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CELL BIOLOGY AND SIGNAL TRANSDUCTION

Ca²⁺ Dynamics in a Pollen Grain and Papilla Cell during Pollination of Arabidopsis¹

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630–0101, Japan (M.I., H.S., T.M., F.-S.C., S.T., A.I.); and The Institute of Physical and Chemical Research, Wako, Saitama 351–0198, Japan (T.N., A.M.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Ca²⁺ dynamics in the growing pollen tube have been well documented in vitro using germination assays and Ca²⁺ imaging techniques. However, very few in vivo studies of Ca²⁺ in the pollen grain and papilla cell during pollination have been performed. We expressed yellow cameleon, a Ca²⁺ indicator based on green fluorescent protein, in the pollen grains and papilla cells of Arabidopsis (Arabidopsis thaliana) and monitored Ca²⁺ dynamics during pollination. In the pollen grain, [Ca²⁺]_cyt increased at the potential germination site soon after hydration and remained augmented until germination. As in previous in vitro germination studies, [Ca²⁺]_cyt oscillations were observed in the tip region of the growing pollen tube, but the oscillation frequency was faster and [Ca²⁺]_cyt was higher than had been observed in vitro. In the pollinated papilla cell, remarkable increases in [Ca²⁺]_cyt occurred three times in succession, just under the site of pollen-grain attachment. [Ca²⁺]_cyt increased first soon after pollen hydration, with a second increase occurring after pollen protrusion. The third and most remarkable [Ca²⁺]_cyt increase took place when the pollen tube penetrated into the papilla cell wall.

In vitro pollen germination requires the presence of Ca²⁺, boron, and an osmoticant such as Suc (Brewbaker and Kwack, 1963). Lack of Ca²⁺ in the growth medium results in morphological abnormalities such as coiling and tip swelling (Shivanna and Rangaswamy, 1992; Taylor and Hepler, 1997). In vitro germination assays using microinjection of a Ca²⁺-sensitive fluorescent probe revealed a [Ca²⁺]_cyt gradient in the tip of the growing pollen tube (Rathore et al., 1991; Franklin-Tong et al., 1993; Malho et al., 1994; Pierson et al., 1994, 1996). Furthermore, it has been shown that such a tip-localized intracellular [Ca²⁺]_cyt gradient, which arises from the influx of Ca²⁺ at the tube tip, is essential for pollen tube elongation (Franklin-Tong et al., 1993; Malho et al., 1994; Pierson et al., 1994, 1996; Holdaway-Clarke et al., 1997; Messerli and Robinson, 1997; Messerli et al., 2000; Feijo et al., 2001; Plieth, 2001; Robinson and Messerli, 2002; Holdaway-Clarke and Hepler, 2003). These in vitro observations suggest that Ca²⁺ dynamics are involved in the pollination process in vivo. However, because of the difficulty in microinjecting a Ca²⁺-sensitive probe into an exine-covered pollen grain, such investigations have not been performed. In addition, there have been few reports concerning Ca²⁺ dynamics in papilla cells, because the physical damage caused by microinjection has been shown to perturb the normal pollination process (Dearnaley et al., 1997). Another approach for real-time imaging of Ca²⁺ involves the expression in plant cells of aequorin, a Ca²⁺-sensitive luminescent protein (Knight et al., 1991). This method, however, requires the incorporation of the cofactor coelenterazine for light emission, and the luminescence is relatively weak. To examine Ca²⁺ dynamics in the pollen grain and papilla cell during pollination, it is important to develop a new, highly sensitive system to monitor real-time Ca²⁺ dynamics in these cells without damaging them.

