PI-PLC

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Characterization of Phosphatidylinositol-Specific Phospholipase C (PI-PLC) ... College of Life Sciences, Agricultural University of Hebei, Baoding, Hebei 071000 ...
Plant Cell Physiol. 46(10): 1657–1665 (2005) doi:10.1093/pcp/pci181, available online at www.pcp.oupjournals.org JSPP © 2005

Characterization of Phosphatidylinositol-Specific Phospholipase C (PI-PLC) from Lilium daviddi Pollen Yan-Yun Pan 1, 2, Xin Wang 1, 3, Li-Geng Ma 1 and Da-Ye Sun 1, * 1 2

Institute of Molecular Cell Biology, Hebei Normal University, Shijiazhuang, Hebei 050016, PR China College of Life Sciences, Agricultural University of Hebei, Baoding, Hebei 071000, PR China ;

The phosphatidylinositol-specific phospholipase C (PIPLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60– 65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP2-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PIPLC activity caused by cholera toxin and it also leads to the decrease of [Ca2+]cyt in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM. Keywords: cDNA cloning — Extracellular calmodulin (CaM) — Heterotrimeric G protein — Lilium daviddi pollen — Phosphatidylinositol-specific phospholipase C. Abbreviations: CaM, calmodulin; CTX, cholera toxin; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate; IP3R, IP3 receptor; PI-PLC, phosphoinositide-specific phospholipase C; PIP2, phosphatidylinositol-4,5-bisphosphate; PTX, pertussis toxin; U73122, (1-[6([(17β)-3-Methoxyestra-1,3,5(10)-trien-17yl]amino)hexyl]-1H-pyrrole2,5-dione); U73343, (1-[6-(-[(17β)-3-methoxyestra-1,3,5(10)-trien-17yl] amino)hexyl]-2,5-pyrrolidinedione). The nucleotide sequences reported in this paper have been submitted to the GenBank database under the following accession numbers: AY735314 (LdPLC1) and AY735313 (LdPLC2).

3 *

Introduction The phosphatidylinositol-specific phospholipase C (PIPLC) signaling pathway has been implicated in a variety of physiological processes in both animals and plants. Many extracellular signals are transmitted across the plasma membrane by PI-PLC, which hydrolyzes the phosphoinositide 4,5bisphosphate (PIP2), a minor membrane phospholipids, and result in the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), both as novel second messengers in plant cells (Meijer and Munnik 2003). In the plant system, PIPLC activity was first identified in oat root plasma membranes (Tate et al. 1989). From then on, the characterization of PI-PLC has been widely studied (Melin et al. 1992, Cho et al. 1995, Huang et al. 1995, Shi et al. 1995a, Brearley et al. 1997). Yamamoto et al. (1995) cloned the first plant EST from Arabidopsis thaliana shoots during floral induction, which matched to the PI-PLC. Hirayama et al. (1995) obtained a cDNA (AtPLC1) from Arabidopsis thaliana shoots under dehydration and salt stress conditions; while AtPLC2 is constitutively expressed in vegetative and floral tissues in Arabidopsis thaliana (Hirayama et al. 1997). The cDNAs of PI -PLC have been cloned in many kinds of plant so far, such as soybean (Shi et al. 1995b), potato (Kopka et al. 1998), pea (Venkataraman et al. 2003), mung bean (Vigna radiata L.) (Kim et al. 2004) and moss Physcomitrella patens (Mikami et al. 2004). Furthermore, a series of studies suggest that plant PI-PLCs, as in animals, play important roles in a wide variety of physiological processes. They are involved in response to a variety of stimuli, including environmental stress (Legendre et al. 1993, Smolenska-Sym and Kacperska 1996, Chapman 1998), gravity (Perera et al. 1999), anaerobic stress (Reggiani and Laoreti 2000), pathogen attack and pollination (Stevenson et al. 2000, Wang 2001) and aluminium (Al) stress (Martinez-Estevez et al. 2003), especially in ABA signal pathway (Assmann and Shimazaki 1999, Staxén et al. 1999, Sanchez and Chua 2001, Cousson 2003, Hunt et al. 2003, Mills et al. 2004). Recently, plant PIPLCs were also verified to be involved in cytokinin and gravity responses (Repp et al. 2004) and the response to ELF-EMF (Piacentini et al. 2004). Pollen germination and tube growth are complicated and hazardous in the fertilization process and many factors involved in this process remain to be identified (Franklin-Tong

