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Plant Cell Physiol. 45(10): 1519–1528 (2004) JSPP © 2004 Gene Expression Profiles of Cold-stored and Fresh Pollen to Investigate Pollen Germination and Growth Min-Long Wang 1, 2, 4, Chia-Mei Hsu 1, 4, Liang-Chi Chang 1, Co-Shine Wang 2, Ting-Ho Su 1, 2, Yih-Jong John Huang 1, Liwen Jiang 3 and Guang-Yuh Jauh 1, 5 1 Institute of Botany, Academia Sinica, Nankang, Taipei 11529, Taiwan, R.O.C. Graduate Institute of Biotechnology, National Chung Hsing University, Taichung 40227, Taiwan, R.O.C. 3 Department of Biology, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China 2 ; In lily (Lilium longiflorum cv. Avita) pollen cold-stored (–20°C) for 2 months, typical in vitro germination/growth was delayed by about 1 h compared with fresh pollen. We hypothesized that some proteins and mRNAs stored in mature pollen were degraded during storage periods and that re-synthesis of them was essential to resume normal germination and growth. Cold-stored and fresh pollen grains were used to investigate the regulatory mechanism of pollen germination and tube growth in terms of both total protein profile and gene expression. Total protein profiles of cold-stored pollen differed qualitatively and quantitatively from fresh pollen. Actinomycin D significantly inhibited both germination and tube growth of cold-stored pollen and later tube growth of fresh pollen but had no effect on fresh pollen germination and early tube growth. Suppression subtractive hybridization screening revealed 99 cDNAs enriched in fresh mature pollen, and 22 were selected for further characterization. Most of these 22 cDNAs gradually disappeared during cold storage, but full recovery was achieved by incubating the cold-stored pollen in culture medium for 2 h. Because of different sensitivities to cold storage and actinomycin D, the transcripts were divided into three groups according to their possible roles in pollen germination and tube growth. Several cDNAs encoding novel proteins showed pollen-specific expression patterns and may participate in drought tolerance (an Na+/H+ antiporter), endomembrane trafficking (DnaJ), division of the generative cell (Sgt1), pollen wall precursor uptake from stylar exudate (an Na+/myoinositol symporter) and chemotropism of the pollen tube (peptide transporter) during pollination. Keywords: Actinomycin D — Gene expression — Lilium longiflorum — Low temperature — Pollen germination — Pollen tube growth. Abbreviations: ActD, actinomycin D; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis; RT-PCR, reverse transcription-polymerase chain reaction. 4 5 Introduction Pollen development takes place within the anther and provides a good system for investigating the regulation of several fundamental cellular events such as cell division, cell fate determination, gene regulation, cell-cell communication and cellular differentiation during pollination. Mature pollen released from the anther is dehydrated and metabolically quiescent; after reaching a receptive stigma or in vitro cultured medium, the pollen rehydrates and germinates quickly (Mascarenhas 1993, McCormick 1993, Lord and Russell 2002). Pollen germination and tube growth are very rapid events, and the rates of pollen tube growth are extremely high. To support this unusually fast growth, large amounts of protein, rRNA, mRNA, bioactive small molecules and reserved material are stored in mature pollen grains (Mascarenhas 1993, Taylor and Hepler 1997, Twell 2002). Analyses of the expression patterns of a number of pollenexpressed and/or pollen-specific mRNAs have shown that at least two broad classes of genes, early and late, are involved (Mascarenhas 1990). The early genes are first expressed in microspores, but their expression decreases significantly before pollen maturation, whereas the late genes are not expressed until after pollen mitosis I and their transcripts accumulate until pollen maturation. It is believed that the pre-synthesized mRNAs and proteins are mainly products of the late genes of the vegetative cell. The mRNAs in mature pollen grains of Tradescantia (Willing and Mascarenhas 1984) and maize (Willing et al. 1988) are the products of about 20,000–24,000 different genes, and 10% of these appear to be pollen-specific transcripts. Only a small fraction of these genes, approximately 150 genes from 28 species, have been well characterized individually, and most were obtained from the developing pollen grain (Twell 2002). Guyon et al. (2000) showed the rapid and synchronous in vitro germination response of conditional-malefertility pollen to exogenous kaempferol, and isolated several novel germinating pollen cDNAs during the earliest moments of pollen germination in petunia. With the use of novel experimental strategies and the increasing availability of new genetic tools, global gene expression profiles have been explored in Arabidopsis pollen (Becker et al. 2003, Honys and Twell 2003, These authors contributed equally to this work. Corresponding author: E-mail, [email protected]; Fax, +886-2-27827954. 