Yellow cameleons, which are new Ca²⁺ indicators, have been developed to combine the advantages of molecular targeting with a fluorescent readout (Miyawaki et al., 1997, 1999). These indicators are chimeric proteins consisting of an enhanced cyan fluorescent protein (ECFP), calmodulin (CaM), a glycyl-Gly linker, the CaM-binding domain of myosin light chain kinase (M13), and an enhanced yellow fluorescent protein (EYFP). When Ca²⁺ binds to the CaM domain, this domain associates with the M13 peptide, causing the protein to adopt a more compact conformation, which increases the efficiency of fluorescence resonance energy transfer between ECFP and EYFP. Several types of cameleons have been developed with differing affinities for Ca²⁺ due to tunable binding of the CaM domain. Yellow cameleon 2.1 (YC2.1) is a high-affinity indicator and has been shown to be effective in monitoring [Ca²⁺]_cyt in Arabidopsis (Arabidopsis thaliana) guard cells (Allen et al., 1999, 2000, 2001). Yellow cameleon 3.1 (YC3.1) is a low-affinity indicator suitable for monitoring rapid changes in [Ca²⁺]_cyt such as those that occur in the pollen tube.

To investigate Ca²⁺ dynamics during pollination, we genetically transformed Arabidopsis to express the YC3.1 gene in pollen grains and papilla cells. We compared Ca²⁺ dynamics in pollen grains and pollen tubes germinated using in vitro, in vivo, and semi in vivo systems. We also monitored Ca²⁺ dynamics in papilla cells during pollination. These results will be discussed in relation to the physiological relevance of Ca²⁺ dynamics in the pollination process.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Expression of YC3.1 in the Pollen Grains and Ca²⁺ Dynamics in Pollen Germination and Pollen Tube Growth in Vitro

We independently transformed 13 plants with pAct1YC3.1. After selection on kanamycin-containing Murashige and Skoog plates, we confirmed by reverse transcription-PCR that all of the plants were transcribing the transgene. To select the plant with the brightest fluorescence, pollen tubes growing on the germination medium were observed using fluorescence microscopy (Blue excitation, No. 10 filter, Axiophoto; Carl Zeiss, Jena, Germany).

To confirm that green fluorescence was due to the ECFP and EYFP of the genetically expressed YC3.1, we obtained the fluorescence spectra of the growing pollen tubes using a spectral-imaging microscope system with excitation at 458 nm. The YC3.1 spectrum comprised both spectra typically observed from recombinant ECFP and EYFP proteins. Photobleaching with 514-nm light in a 5 µm x 10-µm area of the pollen tube tip induced an increase in ECFP fluorescence of about 15% and a decrease in EYFP fluorescence (data not shown). This demonstrated that full-length YC3.1 was expressed in the transgenic pollen tubes and that fluorescence resonance energy transfer (FRET) occurred between ECFP and EYFP. To investigate [Ca²⁺]_cyt distribution in the pollen tube, the ECFP and EYFP components of the YC3.1 fluorescence spectra were separated and the EYFP:ECFP ratio was calculated. After digital low-pass filter treatment, the gray-level value of each pixel was expressed with a pseudo-color as shown in Figure 1. Corroborating a previous report, [Ca²⁺]_cyt was found to be higher at the tip region. To investigate Ca²⁺ dynamics at the pollen tube tip, fluorescence images were acquired at 1.2- to 6-s intervals in the 463- to 543-nm range. These images were separated into ECFP and EYFP images. We measured the ECFP and EYFP fluorescence intensities in a 5 x 5-µm² region at the tip and calculated the EYFP:ECFP ratio. At the tip, the value of this ratio oscillated periodically around a mean value of 1.32 ± 0.24 (n = 16; Fig. 2). Furthermore, we converted the YC3.1 ratios into approximate [Ca²⁺]_cyt according to the previous report (Allen et al., 1999), and then we estimated that [Ca²⁺]_cyt at the tip region ranged from 0.15 to 0.68 µM and changed periodically with a period between 12 and 21 s (Table I).

View larger version (35K): [in this window] [in a new window]   Figure 1. An EYFP:ECFP ratiometric image of the growing pollen tube. ECFP and EYFP images were separated from the total YC3.1 fluorescence image using their individual spectra, and an image depicting their ratio was obtained.

