intensity
of spots for phosphorylated NtEPs in the two-dimensional
gel electrophoretic pattern was increased (Kyo 1990). The
dedifferentiation was inhibited in the presence of BA in the
medium even in stage III pollen, and at the same time the
intensity of the spots was decreased (Kyo and Harada
1990). We examined the effects of these factors on the level
of mRNA for NtEPc by RT-PCR and Southern hybridization (Fig. 6). Stage II, III and IV pollen were isolated
from fresh flower buds with corolla lengths of 15-17, 20-22
and 25-27 mm, respectively. To prepare a highly homogeneous cell population, we performed two-step Percoll
density gradient centrifugation (40/50, 50/60, 60/70% for
stage II, III and IV, respectively) as described previously
(Kyo and Harada 1987). The fractionated pollen was cultured in 9 or 5-cm Petri dishes (3 to 5 x 104 grains ml"1)
without agitation. Because of the limited amount of cells,
we adopted RT-PCR in the expression analysis.
With stage II pollen cultured in medium B", the level
of mRNA for NtEPc decreased with culture time. With
stage III pollen in medium B", the level increased 12 h after
the start of culture and remained high during the 1st cul-
lh 12h 24h 48h lh 12h 24h 48h 12h 24h 48h lh 12h 24h 48h 12h 24h 48h
135
ture. Such increase was inhibited under the effect of BA in
the medium. With stage IV pollen, the level increased 12 h
after the start of culture in both media B and E, but the
level was much higher and was maintained for a longer
period in medium E. The expression of NtEPc gene are
well coincident with the appearance of NtEPc and with the
occurrence of the pollen dedifferentiation.
Sequence analysis ofcDNA for NtEPc—We identified
four possible phosphorylation sites, one potential copper
binding site, and one N-glycosylation site. The possible
sites phosphorylated by protein kinase C, [ST]-x-[RK]; tyrosine kinase, [RK]-x(2,3)-[DK]-x(2,3)-Y; cAMP- or cGMPdependent protein kinase, [RK](2)-x-[ST] and casein kinase
II, [ST]-x(2)-[DE] are found in 3-SSK-5, 39-KVKDDQVY46, 66-KKDS-69, and 74-TKEE-77, in the order given.
These results are coincident with the fact that NtEPc had
been identified as one of 32Pj-labeled phosphoproteins. A
N-glycosylation site, N-{P}-[ST]-{P} was found in 136NQTS-139. A multicopper oxidase 1 signature (PS00079,
PROSITE), G-x-[FYW]-x-[LIVMFYW]-x-[CST]-x(8)-G[LM]-x(3)-[LIVMFVW] is found in 103-GLYYFISGVSGHCERGLKMII-123. In plant this signature is found in
laccase (EC 1.10.3.2) and ascorbate oxidase (EC 1.10.3.3)
which possess all of the three type copper centers (type I,
II and III). The two enzymes possess another signature,
H-C-H-x(3)-H-x(3)-[AG]-[LM] (multicopper oxidase 2,
PS00080, PROSITE) which is specific to copper-binding
domains, but the putative amino acid sequence of NtEPc
BCB
UMEC
CPC
BCB
UMEC
CPC
MAVI
STEL
NtEPc
N3 15
t ' MAG-VFKTVTFLV
LVFAAVVVFAEDYDVGDD-TEWTRP—MDPEFYTSWATGKTFRV 53
i .
EDYDVGGD-MEWKRP--SDPKFY I TWATGKTFRV 31
| .
QSTVHIVG-DNTGWSVP — SSPNFYSQWAAGKTFRV 33
.
ATVHKVG-DSTGWTT--LVPYD-YAKWASSNKFHV 31
.
TVYTVG-DSAGWKVPFFGDVDYDWKWASNKTFHI 33
:MDS--SKAFLYLVFFLFSLHFFTVFATEFAVGGD-KGWAVPKVKDDQVYDQWAGKNRFKI
57
:MASCLPNASPFLVMLAMCLLISTSEAEKYVVGGSEKSWKFPLSKPDSL-SHWANSHRFKI
59
**
»
**
•
54:GDELEFDFAAGRM)V-AVVSEAAFENCEKEKPISHMT-VPPVKIMLNTTG.
32:GDELEFDFAAGM«DV-AVVTKDAFONCKKENPISHMT-TPPVKIMLNTTGPaVXL<
34 rGDSLQFNFPANAWNVHEMETKQSFDACNFVNSDNDVERTSPVIERLDElt
32:GDSLLFNYNNKFWNVLQVDQEQFKS-CNSSSPAASY-TSGADSIPLKRPG1EXF
34:GDVLVFKYDRRF«NVDKVTQKNY0S-CNDTTPIASY-NTGDDRINLKTVG_flKjnLLCG_yPJ(
91
58:GDTLSFEY-KK-DSVLVVTKEEYEK-CKSS-HPIFFSNNGKTIYKLEQPGLYYFISGVSG
60:GDTUFKYEKRTESVHEGNETDYEG-CNTVGKYHIVFNGGNTKVMLTKPGFRHFISGNQS
NtEPc
BCB
C24
Fig. 6 RT-PCR/Southern hybridization analysis. Total RNA
samples were prepared from stage II pollen cultured in medium
B", stage III pollen cultured in medium B" with or without BA
(10 6 M) and stage IV pollen grains cultured in medium B" or E
for the indicated periods. Each 1st strand cDNA sample for the
templates in PCR was prepared by reverse-transcription using
oligo dTn-ig. PCR was performed using pair of primers specific to
the cDNA for NtEPc (top) or specific to the cDNA of clone C24
as an internal standard (bottom). Clone C24 was isolated from the
cDNA library described in Materials and Methods and encoded
polyubiquitin homolog.