1519 1520 Gene expression profile in germinating lily pollen Lee and Lee 2003), maize sperm cells (Engel et al. 2003) and tobacco sperm cells (Xu et al. 2002). These studies greatly broaden our current knowledge of transcripts present in the mature pollen grain. However, further gene expression and functional analyses of individual transcripts (da Costa-Nunes and Grossniklaus 2003), as well as the establishment and characterization of the gene expression profiles in germinating pollen and/or pollen tubes, can expand our comprehension of the complex events during pollination (Mascarenhas 1993). In this study, Lilium longiflorum cv. Avita pollen stored at –20°C for 2 months showed retarded (by 1 h) germination and growth rate compared with fresh pollen. Some pre-synthesized proteins and mRNAs stored in mature pollen might have degraded gradually during cold storage and had to be resynthesized to carry on pollination. To test this hypothesis, cold-stored and fresh pollen grains were analyzed for total protein and gene expression profiles during pollen germination and tube growth. The total protein profile of cold-stored pollen was qualitatively and quantitatively decreased compared with that of fresh pollen. After suppression subtractive hybridization screening, thousands of clones were obtained, 99 cDNAs were enriched in the fresh mature pollen, and expression in both fresh and stored pollen grains and in pollen development was further characterized in 22. The expression of most of the 22 gradually disappeared during cold storage but reappeared on incubation of the stored pollen grains in germination medium for 2 h. Actinomycin D (ActD) significantly inhibited both germination and tube growth of cold-stored pollen and later tube growth of fresh pollen but had no effect on fresh pollen germination and early tube growth. Because of different sensitivities to cold storage and ActD, these 22 transcripts were divided into three groups according to their possible roles in pollen germination and tube growth. Several novel cDNAs (i.e. the first to be identified in pollen) were specifically expressed in the mature lily pollen grains and germinating pollen tubes. Their possible functions during pollination are discussed. Results Cold storage significantly altered lily pollen germination, growth and total protein composition. Lily pollen freshly collected or stored at –20°C for 1 and 2 months was germinated in vitro to evaluate the effect of cold storage on germination and growth rates (Fig. 1). Compared with fresh pollen, cold-stored pollen was delayed in germination (Fig. 1A) and initially had a slower tube growth rate (Fig. 1B). These consequences were correlated with the length of storage. However, stored pollen grains were still capable of pollination, as shown by the in vivo pollen tubes isolated from pollinated 24-h styles (Fig. 1C), and fertilization, as evidenced by ovule development (data not shown). To examine the possible causes of growth retardation at the protein level, the total protein profiles of fresh pollen and pollen cold-stored for 2 Fig. 1 Effects of cold storage on lily pollen germination and pollen tube growth. Lily pollen grains freshly collected or stored at –20°C for 1 or 2 months were cultured in vitro. Pollen germination (A) and tube growth (B) rates were measured. Compared with freshly collected pollen grains, cold-stored pollen showed greatly decreased germination and tube growth rates, which increased with longer storage time. (C) Pollen grains stored for 2 months at –20°C were checked for viability by in vivo pollination. After 24 h, the pollen tubes were pulled out of the style. The arrowhead and arrow indicate the stigma and aggregated in vivo pollen tubes, respectively. All results from three independent experiments were similar. Bar = 1 cm. months were compared on 2D-PAGE separation (Fig. 2). The profiles of fresh (Fig. 2A) and stored (Fig. 2B) pollen differed quantitatively and qualitatively. Many proteins were no longer detectable (solid arrows in Fig. 2A) or had greatly decreased levels (open arrows in Fig. 2A) in pollen cold-stored for 2 months. In addition, several new proteins and/or the products of the degraded proteins were present in the pollen (open arrowheads in Fig. 2B). Gene expression profile in germinating lily pollen Fig. 2 Protein degradation in cold-stored lily pollen grains. A total of 200 µg of total proteins extracted from freshly collected lily pollen grains (A) and those stored at –20°C for 2 months (B) were separated by IEF gel electrophoresis (pH 4.2–7.5) and 10% SDS-PAGE. Quantitative (open arrow) and qualitative (solid arrow) differences were observed. Several new proteins (open arrowhead) were also found in cold-stored pollen. The open boxes are the coordinates for comparison of the corresponding protein spots on the gels. M is the middle range protein molecular weight marker (kDa). De novo synthesis of essential