View larger version (12K): [in this window] [in a new window]   Figure 2. Periodic change of the EYGP:ECFP ratio in a 5 x 5 µm2 region of the growing pollen-tube tip observed during in vitro pollination.

View larger version (32K): [in this window] [in a new window]   Figure 3. Distribution of Ca2+ in the pollen grain during in vitro germination. a, A ratiometric image of the pollen grain after hydration. b, A ratiometric image of a protrusion on a pollen grain. [Ca2+]cyt increase was observed not only at the potential germination site (arrow) but also at another site (arrowhead). c, A ratiometric image of a nifedipine-treated pollen grain after hydration.

To examine Ca²⁺ dynamics in a pollen grain during pollination, YC3.1 fluorescence was monitored at 1.2- to 30-s intervals after a pollen grain from a pAct1YC3.1-expressing plant was pollinated onto a papilla cell under micromanipulation. During pollination, the YC3.1 fluorescence in the pollen grain and pollen tube were captured and split into ECFP, EYFP, and autofluorescence images. Figure 4 shows a time course of ratiometric images.

View larger version (121K): [in this window] [in a new window]   Figure 4. Ca2+ dynamics in the pollen grain during in vivo pollination. Monitoring was performed under dry conditions. [Ca2+]cyt was high at the center of the pollen grain before hydration. [Ca2+]cyt was increased at the potential germination site during hydration (9'30''–15'30''). The accumulation continued until germination (approximately 22'30'').

During the period from germination to penetration of the papilla cell wall, [Ca²⁺]_cyt changed frequently at the tip of the pollen tube. Furthermore, when the pollen tube elongated within the papilla cell after penetration, changes in [Ca²⁺]_cyt were observed not only at the tip, but also throughout the elongated pollen tube (Fig. 5). The mean value of the ratio at the tube tip was 1.69 ± 0.16 (n = 20), representing a mean [Ca²⁺]_cyt of 0.86 µM (range, 0.64–1.14 µM; Table I). The period of the [Ca²⁺]_cyt oscillation was shorter (3.6–6 s; Fig. 6) than that observed in vitro, although a faster monitoring system with higher resolution, such as a cooled charged coupled device (CCD) camera, would be required to determine the oscillation period more precisely. The [Ca²⁺]_cyt near the penetration site, however, was frequently elevated (Fig. 7). At this site, the pollen tube changed its direction of elongation.

View larger version (90K): [in this window] [in a new window]   Figure 5. Ca2+ dynamics during the elongation of a pollen tube in the papilla cell wall. In the growing pollen tube, frequent changes of [Ca2+]cyt throughout the tube were observed.

View larger version (14K): [in this window] [in a new window]   Figure 6. Periodic changes in the EYGP:ECFP ratio in a 5 x 5 µm2 region at the tip of the growing pollen tube within the papilla cell wall observed during in vivo pollination.

View larger version (64K): [in this window] [in a new window]   Figure 7. Ca2+ distribution in the elongated pollen tube within the papilla cell wall. This image was constructed from a series of optical sections taken at 1.0-µm intervals. An increase in [Ca2+]cyt was observed near the penetration site (an arrow) into the papilla cell.

As demonstrated by the observed changes in [Ca²⁺]_cyt, the [Ca²⁺]_cyt oscillation period in the pollen grain was shorter in vivo than in vitro. Therefore, we speculated that molecules from the papilla cell affect [Ca²⁺]_cyt distribution in the pollen tube. To study whether Ca²⁺ dynamics in a pollen tube passing through the pistil are different from those in vitro, we monitored [Ca²⁺]_cyt in the growing pollen tube under semi in vivo conditions. Figure 8 shows the tip of the growing pollen tube at 3-s intervals. The mean value of the EYFP:ECFP fluorescence ratio at the tip was 1.98 ± 0.26 (n = 20), representing a mean [Ca²⁺]_cyt of 1.42 µM (range, 0.91–2.18 µM; Table I), which was higher than that observed in vitro. The oscillation period varied between 9 and 15 s (Fig. 9).

