Plant Cell Physiol.

44(3): 262268 (2003) JSPP 2003

Sepal Color Variation of Hydrangea macrophylla and Vacuolar pH Measured with a Proton-Selective Microelectrode
Kumi Yoshida 1, 4, Yuki Toyama-Kato 2, Kiyoshi Kameda 2 and Tadao Kondo 3
1 2

Graduate School of Human Informatics, Nagoya University, Chikusa, Nagoya, 464-8601 Japan School of Life Studies, Sugiyama Jogakuen University, Chikusa, Nagoya, 464-8662 Japan 3 Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601 Japan ;

Sepal color of hydrangea varies with the environmental conditions. Although chemical and biological studies on this color variation have a long history, little correct knowledge has been generated about color development. All colored sepals contain the same anthocyanin, delphinidin 3glucoside. Thus, there must be some other system for developing the wide variety of colors. In hydrangea sepals the cells of the epidermis are colorless and only the second layer of cells contain pigment. We prepared protoplasts without any color change during enzyme treatment of sepals and measured the vacuolar pH of each of the colored cells. We could correlate the color of a single hydrangea cell with its vacuolar pH using a combination of micro-spectrophotometry and a proton-selective microelectrode. Values for the vacuolar pH of blue (lvismax: 589 nm) and red cells (lvismax: 537 nm) were 4.1 and 3.3, respectively, the vacuolar pH of blue cells being significantly higher. Keywords: Anthocyanin Color variation Hydrangea macrophylla Micro-spectrophotometry Proton selective microelectrode Vacuolar pH.

Introduction
The sepal color of Hydrangea macrophylla (Fig. 1A, B) is very famous for ready change of hues (Allen 1943). However, the underlying mechanism remains unclear. Although it is well known that acidity of the soil and content of Al3+ influence sepal color; the more acidic the soil and the higher the Al3+ content in sepals, the bluer they become (Chenery 1937, Allen 1943, Ma et al. 2001). All colored hydrangea sepals have only one anthocyanin, delphinidin 3-glucoside (Lawrence et al. 1938, Robinson and Robinson 1939, Hayashi and Abe 1953, Asen et al. 1957) (Fig. 2, 1). Therefore, the question of why this single anthocyanin should give a wide variety of color, from red through purple to blue in hydrangea, has attracted much interest. Anthocyanin changes its color depending on pH (Goto 1987, Brouillard 1988, Goto and Kondo 1991). In strong acidic conditions it shows red, in neutral, purple and in alkaline, blue.
4

But generally the vacuolar pH of plant cells is weakly acidic and, under such conditions, almost all anthocyanins are purple and very unstable. From recent work, the following three mechanisms have been proposed for flower color variation: metal chelation, vacuolar pH change and molecular associations (Goto 1987, Goto and Kondo 1991, Brouillard and Dangles 1994, Mol et al. 1998). In the blue dayflower, Commelina communis, self-assembled supramolecular pigments develop the blue petal color (Kondo et al. 1992). In the blue morning glory, Ipomoea tricolor, increase of vacuolar pH causes color change from reddish purple to blue during blooming (Yoshida et al. 1995). In hydrangea, delphinidin 3-glucoside gives a stable blue solution co-existing with 5-O-caffeoyl and/or 5-O-pcoumaroylquinic acid and Al3+ (Takeda et al. 1985a, Takeda et al. 1985b, Takeda et al. 1990, Kondo et al. 1999, Yoshida et al. 2002). But the in vivo mechanism of color variation is still ambiguous. In attempts to clarify the mechanisms of blue and red color development of hydrangea, we have isolated delphinidin 3-O-b-D-glucopyranoside (1) and co-pigment components, 5O-caffeoyl quinic acid (2), 5-O-p-coumaroyl quinic acid (3) and 3-O-caffeoyl quinic acid (4) from hydrangea sepals. We mixed 1 and co-pigment, 2, 3 and 4, co-existing with several metal ions under various pH conditions. We found that the pH of the solution exerted a considerable effect on color development, suggesting that the vacuolar pH may be different between blue and red sepals (Kondo et al. 1999, Yoshida et al. 2002). However, there are only two reports describing hydrangea color and flower pH. Robinson and Robinson (1939) suggested that red flowers were slightly more acidic than their blue counterparts and Stewart et al. (1975) reported that the pH of pink colored sepals was 4.0. Therefore, we tried to measure the true vacuolar pH of living hydrangea cells directly. For this purpose we prepared protoplasts from blue and red hydrangea sepals and analyzed color and vacuolar pH of each individual cell by micro-spectrophotometry and an intracellular microelectrode method.