mRNAs/proteins allows resumption of normal germination and tube growth in cold-stored pollen. The finding that cold-stored pollen needs a longer incubation time (about 1 h) than does fresh pollen to resume normal pollen germination and tube growth suggests that de novo synthesis of mRNA/protein, perhaps degraded during storage, may occur in cold-stored pollen during incubation. To test this hypothesis, fresh and cold-stored (2 months) pollen grains were germinated in medium with or without ActD and with or without cycloheximide, which are effective transcriptional and translational inhibitors, respectively. ActD treatment had no effect on fresh pollen germination (Fig. 3A) and early tube growth (the first 3 h in Fig. 3B), but later tube growth was greatly inhibited (Fig. 3B). ActD had a slight effect on germination of cold-stored pollen (Fig. 3A), but pollen tube growth was almost completely inhibited (Fig. 3B). Another efficient 1521 Fig. 3 Effects of ActD and cycloheximide on the restoration of germination competence of the cold-stored pollen grains. A total of 100 µg ml–1 of ActD or 100 µM of cycloheximide (CHX) was added to germination medium to determine the effect on pollen germination (A) and tube growth (B) rates of freshly collected and cold-stored (2 months) lily pollen grains. Cycloheximide completely inhibited both pollen germination and growth. ActD had no effect on fresh pollen germination (A) and early tube growth (B), but later tube growth was greatly inhibited (B). ActD had a stronger effect on the germination of cold-stored pollen (A), and pollen tube growth was almost completely inhibited (B). Results from three independent experiments were similar. transcriptional inhibitor, cordecypin (100 µg ml–1), gave a similar effect (data not shown). Cycloheximide efficiently inhibited both germination and tube growth of both fresh and cold-stored pollen (Fig. 3). The differences in germination rates of fresh pollen shown in Fig. 1, 3 are a consequence of different samples of pollen, but results from three independent experiments with either the same or different batches of collected pollen grains were similar. All these results supported the hypothesis, and suppression subtractive hybridization screening was conducted to clone the expressed and functional transcripts persisting in mature pollen grains. 1522 Table 1 Gene expression profile in germinating lily pollen Identity of selected cDNAs obtained from suppression subtractive hybridization screening Clone Amino acid identity (%) Amino acid Homolog in other organism similarity (%) (with Best Blast P) 69 (118/169) a 66 (18/27) 70 (129/183) 56 (53/94) 48 (46/94) 67 (105/156) 82 (140/169) 77 (21/27) 82 (151/183) 71 (68/94) 62 (60/94) 74 (117/156) Arabidopsis thaliana (AP000603) Medicago truncatula (AJ249611) A. thaliana (AP000735) Solanum tuberosum (AJ133765) A. thaliana (AC005825) A. thaliana (AL162691) 27 (32/115) 64 (90/139) 46 (54/115) 74 (105/139) Peptide transporter Plasma membrane H+-ATPase Monosaccharide transporter Na+/H+ antiporter 54 (92/168) 77 (35/45) 75 (90/119) 39 (101/254) 68 (117/168) 88 (40/45) 86 (104/119) 54 (141/254) A. thaliana (AL050352) Mesembryanthemum crystallinum This study (AF280432) A. thaliana (AL161555) This study Kosteletzkya virginica (AF029258) A. thaliana (AL161555) b A. thaliana (AC007063) This study RNA binding protein Acid phosphatase DnaJ Sgt1 COP8 Carbonic anhydrase NTP303 precursor 26S proteasome regulatory subunit 36 (22/60) 59 (65/109) 53 (68/126) 52 (62/119) 90 (147/162) 46 (36/77) 81 (63/77) 85 (91/106) 51 (31/60) 73 (81/109) 72 (93/126) 67 (81/119) 95 (156/162) 65 (51/77) 92 (72/77) 94 (101/106) A. thaliana (AC011437) A. thaliana (AJ243527) A. thaliana (U95973) Oryza sativa (AF192467) A. thaliana (AF176089) A. thaliana (U73462) Nicotiana tabacum (P29162) A. thaliana (AC073395) Unknown protein Hypothetical protein 65 (122/186) 54 (73/135) 79 (149/186) 65 (89/135) A. thaliana (AC018907) A. thaliana (AL035602) Name Cell wall/plasma membrane protein LLPB7 Pectin esterase LLPB9 Pectin methylesterase LLPC8 Extensin LLPD9 Invertase LLPD16 β-Galactosidase LLPE21 Cellulase Special transporter LLPC12 Ca2+-transporting ATPase LLPD5 Na+/myo-inositol symporter LLPD11 LLPD21 LLPD22 LLPE49 Others LLPB3 LLPC1 LLPC3 LLPC4 LLPC14 LLPD2 LLPD13 LLPD27 Unknown LLPB8 LLPD3 a b Pollen specificity b This study This study b This study b This study Parentheses indicate the number of amino acids for each clone used for the comparison. Pollen specificity was obtained from the Arabidopsis pollen transcriptome (Honys and Twell 2003). Cloning and characterization of the transcripts degraded in stored pollen by suppression subtractive hybridization screening. The cDNAs from cold-stored (2 months) pollen (driver) were used to subtract those from the fresh pollen (tester). Thousands of clones were obtained after nested PCR amplification, and 99 cDNAs greatly enriched in the fresh pollen (supplementary Table), as confirmed by reverse Northern blotting (data not