View larger version (52K): [in this window] [in a new window]   Figure 8. Ca2+ distribution in the growing pollen tube under semi in vivo conditions. Periodic changes in [Ca2+]cyt were observed at the tube's tip.

View larger version (15K): [in this window] [in a new window]   Figure 9. Periodic changes in the EYGP:ECFP ratio in a 5 x 5-µm2 region at the tip of the growing pollen tube under semi in vivo conditions.

The in vitro, nifedipine-induced inhibition of [Ca²⁺]_cyt increase and pollen germination suggested that the Ca²⁺ required for pollen germination is internalized from the external medium. We hypothesized, then, that Ca²⁺ in the papilla cell would affect germination in vivo. To examine Ca²⁺ dynamics in the papilla cell during pollination, a construct encoding the YC3.1 gene driven by the SLG promoter, which is highly expressed in the stigma of Brassica, was transformed into Arabidopsis. We obtained five transgenic plants and selected the plant with the brightest YC3.1 fluorescence.

Before pollination, no local increase of [Ca²⁺]_cyt was observed (before, Fig. 10). [Ca²⁺]_cyt near the surface was estimated to be below 0.1 µM. During pollination, however, remarkable [Ca²⁺]_cyt increases in the papilla cell directly apposing the pollen grain occurred several times (Fig. 10). Later in the hydration period (6'00'' in Fig. 10), increases in [Ca²⁺]_cyt near the pollen attachment site were prominent. During this hydration period, [Ca²⁺]_cyt in the pollen grain increased at the potential germination site (Fig. 4). Before pollen germination (9'00'' and 10'00'' in Fig. 10), [Ca²⁺]_cyt was increased locally near the pollen attachment site, and in the pollen grain itself, [Ca²⁺]_cyt was highest at the tip and decreased from there (Fig. 4). Furthermore, before penetration of the pollen tube into the papilla cell (12'00'' in Fig. 10), [Ca²⁺]_cyt increased at the site of pollen tube attachment. The increase in [Ca²⁺]_cyt continued during the elongation of the pollen tube. The maximum [Ca²⁺]_cyt after pollination was estimated to be 0.8 µM. We observed a similar increase in [Ca²⁺]_cyt during in vivo pollination in 20 independent experiments. These observations indicate that the increase in [Ca²⁺]_cyt in the papilla cell is induced by pollination and that interaction between a pollen grain and a papilla cell during pollination is mediated through Ca²⁺.

View larger version (94K): [in this window] [in a new window]   Figure 10. Ca2+ distribution in the papilla cell during pollination. Arrows represent notable increases in [Ca2+]cyt, which were typically observed 6, 10, and 12 min after pollination.

We examined the mean growth rates of the growing pollen tubes using in vitro, in vivo, and semi in vivo techniques (Table I). The mean growth rate was fastest in vivo and slowest in vitro. Thus, the growth rate was inversely proportional to the period of [Ca²⁺]_cyt oscillation at the tip of the pollen tube.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Using transgenic Arabidopsis expressing YC3.1, we have analyzed here for the first time, to our knowledge, the Ca²⁺ dynamics of a single pollen grain and a papilla cell during pollination. In the pollen grain, [Ca²⁺]_cyt was increased at the potential germination site after hydration, and a tip-focused gradient of [Ca²⁺]_cyt was formed before germination. After penetration into the papilla cell wall, rapid oscillation of [Ca²⁺]_cyt was observed at the tip of the growing pollen tube. In the papilla cell, on the other hand, [Ca²⁺]_cyt increased periodically during pollen hydration, after pollen protrusion, and during pollen tube penetration. These increases in [Ca²⁺]_cyt in the papilla cell were correlated with the behavior of the attached pollen grain.