Present address: Institute of Genetics and Developmental Biology, Chinese Academy of Science, Beijing, People’s Republic of China. Corresponding author: E-mail, [email protected]; Fax, 86-311-85820649. 1657

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Phospholipase C pathway in pollen

Fig. 2 The phylogenetic analysis of selected plant PI-PLCs. The accession and organism of each PI-PLC are U25027.1(GmPLC), Glycine max (soybean); Y15253.2 (PsPLC), Pisum sativum (pea); X94289.1 (StPLC3), Solanum tuberosum (potato); AF223573.1 (NtPLC2), Nicotiana tabacum (common tobacco); D50804 (AtPLC2), Arabidopsis thaliana, respectively (from http://www.ncbi.nlm.nih.gov).

detected in pollen using the method of Melin et al. (1992). It was found that the PIP2-dependent PI-PLC activity increased linearity for the first hour and then became stable (Fig. 1). Fig. 1 Detection of PI-PLC activity in Lilium daviddi pollen protoplasts. Protoplasts were incubated with authenticated [3H]-PIP2, and the release of IP3 was measured. The data are from three independent experiments; vertical bars indicate standard error range.

1999). Helsper et al. (1987) first demonstrated the presence of PI-PLC activity in pollen tubes of Lilium longiflorum; while Franklin-Tong et al. (1996) provided pharmacological evidence for the presence of a Ca2+-dependent PI-PLC activity in P. rhoeas pollen, and their result indicated the correlation between PI-PLC activity and pollen tube growth during the self-incompatibility process (Franklin-Tong et al. 1996). However, no direct evidence has been found to show the existence of PI-PLC transcripts in plant pollen system. In previous work, we found that U-73122, an inhibitor of PI-PLC, could inhibit germination and tube growth of Lilium daviddi pollen in a dose-dependent manner (Ma et al. 1998). Further experiments by means of microinjection showed that anti-PLC or anti-IP3R antibodies inhibited the Lilium daviddi pollen tube elongation, whereas, pollen tube elongation was stimulated by microinjecting IP3 (Wang et al. 2000). These results suggest that PI-PLC-IP3-IP3R signaling pathway might be present in Lilium daviddi pollen system and be involved in pollen tube growth (Wang et al. 2000). In the present study, we first isolate the two full-length PI-PLC cDNAs from Lilium daviddi pollen, detect their PIP2-hydrolyzing activity, and provide evidence that the PI-PLC activity is present and might be regulated by heterotrimeric G-protein and exogenous CaM in pollen.

Results Analysis of PI-PLC activity in the Lilium daviddi pollen protoplasts To measure the PI-PLC activity directly, the pollen protoplasts were prepared according to the method of Tanaka et al. (1987), and those protoplasts were used to measure the PI-PLC activity in the pollen. The PIP2-dependent PI-PLC activity was

Cloning of PI-PLC cDNA from Lilium daviddi pollen The above mentioned results suggest the presence of PIPLC transcript in Lilium daviddi pollen. In order to clone the PLC gene in Lilium daviddi pollen, two degenerate oligonucleotides designed according to the X domain conserved amino acid sequence were used in a PCR analysis with cDNA from Lilium daviddi pollen as a template. Two DNA fragments with about 400 bp in length were amplified. Blast analysis indicated that these two fragments were homologous to plant PI-PLCs. The nested primers were designed according to the two fragment nucleotide sequences and the two full length cDNAs corresponding to the two PI-PLCs were obtained by 5′RACE and 3′RACE, and they were named as LdPLC1 and 2. The fulllength cDNA of LdPLC1 has 1878bp with an open reading frame representing 475 amino acids, and 1785bp cDNA of LdPLC2 codes a protein with 536 amino acids in length. Relative molecular weights of Lilium daviddi PI-PLC enzymes are 54 kDa and 61 kDa, respectively. The pIs are 8.572 (LdPLC1) and 5.978 (LdPLC2), respectively. BLAST results indicate that the primary structures of LdPLC1 and LdPLC2 are similar to those of currently known plant PI-PLC homologs (http:// www.ebi.ac.uk/clustalw). Homology analysis among some plants’ PI-PLC protein sequences indicate that LdPLC1 and LdPLC2 are more closely related to each other than to any other plants’ PI-PLCs (Fig. 2 and supplemental Fig. 1) and they show 60–65% identities to other plants’ PI-PLCs (supplemental Fig. 1). The deduced amino acid sequences of LdPLCs include the X, Y and C2-domains, which are ubiquitous in other PI-PLCs, but N termini of LdPLCs are shorter than the PI-PLCs from other plant species. The recombinant protein of LdPLC possesses PIP2-hydrolyzing activity To confirm that the LdPLCs genes cloned from Lilium daviddi pollen encode the proteins with PI-PLC activity, we introduced the full length cDNA of LdPLCs into pET28b vector. The recombinant protein of AtPLC1 expressed in E. coli was used as a positive control, which had showed the PI-PLC activity as described by Hirayama et al. (1995). Both