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Results and Discussion

Sepal and cell color measurement of hydrangea In the past flower petal color was measured first using the

Corresponding author: E-mail, yoshidak@info.human.nagoya-u.ac.jp; Fax, +81-52-789-5638. 262

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Fig. 1 Typical blue and red colored hydrangea, Hydrangea macrophylla: (A) blue cultivar, Narumi blue; (B) red cultivar, Kasterin; (C) transverse section of a blue sepal; (D) transverse section of a red sepal; (E) a protoplast mixture from blue sepals; (F) a protoplast mixture from red sepals. Scale bars: 50 mm.

opal glass transmission method (Saito 1967) and subsequently (Asen et al. 1971a, Asen et al. 1971b, Asen et al. 1975) and his group (Stewart et al. 1975) reported micro-spectrophotomeric measurement with peeled epidermis. It is also possible to use

the integral sphere as a reflection spectrum. However, petal color tone could be affected by the shape of epidermal cells (Noda et al. 1994) and reflection spectra suffer from light scattering due to the structure of petal tissue. To reveal the true

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Fig. 2

Structures of anthocyanin and co-pigments in hydrangea sepals.

Fig. 3 Reflection spectra of blue and red hydrangea sepals measured with the integral sphere apparatus and absorption spectra of colored protoplasts with micro-spectrophotometry: (A) blue hydrangea sepal (cv. Narumi blue) and its protoplasts; (B) red hydrangea sepal (cv. Kasterin) and its protoplasts.

color absorption spectrum, an individual cell must be measured. Reflection spectra of hydrangea sepals are shown in Fig. 3A, B. Blue and red sepals showed a lvismax at 585 nm and 535 nm, respectively. Fig. 1C, D shows transverse sections of individual colored sepals. In general, colored cells of flower petals are located in both abaxicial and adaxicial epidermis. However, in hydrangea sepals colored cells are not located in the epidermis but only in the second layer. Thus, it is impossible to introduce an electrode into the colored sepal cells through the tissue directly as we did for measurement of blue morning glory (Yoshida et al. 1995). To overcome this problem we prepared protoplasts from the sepals. Hydrangea sepals treated by Nishimuras procedure (Nishimura et al. 1987) with a modification gave a mixture of colored and colorless protoplasts (Fig. 1E, F). The cell suspensions obtained were directly poured onto poly-L-lysine precoated cover glass to fix the cells. In the stored buffer the protoplasts did not show any color change for a couple of days under 5C. Since the diameter of hydrangea protoplasts is ca. 2030 mm, the light beam for micro-spectrophotometry was set

at 10 mm. Absorption spectra of blue (cv. Narumi blue) and red (Kasterin) hydrangea protoplasts are shown in Fig. 3A, B. The lvismax of the blue cell is 586 nm and that of the red cell 535 nm. No difference was observed between the reflection spectra of sepals and absorption spectra of colored cells, indicating that during the enzyme treatment no color change occurred. Other blue hydrangeas (cv. Blue diamond and a wild blue hydrangea collected at Mt. Chausu of Aichi Prefecture) and a red one, LK-49, were also compared. Independent of the cultivar, the same colored sepals and derived protoplasts showed similar reflection and absorption spectra. Vacuolar pH measurement of colored cells of hydrangea Vacuolar pH measurement of flower cells by microelectrode (Felle and Bertl 1986, Felle 1987, Felle 1993) has advantages over measurement by fluorescent dye (Kurkdjian and Guern 1989, Roos 2000) because it can directly obtain vacuolar pH data for single cells containing anthocyanins. However, it is not so easy to prepare an electrode which has a good response as well as sufficient resistance to a turgor pressure of more than

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265

Fig. 4 A blue protoplast fixed on a cover glass pre-coated with polyL-lysine into which a double-barreled microelectrode has been introduced. The tip of the electrode is located in the vacuole.