shown), were sequenced and BLAST analyzed. The cDNAs were divided into five categories according to the nature of deduced amino acid sequences: 17% are involved in cell wall metabolism, 12% are various transporters, 6% participate in signaling pathways, 34% are components of other cellular processes and 30% are hypothetical proteins or clones with no significant similarity. Twenty-two cDNAs (Table 1) with higher expression levels in fresh than in cold-stored pollen, as examined by use of reverse Northern blotting (data not shown), were further investigated for expression patterns using Northern blotting (Fig. 4). For the cDNAs that encoded wall and/or plasma membrane proteins, most of the transcripts, except for that for LLPB7, were degraded after cold storage for 1 month (Fig. 4A, lane 2) and 2 months (Fig. 4A, lane 3), as compared with fresh pollen (Fig. 4A, lane 1). Those degraded mRNAs, however, reappeared and accumulated to normal or even higher levels in some cases after 2 h in vitro incubation (Fig. 4A, lane 4). These results suggest that the cold-stored pollen still has the ability, but needs a longer culture time, to re-synthesize the mRNAs gradually degraded during cold storage. Similar results were obtained for most of the cDNAs that encode several transporters (Fig. 4B), as well as novel ones (Fig. 4C), or proteins with unknown function (Fig. 4D). Some transcripts, including pectin esterase (LLPB7), acid phosphatase (LLPC1) and carbonic anhydrase (LLPD2), showed high stability even after 2 months’ storage. Also, ActD produced different responses during the 2h incubation (Fig. 4, lane 4 vs. lane 5): (i) a significant inhibitory effect on the message levels of LLPD21 (Fig. 4B), LLPB3, LLPC14 (Fig. 4C) and LLPB8 (Fig. 4D); (ii) a moderate effect on the levels of LLPB9, LLPC8, LLPE21 (Fig. 4A), LLPD5, LLPC12 (Fig. 4B) and LLPC3 (Fig. 4C); (iii) no effect on the Gene expression profile in germinating lily pollen Fig. 4 Low-temperature storage results in significant decreases in the expression of several cell wall/plasma membrane, transporter and novel genes in lily pollen. Twenty micrograms of total RNA from fresh pollen grains (lane 1) and pollen grains stored at –20°C for 1 month (lane 2), 2 months (lane 3) or 2 months, then germinated in vitro without (lane 4) or with (lane 5) actinomycin D for 2 h were analyzed by Northern blotting. (A) Wall/plasma membrane cDNAs used as probes: LLPB7 (pectin esterase), LLPB9 (pectin methylesterase), LLPC8 (extensin-like protein), LLPD9 (invertase), LLPD16 (β-galactosidase) and LLPE21 (cellulase). (B) Transporter cDNAs used as probes: LLPC12 (Ca2+-transporting ATPase), LLPD5 (Na+/myo-inositol symporter), LLPD11 (peptide transporter), LLPD21 (plasma membrane H+-ATPase), LLPD22 (monosaccharide transporter) and LLPE49 (Na+/H+ antiporter). (C) Novel cDNAs used as probes: LLPB3 (RNA binding protein), LLPC1 (acid phosphatase type 5), LLPC3 (DnaJ), LLPC4 (Sgt1), LLPC14 (COP8), LLPD2 (carbonic anhydrase) LLPD13 (1-ascorbate oxidase) and LLPD27 (26S proteasome regulatory subunit S12). (D) Two cDNAs of unknown function were used as probes. EtBr-stained rRNA was used as an internal standard to monitor equal loading of total RNA. levels of LLPB7, LLPD16 (Fig. 4A), LLPD11 (Fig. 4B), LLPC1, LLPC4, LLPD2, LLPD13, LLPD27 (Fig. 4C) and LLPD3 (Fig. 4D); and (iv) a slightly enhanced effect on the levels of LLPD9 (Fig. 4A), LLPD22, LLPE49 (Fig. 4B). The variable expression patterns of the characterized transcripts were probably caused by the different strengths of their promoters or mRNA stability and would be interesting for further study. Since pollen tube growth of cold-stored pollen was almost completely inhibited by ActD (Fig. 3B), the cDNA expression patterns sensitive to ActD as shown in Fig. 4 may play an 1523 Fig. 5 Expression of several genes in low-temperature-stored pollen is steadily inhibited by transcription inhibitor during pollen germination. mRNAs were purified from low-temperature-stored pollens cultured in the media with or without (control) 100 µg ml–1 cordecypin for 0, 3, 6 or 9 h and were used as templates for corresponding cDNA synthesis. Primer pairs specific to each cDNA were used for RT-PCR amplification to determine cDNA expression. Actin was the internal control. important role in pollen tube growth. To test this idea, 2-month low-temperature-stored pollen was cultured in media with or without another efficient transcription inhibitor, cordecypin, for 0, 3, 6 and 9 h and was used for total RNA extraction, mRNA purification and corresponding cDNA synthesis. Primer pairs designed specifically for each cDNA (listed in Table 2) were used for semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) amplification to determine their expression profiles after different incubation intervals. LLPD21 cDNA was too short for proper RT-PCR amplification and was not included in this experiment. For the pollen cultured in normal medium, all the cDNAs, including the ActD-insensitive LLPD27 and LLPC1 cDNAs shown in Fig. 4, started to accumulate after 3 h of incubation and continued to 9 h incubation (control in Fig. 5). However, this increased expression was significantly blocked by cordycepin, as evidenced by the steady expression levels of these cDNAs during all incubation periods (cordycepin in Fig. 5). In addition, the RT-PCR expression patterns correlated well with the results of Northern blotting (compare 0 and 3 h in Fig. 5 with lanes 1 and 2 in Fig. 4, respectively). All these results suggest that these ActD- and cordecypin-sensitive transcripts are essential for normal pollen tube growth. 