Phospholipase C pathway in pollen

Fig. 3 Detection of PIP2-hydrolyzing activity for recombinant HisPI-PLCs protein. PIP2-hydrolyzing activity of the recombinant PI-PLC proteins are assayed in a crude extracts (10 µg protein) from IPTGinduced E. coli cells. Control is the crude extracts from IPTG-induced E. coli cells that harbored pET28b vector.

recombinant LdPLCs and AtPLC1 expressed in E. coli BL21 cells contained a 6-His-tag in the N-terminal, and they showed the expected electrophoretic motilities for the relative molecular weights predicted from the cDNA sequences (data not shown). Four kinds of crude extracts of E. coli cells (induced by IPTG) including harbored pET28b vector (negative control), pET28b-AtPI-PLC1 (positive control), pET28b-LdPLC1 and pET28b-LdPLC2, were detected for PIP2-hydrolyzing activity. It was shown that both LdPLCs possessed PIP2-hydrolyzing activity, while no PI-PLC activity was detected in the negative control (Fig. 3). Compared with the positive control AtPLC1, the PIP2-hydrolyzing activity for LdPLC1 was lower, but LdPLC2 was higher than that of AtPLC1 (Fig. 3). Those results suggest that both of the two LdPLCs we cloned from Lilium daviddi pollen have PI-PLC activity. Analysis of PI-PLC genes expression in Lilium daviddi pollen grains and pollen protoplasts To assess whether the two LdPLCs are related to pollen germination, we first check the expression pattern of LdPLCs in the pollen by RT-PCR. The results show that both LdPLC1 and LdPLC2 are expressed in both pre-germinated and germinating pollen. The transcript level of LdPLC2 was induced in germinating pollen, whereas LdPLC1 transcript level did not change during pollen germinating process (Fig. 4A). LdPLCs were also expressed in the leaf (data not shown), suggesting that they are not specifically expressed in pollen. In order to assess whether the protoplasting process affect LdPLCs expression, the transcription levels of LdPLCs from pollen grains and pollen protoplasts are compared. It was shown that there was not obvious different for LdPLCs expression level between pollen grains and pollen protoplasts, suggesting that protoplasting process may not affect LdPLCs expression (Fig. 4B).

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Fig. 4 Analysis the expression of LdPLC1 and 2 in Lilium daviddi pollen by RT-PCR. (A) Expression of LdPLC1 and 2 in pre-germinated or germinating pollen. a: pre-germinated pollen; b: germinating pollen (1 h); c: germinating pollen (2 h); (B) Expression of LdPLC1 and 2 in pollen gains or pollen protoplasts. a: pollen grains; b: pollen protoplasts. Actin-exon: both up-stream and downstream primers are from actin exon sequence; actin-intron: up-stream primer is from actin intron sequence, and down-stream primer is from actin exon sequence. PCR of actin-intron have no production using the total RNA as a template.

Regulating factors of PI-PLC activity in the Lilium daviddi pollen protoplasts Our previous work indicated that extracellular CaM was able to regulate pollen germination and tube growth (Ma and Sun 1997), a heterotrimeric G protein might be involved in the regulation of pollen tube growth and extracellular CaM was able to activate intrinsic GTPase activity of the heterotrimeric G protein in pollen (Ma et al. 1999). To assess the relationship among extracellular CaM, G protein and PI-PLC in the regulation of pollen germination and tube growth, we measured the effect of exogenous CaM and heterotrimeric G protein on the PI-PLC activity by supplying with exogenous purified CaM or G protein modulators into the incubated pollen protoplast system. It was found that the PI-PLC activity was increased by adding exogenous CaM in a dose-dependent manner, whereas a control protein, BSA, had no effect on the activity of PI-PLC (Fig. 5A). The stimulation effect increased about three-fold when the exogenous CaM concentration was 10–9 mol liter–1, but began to decrease when exogenous CaM concentrations were higher than 10–9 mol liter–1 (Fig. 5A). Anti-CaM antibody inhibited the stimulation effect of PI-PLC activity caused by exogenous CaM in a dose-dependent manner (Fig. 5B). Using G protein modulators, we addressed the relationship between PI-PLC and heterotrimeric G protein activities, and we observed that the activity of PI-PLC was increased by G protein agonist cholera toxin (CTX) and inhibited by G protein antagonist pertussis toxin (PTX) (Fig. 6). PI-PLC activity was stimulated by CTX at the range from 200 to 800 ng ml–1, and it was enhanced about 3-fold when the CTX concentration was at 600 ng ml–1 (Fig. 6A). Whereas, PI-PLC activity was inhibited by PTX at a concentration from 400 to 800 ng ml–1 in a dosedependent manner (Fig. 6B). Those results suggest that a heter-