6 atms. These two are a question of trade-off. For a high enough response, the aperture of the electrode tip must be large, but to keep cell damage to a minimum the tip must be thin. Furthermore, the position of a tip with a diameter less than 1 mm cannot be observed under light microscopy. Therefore, evidence that the tip is inserted into the vacuole must be proven with membrane potential and obtained pH values (Felle Table 1

1993, Miller et al. 2001). However, previously reported membrane potentials for plant protoplasts varied; negative values as intact cells (Abe et al. 1983, Abe and Takeda 1986), low values near zero (Pantoja and Willmer 1986, Bouteau et al. 1993, Bouteau et al. 1999, Duijn and Hemimovaara-Dijkstra 1994, Shabala et al. 1998) and positive values (Racusen et al. 1977). The positive potential may be caused by membrane damage during enzymatic treatment (Cornel et al. 1983). Furthermore, potential value may also change depending on the concentration of outer ions (Saftner and Raschke 1981, Abe and Takeda 1986) and suction pressure (Pantoja and Willmer 1986). In order to observe tip impalement and the quick response of an electrode, an electrode was prepared with a thick tip which would damage the cell. The diameter of the hole caused by the electrode tip was thus expected to be larger than 1.0 mm. Using this double-barreled electrode, electrophysiological experiments with hydrangea sepal protoplasts were carried out (Fig. 4). In contrast to previous reports (Felle 1993, Miller et al. 2001), the microelectrode tip was always impaled into the vacuole as in the case of morning glory petals (Yoshida et al. 1995) and never retained in the cytosol, because the volume of central vacuoles of mature flower and sepal cells is nearly the same as the cell volume. When the electrode was withdrawn, the colored sap exuded and the protoplast shrunken, indicating that the tip was indeed inserted into the vacuole. At the concentration of 10 mM KCl the mean value for

Membrane potential of blue and red colored hydrangea protoplast a Conc. of KCl (mM) 10 0.1 10 0.1 Membrane potential (mV) b 6.26.6* 1.56.1** 1.56.4 8.22.1 No. of experiments 12 4 16 5

Sepal color Blue

c

Red d
a

Protoplasts prepared from blue sepals cv. Narumi blue and red sepals cv. Kasterin were studied. Membrane potential was measured by a double-barreled microelectrode, the same as for the pH measurement. b Means SD of membrane potential measured by a proton-selective microelectrode. Significant differences (P<0.05) and (P<0.001) were obtained between * and ** and and , respectively, by Students t test. c Average vacuolar pH value in 10 mM KCl was 3.6 and that in 0.1 mM KCl was 3.7. d Average vacuolar pH value in 10 mM KCl was 3.2 and that in 0.1 mM KCl was 3.5.

Table 2

Vacuolar pH of blue and red hydrangea protoplast Cultivar Narumi blue Blue diamond Mt. Chausu Kasterin LK-49 lvismax (nm) a 5863.05 5884.17 5951.69 5394.23 5302.07 Vacuolar pH b 3.60.27* 4.50.36*, , 4.30.29*, , 3.30.24** 3.50.35**
,

Sepal color Blue

No. of experiments 16 16 9 21 5

Red
a b

Wavelength of peak of visible absorption spectra of protoplast measured by micro-spectrophotometry. Means SD of vacuolar pH value measured by proton-selective microelectrode. Significant differences (P<0.001) were obtained between * and ** and and and significant differences (P<0.05) were obtained between and by Students t test.

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Fig. 5 Vacuolar pH and membrane potential profiles of colored hydrangea protoplasts measured by a double-barreled microelectrode. Downward-pointing arrows indicate insertion and upward-pointing arrows withdrawal of the electrode. (A) Blue protoplast from blue hydrangea sepals (cv. Narumi blue); (B) red protoplast from red hydrangea sepals (cv. Kasterin).