112: //CRFGQKLS ITVVAAGATGGATLGAGATPALGSTPSTGGTTPPTA
UMEC
90: flCRVGOKLSINVVGAGGAGGGATPGA
CPC
9 4: WCSNGOKLSINVVAANATVSMPPPSSSPPSSVMPPPVMPPPSPS
MAVI
90:WCOLGQKVEIKVDPGSSSA
STEL
92 :/<CDLGOKVHINVTVRS
N3I5
119:HCaMGLKLAVLVISSNKTKKNLLSPSPSPSPPPSSLLSPSPSPLPNN0GVTSSSGAGFIG••
• *
*
Fig. 7 Alignment of amino acid sequences of blue copper binding protein (BCB), umecyanin (UMEC), cucumber peeling
cupredoxin (CPC), mavicyanin (MAVI), stellacyanin (STEL),
NtEPc and GmN#315 (N315). Single- and double-underlined
regions indicate copper blue signatures and a multi-copper oxidase 1 signature, respectively. Two His, Cys and Gin indicated by
italics are probable ligands for a single copper atom. Asterisks
indicate the 19 residues conserved in the listed proteins.
136
NtEPc, a marker for pollen dedifferentiation
had no sequence corresponding to such a signature.
Homology search by a BLASTX computer program
using a SWISS-PROT protein sequence database indicated
that the putative NtEPc sequence exhibits moderate and
significant similarity with some copper binding glycoproteins, stellacyanin from Japanese lacquer tree (Bergman et
al. 1977), mavicyanin from zucchini peelings (Schinina et
al. 1996), umecyanin from horseradish roots (Van Driessche et al. 1995), cupredoxin from cucumber peelings
(Mann et al. 1992), blue copper binding protein of Arabidopsis thaliana (Van Gysel et al. 1993), and also with a
kind of early nodulin, GmN#315 (Kouchi and Hata 1993).
Other than the proteins described above, the putative
NtEPc sequence exhibits a low similarity with rat liver
Nuclear Factor 1, which shows a specific DNA-binding
activity (Monaci et al. 1995) and novel lamin-like protein in
Brassica (accession No. X97022).
Those copper binding glycoproteins listed above possess a copper blue signature; [GA]-x(0,2)-[YSA]-x(0,l)[VFY]-x-C-x(l,2)-[PG]-x(0,l)-H-x(2,4)-[MQ] (PS00196,
PROSITE) and four amino acid residues, Cys, Gin and
two His (indicated by italics in Fig. 5) which probably
function as ligands for a copper atom (Fields et al. 1991,
Van Driessche et al. 1995). However, the putative amino
acid sequence of NtEPc possesses a multicopper oxidase
signature which is similar but not identical to type-I copper
protein signature. GmN#315 also has a sequence very similar to multicopper oxidase signature except for Arg110 in
the corresponding site. In NtEPc and GmN#315, only one
of the four ligands for a copper atom in those copper
binding glycoproteins are found at His114 in NtEPc and at
His119 in GmN#315, suggesting no ability for binding to a
copper atom.
As shown in Figure 7, 19 intermittently localized amino acid residues (indicated by asterisk) were conserved in
all of the proteins listed. Similar observations have been
reported among some copper-binding glycoproteins and
those conservative amino acid residues seem to be important to form a certain three-dimensional structure required for their function (Schinina et al. 1996). For example, the two Cys in stellacyanin (Cys59 and Cys93) and
umecyanin (Cys57 and Cys91) contribute to make disulfide
bonds (Engeseth et al. 1984, Van Driessche et al. 1995).
These suggest that all of the proteins listed here possess
similar three-dimensional structure. The three-dimensional
structure models for stellacyanin and umecyanin were
previously proposed (Fields et al. 1991, Van Driessche et
al. 1995). We speculated that the proteins listed in Figure 7
may have originated from a common ancestral protein
which possess a type I (blue) copper center and that NtEPc
and GmN#315 have lost their copper binding ability during
the evolutional process but obtained new different functions conserving the 19 amino acid residues.
The functions of the copper binding glycoproteins
listed above are still unclear. As for the function of plant
proteins which possess type I (blue) copper center, chloroplastic plastocyanin is an electron carrier and laccase is
an oxidase. GmN#315 is a kind of early nodulin of soybean
and its mRNA appeared 6-7 d after the inoculation with
Bradyrhizobium, just before nodule emergence (Kouchi
and Hata 1993), but its function is unknown. The function
of NtEPc remains to be elucidated.
This work was supported in part by a Grant-in-Aid for Special Research on Priority Areas (No. 07281101; Genetic Dissection
of Sexual Differentiation and Pollination Process in Higher
Plants) from the Ministry of Education, Science, Sports and
Culture, Japan. We thank Dr. K. Nagao (Nihon Millipore Ltd.)
and Prof. H. Hirano (Yokohama City University) for their technical support in the N-terminal amino acid sequencing. We also
thank Dr. P. Pechan (Technical University Munich) for the revision of this manuscript.
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(Received July 29, 1999; Accepted November 9, 1999)