1524 Gene expression profile in germinating lily pollen Table 2 Primers of chosen cDNAs and length of corresponding amplified products after RT-PCR analysis Clone Primer sequence Product size (bp) LLPB3 (RNA binding protein) Sense: 5′-TAGCATGGCACATATGGACCACGG-3′ Antisense: 5′-CGGAAGTGCCTTGCTATCCATTTGAG-3′ LLPB8 (unknown protein) Sense: 5′-CTGCATAAAGGTCGAAGATGTGGAGCC-3′ Antisense: 5′-CGAGGATAAGGTTGGATACGTC-3′ LLPB9 (pectin methylesterase) Sense: 5′-GGGCAGGTACGGCAACCGTGGC-3′ Antisense: 5′-TTCATAGCCTCGGCCCTGCTG-3′ LLPC3 (DnaJ) Sense: 5′-GATCACATTCGAAGGGATGGGG-3′ Antisense: 5′-GCTTAGCCTCTTAGGGAAGACAACAC-3′ LLPC4 (Sgt1) Sense: 5′-GCGTCGCAGG ATTTTGAAAAGCTG-3′ Antisense: 5′-GAGAGATTGATGCCCACATAG-3′ LLPC1 (acid phosphatase) Sense: 5′-GCGTCGCAGG ATTTTGAAAAGCTG-3′ Antisense: 5′-GAGAGATTGATGCCCACATAG-3′ LLPC8 (extensin) Sense: 5′-CCGACGAAGCGGTTGTTGCTCA-3′ Antisense: 5′-TGCCCTATCCGATGCCGAAGTC-3′ LLPC12 (Ca2+-transporting ATPase) Sense: 5′-GGATGGATTAGCTTCTCCCGAA-3′ Antisense: 5′-GGAAAGCCTTCGGAGAGTAGTTTG-3′ LLPC14 (COP8) Sense: 5′-GCTCGTCTCTATCTTGAGGATG-3′ Antisense: 5′-GTAGCAAGAACCCGAGAGCGTTGTG-3′ LLPD5 (Na+/myo-inositol symporter) Sense: 5′-ACGAATGGTCTGTTGATCACC-3′ Antisense: 5′-ACATGACTGTGTTGATACCAAC-3′ LLPD11 (peptide transporter) Sense: 5′-GGCTATCAGCCATCAATTGC-3′ Antisense: 5′-CTCCATGGATTGGTTTTCTCTG-3′ LLPD27 (26S proteasome regulatory subunit S12) Sense: 5′-CTCCCGAAGTTTGGGACCAGTGCTAT-3′ Antisense: 5′-GCACCCTCTCGTTCTGCTTTCC-3′ LLPE49 (Na+/H+ antiporter) Sense: 5′-GGGTGGGATGCCGCTCGG-3′ Antisense: 5′-CCTCGTCCAGTCGTGCTC-3′ actin Sense: 5′-GGCY*GGHTTYGCNGGNGAYGATGC-3′ Antisense: 5′CCKGATRTCVACRTCACACTTCAT-3′ 358 415 93 334 261 327 459 371 311 415 488 320 1,000 761 *IUB codes: H = A, C or T; K = G or T; N = any base; R = A or G; V = A, C or G; Y = A or G. Spatial and temporal expression of several novel cDNAs in lily pollen Several cDNAs coded for novel proteins, including DnaJ (LLPC3), Sgt1 (LLPC4), Na+/myo-inositol symporter (LLPD5), peptide transporter (LLPD11), Na+/H+ antiporter (LLPE49) and the 26S proteasome regulatory subunit S12 (LLPD27). These cDNAs were chosen to explore expression profiles during pollen development. Their spatial and temporal expression patterns were examined by semi-quantitative RT-PCR of the cDNAs obtained from total RNAs of lily roots (R), leaves (L), pistils (Pi), pollen grains (Po and 0 h), in vivo- (24 h) and in vitro- (12- and –24-h) grown pollen tubes with specific primer sets for each cDNA (Table 2). As shown in Fig. 6A, except for the cDNAs of actin (the internal total RNA-loading quantitative control) and LLPD27 (26S proteasome regulatory subunit S12), all cDNAs were specifically expressed in the pollen grain, and in the in vitro- and in vivo-grown pollen tube. The detailed temporal and spatial expression patterns of LLDNAJ and LLSGT1 transcripts were further investigated by Northern blot analyses. Both transcripts were pollen- (Po) specific, and no signal was detected in total RNA extracted from leaves (L), roots (R), tepals (T), filaments (F) or pistils (Pi, Fig. 6B, upper panel). The transcripts also belong to the late pollen-expressed genes with specific expression of LLDNAJ in anthers of 130mm buds (Fig. 6B, lower panel, lane 6); and for both LLDNAJ and LLSGT1 in mature anthers (Fig. 6B, lower panel, lane 7), mature pollen (Fig. 6B, lower panel, lane 8), and 3-h in vitrogerminated pollen tubes (Fig. 6B, lower panel, lane 9). Discussion A distinctive cold-storage-induced, artificial ‘depleted pollen’ system was used to systematically characterize the general profiles of the genes/proteins participating in lily pollen germination and tube growth. After lily pollen had been cold-stored for 2 months, typical germination and growth was significantly retarded and accompanied by quantitative and qualitative degradation of numerous pre-synthesized proteins and mRNAs (Fig. 2, 4). Thousands of cDNAs were obtained by suppression subtractive hybridization screening, and 99 cDNA clones Gene expression profile in germinating lily pollen enriched in fresh pollen (supplementary Table) were confirmed by reverse Northern blotting. Twenty-two of these with higher expression profiles in fresh pollen were chosen to examine their expression profile in fresh and cold-stored pollen grains. Northern blotting results revealed that most of these 19 cDNAs gradually disappeared during –20°C storage and that full recovery of these degraded