membrane potentials was positive, regardless of the cell color (Table 1). When the concentration of the outer KCl was 0.1 mM, negative membrane potentials were observed, though the values were very low. Whatever, the outer KCl concentration had little effect on membrane potentials and did not have influence on vacuolar pH. Following analysis of cell color by micro-spectrophotometry, the vacuolar pH of the same protoplast was measured. Typical recording profiles are shown in Fig. 5A, B. The data fluctuated within 0.1 pH unit when values over more than 5 s were employed. Two cultivars of blue hydrangea, one wild blue hydrangea and two red cultivars were studied (Table 2). The average pH value of blue cells of cv. Narumi blue was 3.6, while that of cv. Blue diamond was 4.5. The vacuolar pH of a wild hydrangea collected at Mt. Chausu was 4.3. Between blue species significant differences were observed indicating that vacuolar pH might differ depending on specific characteristics. The vacuolar pH of red hydrangea, cv. Kasterin, was 3.3 and that of cv. LK-49 was 3.5. The vacuolar pH values of red cultivars were lower with significant differences (P<0.001) than those of blue hydrangeas. In Fig. 6 lvismax and vacuolar pH values for all the examined protoplasts were plotted. The average pH of all the blue protoplast was 4.1 and that of red cells was 3.3. A positive correlation (R = 0.68) was observed between cell color and vacuolar pH. Consideration of hydrangea color development We mixed 1 and co-pigments, 2, 3 and 4, in the presence of several metal ions under various pH conditions according to the same procedure as Takeda et al. (1985a) and Takeda et al. (1985b). At pH 4.0 the solution of 1 (1.0 mM) by addition of 2 or 3 (1 eq. to 1) and Al3+ (1 eq. to 1), developed the same blue color as the blue hydrangea sepals. On the other hand, the red color, identical to that of the red hydrangea sepals, was real-

ized by mixing 1 (1.0 mM), 4 (1 eq. to 1) and Al3+ (1 eq. to 1) at pH 3.0. However, the color of 1 in the solution was affected by the composition of co-pigments (molar ratio of 2, 3 and 4 to 1) and Al3+ as well as pH. Co-existing large amounts of 2 and/ or 3 (>10 eq.) and Al3+ (1 eq.) 1 gave a blue solution at pH 3.5, while at the same pH 1 gave a red solution with 4 (>10 eq.), a small amount of 2 and/or 3 (<1 eq.) and trace Al3+ (<0.1 eq.) (Kondo et al. 1999, Yoshida et al. 2002). The sepal color difference between Narumi blue (pHV 3.6) and LK-49 (pHV 3.5) might be caused by the same mechanism. In conclusion, we have established a simultaneous system to analyze the absorption spectrum and the vacuolar pH of colored cells in a flower. Preparation of protoplasts followed by absorption spectrum measurement, with micro-spectrophotom-

Fig. 6 Correlation of cell color with vacuolar pH of blue and red hydrangea protoplasts. All the vacuolar pH data (n = 67) were plotted as a function of the lvismax of the absorption spectrum.

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267

etry, and determination of pH by microelectrode lead to a direct correlation being established between the cell color and the vacuolar pH of individual hydrangea cells. The vacuolar pH of blue hydrangea sepal cells was higher than that of red sepals. However, the sepal color is not determined by vacuolar pH only, and the molar ratio of the co-pigments and Al3+may also affect it. Therefore, the color variation of hydrangea sepals may depend on the delicately controlled balance of all those factors. Though the cause of the pH difference between blue and red cells was not known, it might be correlated with the content of Al3+, since blue cells contain 10 the amount of Al3+ compared with that in red cells, as shown in preliminary experiments. Quantitative analysis of vacuolar components, 1, 2, 3, 4 and Al3+, is under progress.