In the rehydrated pollen grain, a [Ca²⁺]_cyt gradient was observed before germination both in vivo and in vitro. Treatment with the Ca²⁺ channel blocker nifedipine was shown to inhibit both [Ca²⁺]_cyt gradient formation and pollen germination in vitro. Such inhibition has also been reported in Narcissus pseudonarcissus (Heslop-Harrison and Heslop-Harrison, 1992a, 1992b). These results suggest that a pregermination [Ca²⁺]_cyt gradient, presumably formed by Ca²⁺ influx through L-type Ca²⁺ channels, is essential for pollen tube germination. However, the pulsatile growth of the pollen tube in Nicotiana and Petunia has been shown to be sensitive to gadolinium and lanthanum but not to nifedipine (Malho et al., 1995; Geitmann and Cresti, 1998; Feijo et al., 2001; Holdaway-Clarke and Hepler, 2003). Furthermore, in the growing hyphae of fungi, which display similar Ca²⁺ dynamics to the growing pollen tube, stretch-activated channels have been discovered on the plasma membrane, and the apical [Ca²⁺]_cyt gradient can be blocked with gadolinium (Garrill et al., 1992, 1993). Finally, it has been suggested that stretch-activated channels are involved directly in pollen tube growth (Holdaway-Clarke and Hepler, 2003). However, it is not clear whether stretch-activated channels exist in the potential germination site of the pollen grain or whether the same Ca²⁺ channels are used both in establishing the pregermination [Ca²⁺]_cyt gradient and in maintaining the tip of the growing pollen tube. To elucidate this issue, these in vitro effects of selective Ca²⁺ channel blockers on [Ca²⁺]_cyt-gradient formation during pollen germination should be examined in vivo using transgenic Arabidopsis expressing YC3.1.

The in vivo-pollinated pollen tube exhibited a higher growth rate than those pollinated under in vitro or semi in vivo conditions. The oscillation frequency of [Ca²⁺]_cyt at the tip was also fastest in the in vivo-pollinated pollen tubes. However, the [Ca²⁺]_cyt level at the tip was intermediate in the in vivo pollination. The elongation rate of a growing neuron has been shown to be correlated with the intracellular calcium level at the growth cone, with maximal outgrowth occurring at an optimal [Ca²⁺]_cyt in the growth cone (Kater and Mills, 1991). By analogy with these results in neurons, [Ca²⁺]_cyt in in vivo-pollinated pollen tubes might be optimal for pollen tube growth. However, these results must be interpreted cautiously due to the wide variety of factors other than Ca²⁺ that presumably affect pollen tube growth.

Under semi in vivo germination conditions, the oscillation frequency of [Ca²⁺]_cyt at the growing pollen tip and the pollen tube's growth rate were both faster than those observed under in vitro conditions. Furthermore, [Ca²⁺]_cyt was higher semi in vivo compared to in vitro. Under semi in vivo conditions, the germination medium contains various substances from the pistil, which modify the growth rate of the pollen tube. In fact, transmitting tissue-specific protein, a substance secreted by the pistil, has been shown in tobacco to attract pollen tubes and stimulate their growth (Cheung et al., 1995). Thus, YC3.1-expressing pollen grains will be useful to search for molecules that mediate [Ca²⁺]_cyt dynamics in the pollen tube.

[Ca²⁺]_cyt in the apical region of the papilla cell was shown to increase from its low level before pollination at the site of pollen grain attachment during pollen hydration. This result suggests that a pollen grain induces a local increase of [Ca²⁺]_cyt in an adjacent papilla cell. In addition, after this local [Ca²⁺]_cyt increase in the papilla cell, a pregermination [Ca²⁺]_cyt gradient formed in the pollinated pollen grain. These results suggest that the [Ca²⁺]_cyt gradient is formed by the influx of Ca²⁺ from the papilla cell, although it is not clear whether the Ca²⁺ comes from the cytoplasm or cell wall of the papilla.