transcripts, as well as resumption of germination and growth competence of the growth-retarded pollen grains, were achieved by immersion in culture medium for 2 h (Fig. 1, 4). Several novel cDNA clones showed pollen-grain- 1525 and pollen-tube-specific expression patterns and were the products of late genes. Thus far, most pollen-expressed genes have been obtained from studies of developing microspore and/or mature pollen, and only a few genes expressed in the germinating pollen and/or pollen tube have been well characterized (reviewed in Mascarenhas 1993, McCormick 1993, Twell 2002). The fact that cold-stored pollen germinated and grew after immersion in culture medium for an extended time (1 h) suggests that de novo synthesis of degraded mRNAs and proteins is essential to resume normal pollen germination and tube growth of coldstored pollen. This suggestion is supported by the significant inhibitory effect of ActD on cold-stored pollen germination and tube growth. For fresh pollen, ActD had no effect on germination and early tube growth, but it significantly inhibited later tube growth, as has been reported for other species (Mascarenhas 1975, Mascarenhas 1993). Cycloheximide prevented pollen germination and tube growth in both fresh and cold-stored pollen (Hoekstra and Bruinsma 1979). All these results imply that the persisting mRNAs in the mature pollen grain are sufficient, but the translation of these mRNAs is obligatory for pollen germination and early tube growth. The transcripts can be divided into three groups according to degree of response to ActD and cold storage, which may have a role in different developmental phases, such as pollen germination/ early pollen tube growth, later tube growth or both events (Fig. 3–5). The first group of transcripts, LLPD9, LLPD16, LLPD11, LLPD22, LLPE49, LLPD13, LLPD27 and LLPD3, is sensitive to cold storage but not ActD and involved in pollen germination and early pollen tube growth. The second group, LLPB9, LLPC8, LLPC12, LLPD5, LLPD21, LLPB3, LLPC3, LLPC14 and LLPB8, is sensitive to both cold storage and ActD (with varying degrees of sensitivity) and may participate in later pollen tube growth. The relationship between the expression of these transcripts and pollen tube growth is supported by Fig. 6 Pollen-specific expression patterns of several novel transcripts. (A) mRNAs isolated from lily root (R), leaves (L), pistils (Pi), pollen grains (P), in vivo-grown (24 h) pollen tubes (Vo) and in vitrogerminated (0, 12 and 24 h) pollen tubes were used as templates for corresponding cDNA synthesis. Actin was the internal control. Primer pairs specific to LLPC3 (DnaJ), LLPC4 (Sgt1), LLPD5 (Na+/myoinositol symporter), LLPD11 (peptide transporter), LLPE49 (Na+/H+ antiporter) and LLPD27 (26S proteasome regulatory subunit S12) cDNAs were used for RT-PCR amplification to determine the cDNA expression. (B) Twenty micrograms of DNase I-treated total RNA were isolated from lily leaves (L), roots (R), tepals (T), filaments (F), pistils (P), pollen (Po) and anthers/stamens of various-sized floral buds: 15 mm (1), 35 mm (2), 65 mm (3), 90 mm (4), 110 mm (5) and 130 mm (6); mature anther (7), mature pollen (8) and in vitrogerminated 3-h pollen tubes (9). Total RNA was denatured, separated on formaldehyde-agarose gels, transferred onto a nylon membrane and hybridized with DIG-labeled LLDNAJ or LLSGT1 cDNA inserts. Almost equal amounts of total RNA were loaded in each lane, as evidenced by ethidium bromide (EtBr) staining of the gel. 1526 Gene expression profile in germinating lily pollen the inhibition of cordycepin on both expression profiles and continuous pollen tube growth. The last group, LLPB7, LLPC1, LLPD2, is insensitive to both ActD and cold storage and may have a role in all phases of pollen germination and pollen tube growth. Nevertheless, we cannot rule out other essential, as yet to be identified, transcripts involved in these events. Another interesting phenomenon is the varying stability of the transcripts characterized here under cold stress. For example, LLPE21 (cellulase), LLPD21 (plasma membrane H+ATPase), LLPE49 (Na+/H+ antiporter), LLPB 3 (RNA binding protein), LLPC14 (COP8), LLPD27 (26S proteasome regulatory subunit S12) and LLPB8 (an unknown protein) were more sensitive to cold storage than the others, but some transcripts were extremely stable, with little or no degradation after long periods of storage, including LLPB7 (pectin esterase), LLPC1 (acid phosphatase) and LLPD2 (carbonic anhydrase) (Fig. 4). The results regarding these mRNAs, with quantitatively different degradation patterns, are consistent with the idea of differential RNA decay mechanisms in pollen as proposed by Ylstra and McCormick (1999). The precise functions of the novel cDNAs listed in Table 1 remain to be experimentally revealed, but their deduced protein sequences give us some clues about the fundamental nature of pollination. For example, LLPE21 cDNA, encoding a cellulase, may be responsible for penetration of