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Fig. 5 The effect of exogenous CaM on the activity of PI-PLC in Lilium daviddi pollen. (A) Purified exogenous CaM or BSA was added into the incubation medium at indicated concentrations. The [3H]-IP3 amount was measured after the addition of CaM or BSA over a period of 20 min. The data are from three independent experiments; vertical bars indicate standard error range. (B) CaM and/or anti-CaM antibody were added into the incubation medium at indicated concentrations. The [3H]-IP3 amount was measured after the addition of CaM and/or anti-CaM antibody over a period of 20 min. The data are from three independent experiments; vertical bars indicate standard error range.

Fig. 6 The effect of membrane-permeable G-protein modulators on the activity of PIP2-dependent PI-PLC in Lilium daviddi pollen. (A) Lilium daviddi protoplasts were treated with CTX. The [3H]-IP3 amount was measured after the addition of CTX over a period of 20 min. The data are from three independent experiments; vertical bars indicate standard error range. (B) Lilium daviddi protoplasts were treated with PTX. The [3H]-IP3 amount was measured after the addition of PTX over a period of 20 min. The data are from three independent experiments; vertical bars indicate standard error range.

otrimeric G protein might be an upstream-regulator of PI-PLC activity. These results led us to predict that extracellular CaM, heterotrimeric G protein and PI-PLC might work in the same signal transduction pathway. Therefore, the relationship between extracellular CaM and the heterotrimeric G protein in the regulation of PI-PLC activity in pollen were further investigated. We found that the stimulation effect of extracellular CaM on PI-PLC activity was inhibited by PTX in a dose-dependent manner (Fig. 7A), suggesting that heterotrimeric G protein acted downstream of extracellular CaM for the regulation of PI-PLC activity. It was also found that the stimulation effect of CTX on PI-PLC activity was inhibited by PI-PLC antagonist U-73122, whereas a weaker PI-PLC antagonist U-73343 showed a less inhibitory effect compared with that of U-73122 (Fig. 7B). This result may further indicate that PI-PLC acts downstream of heterotrimeric G protein.

The effects of membrane-permeable PI-PLC antagonist on the [Ca2+]cyt in pollens To verify whether PI-PLC activity is necessary for [Ca2+]cyt maintaining, we checked the [Ca2+]cyt using confocal laser scanning microscope. We observed an obvious polar distribution (data not show) and weak oscillation of [Ca2+]cyt in intact Lilium daviddi pollen grains (Fig. 8A, B, control curve). Shortly after U-73122 was app1ied to the bathing medium, [Ca2+]cyt decreased slowly during the following 30 min, no apparent [Ca2+]cyt changes were able to be recorded in the control and in U-73343 treatment (Fig. 8A). Our previous evidence suggested that the purified exogenous CaM could elevate the intracellular calcium concentration (Shang et al. 2001). In this study, we observed that the effect of exogenous CaM on [Ca2+]cyt increase was inhibited by U-73122 when both of them were added in the bathing medium (Fig. 8B). These results further suggest that PI-PLC may be involved in the regulation of [Ca2+]cyt in pollen.

Phospholipase C pathway in pollen

Fig. 7 The relationship between extracellular CaM and heterotrimeric G-protein in regulating of PI-PLC activity in pollen protoplasts. (A) The effect of CaM and PTX on PIP2-dependent activity of the PI-PLC in Lilium daviddi pollen. Exogenous purified CaM and/or PTX were added into the incubation medium at indicated concentrations. The [3H]-IP3 amount was measured after the addition of CaM and/or PTX over a period of 20 min. The data are from three independent experiments; vertical bars indicate standard error range. (B) The effect of CTX, U-73122 and U-73343 on the activity of PI-PLC in Lilium daviddi pollen. Pollen protoplasts were treated with CTX, U73122 or U-73343. The [3H]-IP3 amount was measured after the addition of CaM, U-73122 or U-73343 over a period of 20 min. The data are from three independent experiments; vertical bars indicate standard error range. The concentration of CTX is 600 ng ml–1, both U73122 and U-73343 is 10 µM.