Reproduction in flowering plants comprises several sequential steps, from pollination to fertilization. In this study, we have monitored Ca²⁺ dynamics in the pollen grain, pollen tube, and papilla cell during the early steps of the reproductive process using YC3.1-expresseing Arabidopsis. This novel monitoring system makes possible the detailed study of Ca²⁺ dynamics throughout the reproductive process. Furthermore, this system should be useful for investigating mechanisms by which a pollen tube and style communicate.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Transgenic Constructs

A cassette containing the YC3.1-coding region followed by the nopaline synthase polyadenylation signal from pBIYC3.1 was constructed by replacing the -glucuronidase-coding sequence of pBI221 (CLONTECH Laboratories, Palo Alto, CA) with a YC3.1-coding region. The 1.5-kb fragment upstream of the Act1 gene, which is highly expressed in the reproductive tissues (An et al., 1996), was isolated from genomic DNA of a Brassica rapa S9 homozygote, and the 2.2-kb fragment upstream of the SLG9 gene, which is highly expressed in the papilla cell (Suzuki et al., 1997a), was obtained from a P1-derived artificial chromosome clone, E89 (Suzuki et al., 1997b). These fragments were reamplified by PCR using specific primers designed to add HindIII and XbaI, and SphI and SmaI restriction sites to the 5' and 3' ends, respectively, as follows. For amplification of the 5'-flanking region of the Act1 gene: sense primer, 5'-GAAGCTTTCTCTTTAAAAGTTAAGTTTTCTTTGTACATGTCTCTAAGC-3'; and antisense primer, 5'-GTCTAGATTTCTTCTACCTTTATGCAAATCCAAACATTGTTTAAAGATC-3'; for amplification of 5'-flanking region of the SLG9 gene: sense primer, 5'-GGATGCAAAGCATGCATTGAATTATTAGA-3'; and antisense primer, 5'-CCCGGGCTCTCTCCCCACCTTTTTCTTTC-3'. The amplified fragments were subcloned into pBIYC3.1 to yield Act1 promoter-YC3.1 and SLG9 promoter-YC3.1 constructs, respectively. These chimeric genes, comprising the 1.5-kb promoter region of the Act1 gene, the YC3.1 coding sequence, and the nopaline synthase transcription terminator, or the 2.2-kb promoter region of the SLG9 gene, the YC3.1 coding sequence, and the nopaline synthase transcription terminator, were inserted into the binary vector pSLJ1006 (Jones et al., 1992) to create pAct1YC3.1 and pSLG9YC3.1, respectively.

Transformation

pSLJAct1YC3.1 and pSLJSLG9YC3.1 plasmids were electroporated into the Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993). The Agrobacterium infiltration procedure was performed on unopened flower buds of Arabidopsis, ecotype Columbia, as previously described (Bechtold et al., 1993). The transformed seeds were selected on 1/2 Murashige and Skoog plates containing kanamycin (50 µg/mL) and were analyzed by PCR amplification for the presence of YC3.1 genes.

Plant Growth Conditions

Arabidopsis (Arabidopsis thaliana) plants, ecotype Columbia, were grown in mixed soil in a growth chamber. The light intensity was 120 to 150 µmol m^–2 s^–1 during a 12-h daily light period. The temperature was 22°C ± 2°C.

Fluorescence Imaging

For in vitro imaging, pollen grains from freshly dehisced anthers of YC3.1-expressing plants were mounted on germination medium containing 2 mM CaCl₂, 1.65 mM boric acid, 1% (w/v) agar (Ultra-low gelling temperature type IX-A; Sigma, St. Louis), and 17% (w/v) Suc (pH was adjusted with KOH to 7.0; Preuss et al., 1993) in moistened glass-bottomed dishes. After 2 to 6 h at 20°C, [Ca²⁺]_cyt was monitored in pollen tubes growing out of the style using a spectral imaging fluorescence microscope system (LSM510 META, Carl Zeiss). This system is capable of resolving the spectra of fluorescence images; therefore, we could obtain images with no interference between overlapping fluorescence emissions (Haraguchi et al., 2002). Samples were scanned by an argon laser (excitation 458 nm). A Zeiss 63x W.Korr, 1.2 nicotianamine fluorescence objective was used for imaging of pollen tubes and an LD40x Korr, 0.6 nicotianamine fluorescence objective for imaging of papilla cells.