the pollen tube into the stigmatic papillary cells during pollination. For the first time, we report several transcripts coding for novel proteins such as DnaJ, Sgt1, Na+/myo-inositol symporter, peptide transporter and the 26S proteasome regulatory subunit S12, in pollen, and their expression patterns are pollen specific, except for the 26S proteasome regulatory subunit S12 (Fig. 6A). The 26S proteasome is involved in many cellular protein degradation processes, so it is not surprising to find its transcripts present in all organs tested. The pollen-specific pattern of other transcripts suggests that their encoded proteins play important roles during pollination. It is known that style-secreted exudates are enriched with the pectin precursor myo-inositol (Rosen 1971), which is likely taken up into in vivo pollen tubes by the plasma membrane Na+/myo-inositol symporter for pollen wall synthesis during pollination. Lily stigma exudate contains γ-amino butyric acid in a bound form of N-acylated or peptide-like derivatives (Rosen 1971), which may be taken up into in vivo pollen tubes as by the peptide transporter (LLPD11) to support pollen tube growth as found in Arabidopsis (Palanivelu et al. 2003). The expression pattern of the lily Na+/H+ antiporter (LLPE49) transcript may reflect its function under stress conditions to protect the dehydrated mature pollen grain (Chauhan et al. 2000). The DnaJ protein, a member of the Hsp40 family, functions as a co-factor of the heat-shock protein 70 (Hsp70) chaperone machine in facilitation of protein folding (Kelley 1998, Lemmon 2001). SGT1 is a component of SCF ubiquitin ligase and participates in regulation of the cell cycle in yeast (Kitagawa et al. 1999) and meiosis in the Arabidopsis anther (Yang et al. 1999). Lily pollen belongs to the two-celled pollen type, and LLSGT1 may participate in the second mitosis of the generative cell to produce two sperm cells after pollen tube emergence. By taking advantage of the availability of the complete genomic sequence of Arabidopsis and contemporary genetic tools, two different strategies have been conducted recently to investigate the genome-wide gene expression of Arabidopsis pollen: Affymetrix ATH8K Genechip (Becker et al. 2003, Honys and Twell 2003) and serial analysis of gene expression (SAGE; Lee and Lee 2003). In addition, sequence analysis of a cDNA library constructed from maize sperm cells reveals sperm cell-specific transcripts that may be involved in gamete interaction during double fertilization (Engel et al. 2003). These studies significantly increase our current knowledge of the gene expression profile of a pollen grain; however, general and systematic gene and/or protein expression patterns during pollen germination and tube growth are lacking. In addition, the fact that ATH8K GeneChip contains only about 30% of the estimated genes in the Arabidopsis genome suggests that more pollen-expressed and/or -specific genes remain to be identified (da Costa-Nunes and Grossniklaus 2003). In this study, ActD significantly inhibited continuous tube growth of fresh pollen, which suggests that some genes essential for effective later tube growth may not be expressed until the later stages of pollination. Pollination involves very complicated cell-cell interactions, and the characters of pollen tubes in vivo differ significantly from those in vitro (Lord 2000). Pollen grown in vitro does not completely mimic that grown in vivo, and even with highly optimized germination medium, in vitro tubes reach only 30–40% of in vivo lengths (Read et al. 1993). Some transcripts might be up-regulated or induced in later phases of pollination as the in vivo pollen tube travels in the style and during double fertilization. Isolation and characterization of these pollen tube-specific or pollination-induced transcripts will allow us to explore the mechanisms of the complex cellcell interactions during pollination and double fertilization in flowering plants. Materials and Methods Plant materials Easter lily (Lilium longiflorum Thunb. cv. Avita) bulbs obtained from a local farm (Foreport Enterprises Co., Ltd, Taipei, Taiwan) were planted in a greenhouse under ambient conditions at Academia Sinica, Taiwan. Mature pollen grains from anthers were collected 1 day after anthesis and dried on a bench for 2 d. Dried pollen grains were used directly (fresh pollen) or stored at –20°C for later use (cold-stored pollen). To measure germination and growth rates, pollen was placed in germination medium (1.27 mM CaCl2, 0.162 mM H3BO3, 0.99 mM KNO3, 290 mM sucrose, pH 5.2) with or without 100 µg ml–1 ActD, cordecypin or 100 µM cycloheximide for various times. Tube length and germination rate were measured under an Olympus BH2 microscope. For in vivo pollination studies, ‘Snow Queen’ styles were pollinated 2 d after anthesis with Avita pollen stored at –20°C for 2 months, and in vivo pollen tubes were collected as described previously (Jauh and Lord 1995, Jauh and Lord 1996). Gene expression