Discussion The presence of PI-PLC gene and PI-PLC enzyme activity in the Lilium daviddi pollen In mammalian cells, eleven distinct PI-PLCs, which are grouped into four subfamilies as PI-PLCβ, γ, δ and ε, have been identified. There are five domains which are conserved in animal PI-PLC enzymes, these domains include: PH domain in the N-terminus, an EF-hand domain, anX domain, a Y domain which together withX domain to constitute the catalytic domain of the enzyme, and a C2 domain in the C-terminus (Wang 2001). Recently, a novel, sperm-specific phospholipase C, PLCζ, was identified from mouse (Saunders et al. 2002), it triggers Ca2+ oscillations at fertilization and plays a role as the molecular trigger for development of a fertilized egg into an

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Fig. 8 The dynamic of free calcium fluorescence in Lilium daviddi pollen. (A) The effect of different concentrations of U-73122 on cytosolic free Ca2+ in geminating pollen. (B) The effect of CaM (10–7 mol l–1), U-73122 (40 µM), and CaM together on cytosolic calcium concentration. The data are from three independent experiments. Three independent experiments have the same trend, only one of three was shown here.

embryo. PLCζ lacks PH domain compared with the typical animal PI-PLCδ type. Plants’ PI-PLCs appear to have only one gene family, which is similar to the PI-PLCδ subfamily of mammalian PI-PLC family (Wang 2001). In this paper, we isolated two full length cDNA sequences of PI-PLC from Lilium daviddi pollen. BLAST analysis show that both LdPLCs had three domains, X, Y and C2 domain, which are core sequences found in typical PI-PLCs, and they show 60–65% sequence identities to other plants’ PI-PLCs (Fig. 2). Different from other plants PI-PLC, LdPLC2 has shorter EF-hand domain in N-terminus, and LdPLC1 absents EF-hand domain. The similar molecular structure appears in Vr-PLC3 from mung bean, which also contains a short EF-hand (Kim et al. 2004).

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Our previous works also provided some preliminary evidence for the presence and function of a PI-PLC pathway in pollen germination and tube growth (Ma et al. 1998, Wang et al. 2000). Herein we provide two more experimental evidences to show that there is a correlation between the PI-PLC activity and the germination of pollen. First, two full-length of PI-PLC cDNAs (LdPLC1 and LdPLC2) were cloned from Lilium daviddi pollen, and RT-PCR analysis verified that both of them expressed in pollen and the expression of LdPLC2 was induced during pollen germination or tube growth process (Fig. 4). Second, the PI-PLC activities were detected in pollen protoplasts (Fig. 1). Even though we can not provide direct evidence that the PI-PLC activity we measured in the pollen came from the two LdPLCs we cloned from Lilium daviddi pollen due to the lack of Ld-PLC mutants in Lilium daviddi, the recombinant proteins of the LdPLCs expressed in E. coli cells showed the PIP2-hydrolyzing activity (Fig. 3). These results still indicate the presence and involvement of PI-PLC to regulate pollen germination and tube growth. Pollen PI-PLC activity is regulated by heterotrimeric G protein and exogenous CaM In mammalian cells, PI-PLCβ is activated by heterotrimeric G protein and PI-PLCγ is activated by tyrosine kinasecoupled receptors. Both of them play key roles in transducing extracellular signals for regulation of growth, cell proliferation, metabolism and secretion (Wang 2001). Although the sequences of LdPLCs we found, as does the other plants’ PIPLC cDNAs, are more similar to the PI-PLCδ subfamily than other subfamilies in animals structurally (Hartweck et al. 1997, Hirayama et al. 1995, Hirayama et al. 1997, Kopka et al. 1998), there are still many evidences to support that the heterotrimeric G protein, which activates the PI-PLCβ subfamily in animals, might be involved in the regulation of plants’ PI-PLCs (Arz and Grambow 1994, Munnik et al. 1998, van Himbergen et al. 1999, Reggiani and Laoreti 2000, Millner 2001, Apone et al. 2003). Our previous work indicated that a heterotrimeric G protein might be involved in the regulation of pollen tube growth and exogenous CaM was able to activate intrinsic GTPase activity of heterotrimeric G protein in pollen (Ma et al. 1999). In the present study, we further assess whether or not the PIPLC activity is influenced by G protein and extracellular CaM. Due to the inefficient permeation of PIP2 into Lilium daviddi pollen grains cell through the pollen cell wall, we have to prepare the pollen protoplasts system to detect the PI-PLC activity. These results show that PI-PLC activity is increased by the treatment of G protein agonist CTX, and the increase in PIPLC activity caused by CTX is inhibited by PI-PLC inhibitor (Fig. 6A, 7B). Whereas, PI-PLC activity is inhibited by G protein antagonist PTX (Fig. 6B). Thus, these results suggested that Lilium daviddi pollen PI-PLC activity may be regulated by heterotrimeric G protein and PI-PLC is downstream of heterot-