To examine the effect of a Ca²⁺ channel blocker, nifedipine (Sigma, Poole, UK) was dissolved in dimethyl sulfoxide and added to the above germination medium to give a final concentration of 10^–5 to 10^–4 M (Heslop-Harrison and Heslop-Harrison, 1992b). Dimethyl sulfoxide alone had no discernible effect on germination and tube growth.

For in vivo imaging, a pistil was mounted on a coverslip prior to pollination and fixed with double-sided tape. After a pollen grain was mounted on a papilla cell using a micromanipulator, [Ca²⁺]_cyt was monitored under dry conditions using the microscope system described above.

For semi in vivo imaging, flowers of wild-type Arabidopsis were excised before dehiscion and attached to an agar plate after the anthers were removed from the flowers. Pollen grains from freshly dehisced anthers of YC3.1-expressing plants were attached to the wild-type stigma. Thirty minutes after pollination, the upper half of the pollinated pistil was excised and mounted on the above germination medium in a moistened glass-bottomed dish. After 2 h incubation at 20°C, [Ca²⁺]_cyt in the pollen tubes growing through the style was monitored using the microscope system described above.

Ratiometric Imaging

Basic ECFP and EYFP spectra were obtained and registered using recombinant CFP-CaM protein and M13-YFP protein expressed in Escherichia coli or onion cells that transiently express the 35S promotor-CFP-CaM gene or the 35S promotor-M13-YFP gene. The autofluorescence spectrum for pollen grains was captured and registered using wild plants. After YC3.1 fluorescence was captured in each experiment, the images could be split into ECFP, EYFP, and autofluorescence channels based on the registered spectra. Ratiometric images (EYFP/ECFP) were obtained using image processing software (Laser Lixel, Bio-Rad Laboratories, Hercules, CA).

Calibration of YC3.1 Ratiometric Changes

Calibration of [Ca²⁺]_cyt was carried out as described previously (Allen et al., 1999). Serial dilutions of purified YC3.1 protein were made in Ca²⁺ calibration buffer (Molecular Probes, Eugene, OR), in which free Ca²⁺ concentration ranged from 0 µM to 39 µM. YC3.1 dilutions that gave similar intensities to those seen in YC3.1-expressing pollen tubes were used to determine R_min and R_max. And values for R_min of 0.92 and R_max of 3.11 were obtained. These values of R_min and R_max were used to convert YC3.1 fluorescence ratios into [Ca²⁺]_cyt by fitting them to the YC3.1 in vitro calibration curve (see Miyawaki et al., 1999; Fig. 2B).

Growth Rate

We printed images of EYFP fluorescence in the growing pollen tube and measured the length of the tube using a ruler. The growth rate was calculated from the length of elongation between time points and the interval.

	ACKNOWLEDGMENTS

 
We thank Miss Yamaguchi, Mrs. Onishi, Mrs. Yoneyama, Miss Sugita, and Mrs. Ichikawa for their technical assistance.

Received May 25, 2004; returned for revision August 8, 2004; accepted August 9, 2004.

	FOOTNOTES

 
¹ This work was supported in part by a Grant-in-Aid Creative Scientific Research (grant no. 16GS0316, to A. I.) from Japan Society for the Promotion of Science (JSPS), by a Grant-in-Aid for Special Research (C, grant no. 13640649, to M. I.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), and by the 21st Century Centers of Excellence (COE) Program to Nara Institute of Science and Technology from MEXT.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046961.

^* Corresponding author; e-mail m-iwano{at}bs.naist.jp; fax 81–743–72–5459.

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