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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. 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