rimeric G protein in the signal transduction pathway. We also found that exogenous CaM was able to activate the PI-PLC activity (Fig. 5). These results lead us to predict that exogenous CaM, heterotrimeric G protein and PI-PLC might work in the same signal transduction pathway. Because of the stimulation effect of extracellular CaM on PI-PLC activity was inhibited by PTX in dose-dependent manner (Fig. 7A), it is suggesting that heterotrimeric G protein might act downstream of extracellular CaM for regulation of PI-PLC activity. It should be mentioned that all PI-PLC activity data in this study are from pollen protoplasts, but neither from pollen grains nor from pollen tube. While, there is no evidence to show that pollen protoplasts are physiologically identical to pollen grains or pollen tube. The protoplasting process may activate PI-PLC proteins or induce expression of additional PIPLC genes, which contribute toward the PI-PLC activities. Thus, the results from protoplasts may not be necessarily relevant to pollen germination. The PI-PLC pathway regulates mobilization of [Ca2+]cyt in pollen Increase in cytosolic free Ca2+ concentration ( [Ca2+]cyt) is necessary for pollen germination (Song et al. 1998), and PIPLC pathway is involved in the release of free Ca2+ into cytosol from organelles (Rhee 2001, Wang 2001). Several evidences support that PI-PLC-IP3 signaling system is involved in mobilization and oscillation of [Ca2+]cyt in plants (Kashem et al. 2000, Wang 2001). In the pollen system, Franklin-Tong et al. (1996) showed that IP3 could induce the release of Ca2+ to regulate Papaver rhoeas pollen tubes growth. Our previous work provided evidence that exogenous CaM treatment was able to increase [Ca2+]cyt in the pollen system (Shang et al. 2001). In the present study, we showed that the increase effect on [Ca2+]cyt caused by exogenous CaM was repressed by PI-PLC inhibitor (Fig. 8). These results suggest that the Ca2+ release may be the next step of the PI-PLC pathway in the pollen system. Our previous results also showed that pollen tube growth was inhibited by microinjection of IP3R antibody, and increased by microinjection of IP3 (Wang et al. 2000). Based on all the above mentioned results, we can presume that the PIPLC-IP3-IP3R-Ca2+ cascade are involved in regulating the growth of pollen tube.

Materials and Methods Plant materials Pollen of Lilium daviddi was used in this study. The pollen grains were collected from freshly opened anthers and stored at –74°C for use in each experiment. Preparation of calmodulin and anti-calmodulin antibody Cauliflower calmodulin (CaM) was purified by PhenylSepharose 4B affinity chromatography as described previously (Biro et al. 1984). Anti-CaM antibody was raised against potato CaM isoform VI (PCM6), which was expressed and purified from engineering E. coli BL21 (a kindly gift from Professor Poovaiah, Washington Univer-