profile in germinating lily pollen Pollen total protein extraction and 2D-PAGE Total protein extraction, 2D-PAGE and Western blotting were performed according to Wang et al. (1992). The phenol-extracted total protein was precipitated, and the pellet was washed twice with cold acetone, then vacuum-dried and dissolved in solubilization buffer [9.5 M urea, 5 mM K2CO3, 2% (w/v) Triton X-100, 500 µg ml–1 Llysine, 0.5% DTT, 1.6% ampholines, pH 5–7, and 0.4% ampholines, pH 3–10] for 2D-PAGE or in SDS sample buffer for SDS-PAGE and Western blotting. mRNA purification, suppression subtractive hybridization and Northern blotting Total RNA was extracted by use of the Ultraspec™ RNA isolation system according to the manufacturer’s manual (Biotecx Laboratories, Houston, TX, U.S.A.). To avoid DNA contamination, total RNA was treated with of the Message Clean™ kit (Gen Hunter, Nashville, TN, U.S.A.), then dissolved in 20 µl DEPC-H2O and stored at –80°C for further use. mRNAs from fresh and cold-stored pollen were purified from the respective total RNA (1 mg) by use of an mRNA purification kit (Amersham Biosciences, Buckinghamshire, U.K.), and used directly for cDNA synthesis with use of the cDNA subtraction kit (Clontech, Palo Alto, CA, U.S.A.). mRNAs from fresh and cold-stored pollen grains were chosen as tester and driver material, respectively. After double hybridization and nested PCR, the cDNAs were inserted directly into the pGEM-T Easy vector (Promega, Madison, WI, U.S.A.) for sequencing, reverse Northern and Northern blotting analyses. The sequence data were analyzed by use of the Vector NTI Suite (V.7) (IndorMax, Bethesda, MD, U.S.A.) and BLAST program of NCBI. For Northern blotting, 20 µg of DNase I-treated total RNA was separated on a 1% agarose gel, transferred onto a nylon membrane and cross-linked by UV light. Probe synthesis and hybridization were performed by use of a DIG Northern Starter kit according to the manufacturer’s manual (Roche, Mannheim, Germany). After being incubated with anti-DIG antibody at 25°C for 1 h, membranes were washed with buffer twice for 15 min. Membranes were incubated with detection buffer containing CDP-star ready-to-use reagent and exposed to X-ray film. The partial sequences of the transcripts characterized in this study are available upon request. Semi-quantitative RT-PCR analysis of the expression pattern of genes in different organs By using an Absolutely RNA® RT-PCR Miniprep Kit (Stratagene, La Jolla, CA, U.S.A.), total RNA was isolated from 0.1 g of lily tissues: leaves, roots, tepals, filaments, pistils, pollen and anthers/ stamens of various-sized floral buds, including the mature anther and in vitro- and in vivo-grown pollen tubes. First-strand cDNA was synthesized from 3 µg total RNA with oligo(dT) primer and random primers according to the manufacturer’s protocol (M-MLV Reverse Transcriptase; Invitrogen Life Technologies, Carlsbad, CA, U.S.A.). To study the expression pattern of genes in the pollen grain, in vitrogerminated (12 h) pollen tubes and different organs, RT-PCR analyses were performed using the gene-specific primer sets as listed in Table 2. The PCR mix consisted of 1.5 mM magnesium chloride, 0.2 mM dNTPs, 0.5 µM each of sense and antisense primer, 2.5 U of Taq polymerase (MDBio, Taipei, Taiwan) and 1× PCR buffer supplied with the Taq polymerase. The reaction was conducted under the following protocol: 94°C for 10 min; 25–30 cycles at 94°C for 1 min, annealing at 55°C for 1 min, 72°C for 1 min; final elongation at 72°C for 10 min with use of a Biometra® T3 Thermocycler (Whatman Biometra, Goettingen, Germany). The resulting PCR products were run on a 1% agarose gel containing 0.01% ethidium bromide. To monitor the efficacy of cDNA synthesis by PCR amplification, the housekeeping gene actin 1527 was used as a positive control for PCR amplification, and a 761-bp fragment was obtained by use of the primer sets listed in Table 2. Supplementary material Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oupjournals.org. Acknowledgments We thank Drs. E.M. Lord and Yue-Ie Hsing for critically reading and providing comments on the manuscript. This work was supported by grants NSC 91-2317-B-001-014 and NSC 92-2311-B-001-064 from National Science Council, Academia Sinica (Taiwan, ROC) and the Li Foundation (U.S.A.) to G.Y.J. References Becker, J.D., Boavida, L.C., Carneiro, J., Haury, M. and Feijo, J.A. (2003) Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome. Plant Physiol. 133: 713–725. Chauhan, S., Forsthoefel, N., Ran, Y., Quigley, F., Nelson, D.E. and Bohnert, H.J. (2000) Na+/myo-inositol symporters and Na+/H+-antiport in Mesembryanthemum crystallinum. Plant J. 24: 511–522. da Costa-Nunes, J.A. and Grossniklaus, U. (2003) Unveiling the gene-expression profile of pollen. Genome Biol. 5: 205. Engel, M.L., Chaboud, A., Dumas, C. and McCormick, S. (2003) Sperm cells of Zea mays have a complex complement of mRNAs. Plant J. 34: 697–707. 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