Phospholipase C pathway in pollen sity, USA). The antibody was identified to cross-react specifically with plant CaM (Bai et al. 2002). Isolation and purification of protoplasts Protoplasts were prepared by the method described by Tanaka et al. (1987), with some modifications. Pollen grains were directly suspended in White’s medium (White 1963). After being incubated at 30°C for 10 min, the isolated pollen was suspended in the enzyme solution, which included White’s medium supplemented with sucrose (0.6 M), PVP (0.1%), cellulase R-10 (1.5% w/v), and 0.5% potassium dextran sulfate (all reagents are from Calbiochem), with the pH adjusted to 5.8. The enzyme treatment was performed at 30°C for 3 h on a revolving platform shaker operating at 80 rpm After the enzyme treatment, protoplasts were washed 3 times with modified White’s solution containing 0.6 M sucrose by centrifugation at 500×g for 5 min. The final suspension was then layered on a 16% Percoll solution containing the culture medium for purification. The purity of protoplasts was about 90%. Detection of PI-PLC activity Assay of PI-PLC activity was conducted as described by Melin et al. (1992) with some modifications. The standard incubation mixture contained 50 mM Tris–HCl, pH6.0, 0.6 M sucrose, 0.8 mM EDTA, 0.8 mM CaCl2 (25 µM free Ca2+), 10 µl [3H]-PIP2 (1,100 dpm nmol–1, NEN™) in micellar solution and an appropriate amount of purified protoplasts (about 1 µg membrane protein) or the crude extract of E. coli cells (about 10 µg proteins) in a final volume of 60 µl. The [3H]-PIP2 solution was prepared by evaporating the lipids in solvent to dryness under a stream of nitrogen followed by sonication in a water bath for 10 min in Tris–HCl (50 mM pH 6.0) buffer. The above mixture was incubated at 4°C for 4 h, then a reaction was started by the addition of various agonists (ddH2O as a control) performed at 30°C for 20 min. The reaction was stopped by the addition of 750 µl chloroform/methanol (2 : 1, v/v) followed by 250 µl of 0.6 M HCl. After vortexing and 3 min centrifugation (2,000×g), 350 µl of the upper phase was transferred to a scintillation vial, supplemented with 5 ml of liquid (0.3 mg ml–1 POPOP, 4 mg ml–1 PPO, toluene : ethanol 7 : 3, v/v) and radioactivity was measured in a liquid scintillation counter (PACKARD, 2200CA). Values presented have been recalculated to correspond to the total upper phase. The analyses were performed in duplicate when the reaction rate was proportional to incubation time and amount of protein. Preparation of RNA samples One hundred micrograms of Lilium daviddi pollen grains were ground in a mortar with a pestle in the presence of liquid nitrogen. Total RNA was prepared using the RNAwiz (AMBION), as described in RNAWIZ procedure. The total RNA was further digested by DNase and purified using RNeasy Plant Mini Kit (QIAGEN) in order to avoid contamination from genomic DNA. Cloning of PI-PLC cDNAs from Lilium daviddi pollen The full-length cDNA encoding PI-PLCs were obtained by a combination of RT-PCR and RACE cloning. Total pollen RNA (5 µg) was used to synthesize the first stand cDNA using SUPERSCRIPT II RT (GIBCO BRL) reverse transcriptase. Two cDNAs fragments were amplified using two primers, 5′-cayaaywcytayytsacyggk-3′, corresponding to HNS(T)YLT, and 5′-ytcyttyggnggyttngtws-3′, complementary to the coding region of STKPPKE. The primer sequences were selected from X domain conserved regions of PI-PLCs. The temperature program was: 94°C, 5 min; 94°C, 30 s, 55°C, 1 min, 72°C, 1 min, 35 cycles; 72°C, 10 min. The expected size of the X-like product was about 400 bp. Cloning of cDNA 5′-end and 3′-end were per-

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formed using a RACE system (SMART RACE Amplification Kit, CLONTECH). Downstream nested primers of amplification of cDNA 5′-end and 3′-end were designed according to the X-like product sequence, respectively. The primers of LdPLC1 were 5′-aggtggtcctcaagagttatcacaacgg-3′and 5′-accaagatatgaatgccccgctgtc-3′. The primers of LdPLC2 were 5′-gatccacgggagttgtcagcgtc-3′and 5′-cagagtggcgtgagagtcattgagctgg-3′. Upstream primer was provided in kit. The temperature program was: 94°C, 5 s, 72°C, 3 min, 5 cycles; 94°C, 5 s, 70°C, 10 s, 72°C, 3 min, 5 cycles ; 94°C, 5 s, 68°C, 10 s, 72°C, 3 min, 30 cycles. Amplification of the LdPLC cDNAs contained 5′-untranslated region, coding region and partial 3′-untranslated region. Two PCR primers were designed according to the cDNA 5′-end and 3′-end nucleotide sequences in order to test whether 5′-end and 3′-end nucleotide sequences were from the same template. The sequences of primers of LdPLC1 were 5′-acgcgggggatgcggagaaggtgattg-3′ (upstream) and 5′actaaaaccaggcgggctacaaagaacc-3′ (downstream). The sequences of primers of LdPLC2 were 5′-acgcgggagattcggcggaggatgctg-3′ (upstream) and 5′-accaatgcagaaaagccccaaactttcc-3′ (downstream). The temperature program was 94°C, 1 min; 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, 30 cycles; 72°C, 10 min. Sequencing and phylogenetic analysis of LdPLCs DNA sequence was carried out by the dideoxy chain termination method using a Dye terminal cycle sequencing ready reaction kit (Perkin-Elmer) and with a DNA sequencer (model 377, PE Applied BioSystems). The BLAST and OMIGA programs were used for searching homology against databases in GenBank and deducing amino acid, respectively. The multi-alignment amino acid sequence and phylogenetic analysis of selected plant PI-PLCs were performed at http://www.ebi.ac.uk/clustalw. Analysis of recombinant LdPLCs expressed in Escherichia coli Recombinant LdPLC1 and LdPLC2 proteins were expressed in E. coli BL21 as His-LdPLC fusion proteins using the pET28b vector. The complete coding regions of LdPLC1 and LdPLC2 were amplified by PCR using pfu-DNA polymerase. The resulted expression vectors were named pET28b-LdPLC1 and pET28b-LdPLC2, respectively. The primers were specific for each of the two LdPLCs. While the AtPLC1 was amplified by PCR and introduced the pET28b vector as described by Hirayama et al. (1995). E. coli BL21 was transformed with pET28b-AtPLC1, pET28bLdPLC1 or pET28b-LdPLC2 and the transformants (10 ml) were grown in LB culture medium (plus 100 mg ml–1 Amp) at 37°C to A600=0.6, then they were subsequently induced for 1 h~3 h with 1 mM isopropyl-β-D-galactopyranoside (IPTG). The cells were harvested, washed and suspended in extraction buffer (PBS). The cellular extracts were analyzed by SDS–PAGE and crude extracts were assayed the PIP2 activity using the method described above. Analysis of LdPLCs expression RNA samples (500 ng) isolated from pollen of Lilium daviddi were used as the template. One step RT-PCR kit (TaKaRa) was used to examine LdPLCs expression in pollen or germination pollen. The sequences of primers of LdPLC1 were 5′-acgcgggggatgcggagaaggtgattg-3′ (upstream) and 5′-actaaaaccaggcgggctacaaagaacc-3′ (downstream). The sequences of primers of LdPLC2 were 5′-acgcgggagattcggcggaggatgctg-3′ (upstream) and 5′-accaatgcagaaaagccccaaactttcc3′ (downstream). The temperature program was 94°C, 1 min; 94°C, 30 s, 60°C, 30 s, 72°C, 1 min, 30 cycles; 72°C, 10 min. The lengths of LdPLCs were about 1.8 kb, respectively. As the control, actin cDNAs fragment was amplified using the same RNA samples in the program of 94°C, 1 min; 94°C, 30 s, 52°C, 30 s, 72°C, 1 min, 30 cycles; 72°C, 10 min. The primers of actin-exon cDNA were 5′-agcaactgggatga-

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tatgg-3′ (forward primer) and 5′-tccacatcacacttcatgat-3′ (reverse primer), and length of fragment was about 600bp. The up-stream primer of actin-intron DNA was 5′-aaccttagaaaatttcaatat-3′, the down stream primer was same to the reverse primer of the actin-exon. The length of actin-intron fragment was about 1,000 bp when using genomic DNA as the template Calcium fluorescent indicator loading and the measurement of [Ca2+]cyt Pollen grains were hydrated for 30 min with BK solution which consists of 1 mmol l–1 Ca(NO3)2, 1 mmol l–1 KNO3, 1 mmol l–1 MgSO4, 1 mmol l–1 boric acid, 0.5 mol l–1 sucrose. The method for loading fluorescent indicator under low-temperature was accorded to Zhang and Rengel (1998). Adding the stock fluo-3AM solution (Molecular Probes, dissolved in DMSO) into pollen grain suspension to a final concentration of 20 µmol l–1. Then pollen grains were incubated in dark at 4°C for 1 h, centrifuged and washed with BK solution twice, then incubated in dark at 25°C for 30 min. A confocal laser scanning microscope (Bio-Rad, MR/A-2) was used to measure calcium distribution and fluorescent intensity. During the scanning period, U-73122 was added in bathing medium to trace cytoplasmic calcium dynamics in pollen cells. The experiment was repeated more than 3 times, in each treatment, data from 20~30 cells were recorded.

Supplementary material Supplementary material mentioned in the article is available to online subscribers at the journal website www.pcp.oupjournals.org.

Acknowledgments We thank Jessica Habashi (Department of Molecular, Cellular & Developmental Biology, Yale University) for reading and commenting on this manuscript. This work was supported by The Outstanding Younger Scientist Foundation of China (No. 30025024), The National Key Basic Research Special Funds (No.G1999011700 and No.90208004) of China, The National Natural Science Foundation of China Grants (No. 39730230 and No. 39870365)

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(Received February 21, 2005; Accepted July 25, 2005)