into the dish. It was then set on a microscope (IX70; Olympus), equipped with a micro-spectrophotometer (MCPD-7000; Photal). The absorption spectrum of colored protoplasts was measured in the visible region (400800 nm) with a 10-mm-diameter optical light beam. Preparation of proton-selective microelectrodes Proton-selective microelectrodes were prepared according to the method of Okazaki et al. (1994) and Yoshida et al. (1995), with a slight modification. For measurement of the vacuolar pH of protoplasts double-barreled pipettes (IB 100 F-4, World Precision Instruments Inc.) were prepared instead of triple-barreled ones. The diameter of the tip of the pipette was confirmed to be about 1 mm by observation by scanning micro-electrophotometry. After the pipette was treated with chlorotrimethylsilane [0.1% v/v chlorotrimethylsilane in chloroform] the proton-ionophore solution [5% v/v Hydrogen Ionophore II-Cocktail A, 0.1% w/v nitrocellulose in THF] was filled to the longer pipette to strengthen against turgor pressure. Then, the pipettes were stored in a dry box for at least 1 d. Before measurement, the longer pipette was filled with the 0.2 ml of proton-ionophore cocktail, then filled to the top with the reference solution [100 mM MES-Tris, 500 mM KCl, pH 6.0]. The shorter pipette for measuring membrane potential was filled with 500 mM KCl. Calibration of the pH microelectrode was made before and after pH measurement using buffers [10 mM DMG-Tris, 10 mM KCl, pH 3.0, 4.0 and 5.0, and 10 mM MES-Tris, 10 mM KCl, pH 6.0]. Electrodes with a slope more than 45 mV per pH unit were selected for measurement. Measurement of vacuolar pH A high input impedance electrometer (FD223; World Precision Instruments Inc.) was employed for pH measurement. The back part of the longer pipette was held in a microelectrode holder (MEH3SF; World Precision Instruments. Inc.) while it was filled with 3 M KCl. The shorter pipette was bridged to the other holder with a salt bridge [500 mM KCl, 2% w/v agarose] prepared with a thin plastic tube. Each holder was connected to the tip of the electrometer through first stage amplifiers. A reference electrode (FLEXREF; World Precision Instruments. Inc.) was set into the dish. After measurement of the absorption spectrum of a protoplast the microelectrode was impaled into the same cell at an angle of 3040 under light microscopic observation using a mechanical micromanipulator. The data were collected into a personal computer with Chart v. 4.0 software (AD Instruments Inc.) and processed according to the calibration data recorded after vacuolar pH measurement each time. All the data with the drift within 5 mV 5 s1 were adopted, giving reference to the calibration after the measurement. Reproduction of sepal color in vitro Reproduction experiments were carried out according to the procedure reported by Takeda et al. (1985a) and Takeda et al. (1985b) with a modification. Aqueous solution of 1 (1 mM), 2, 3, 4 and AlNH4(SO4) 2 were mixed in 100 mM acetate buffer (pH 3.0, 3.5 and 4.0) in various molar ratios in a quartz cuvette (path length, 1.0 mm). The UV/VIS spectra of the mixture were recorded by a JASCO Ubest55 spectrometer.

Materials and Methods

Plant materials Hydrangea macropylla cv. Narumi blue and cv. Kasterin were obtained as presents from Okumura Farm. Cv. Blue diamond and LK49 were supplied from Gunma Horticultural Experimental Station and by Mr. Sakamoto of Sakamoto Farm. Wild hydrangea branches were collected at Mt. Chausu of Aichi prefecture. Pots were kept in an incubator under 12 h dark and 12 h light (15,000 lux) conditions. The temperature in the dark was 12C and in the light 18C. The branches were kept in water vases under the same conditions as pot hydrangeas. Transverse section of sepals Fresh sepals were cut with a razor blade into ca. 1010 mm squares which were put between the two halves of a split elder stem. The pith was set in a plant microtome (MT-3, Nihon Ikakikai) and cut at 50 mm. The sections were placed on glass slides and observed by micro-spectrophotometry. Reflection spectrum measurement of hydrangea sepals Fresh sepals were cut into ca. 1010 mm squares and fixed on a cover glass, then put on a white plate of BaO. The plate was set in the integral sphere apparatus of a JASCO Ubest-55 spectrometer and the reflection spectrum from 400 to 800 nm was recorded. Preparation of protoplast Fresh sepals (ca. 0.5 g) were cut at 2 mm thickness with a razor blade. To the fragments were added a buffer [20 mM MES-Tris (pH 6.3), 0.6 M mannitol] containing 0.2% (w/v) Macerozyme R-200 and 2.0% (w/v) Cellulase Y-C. The mixture was evacuated for 2 min, incubated at 30C for 2 h, then filtered through Miracloth (Calbiochem), washed with buffer [20 mM MES-Tris (pH 6.3), 0.6 M mannitol] and centrifuged (70g for 5 min). This procedure was repeated three times, then the protoplasts obtained were suspended in ca. 1 ml of storage buffer [20 mM MES-Tris (pH 6.0), 0.6 M mannitol, 2 mM MgSO4, 5 mM EGTA, 0.1 mM or 10 mM KCl]. All the treatments after filtration were conducted under 4C Measurement of visible absorption spectrum of vacuole Two hundred microliters of protoplast suspension was poured onto a poly-L-lysine pre-coated cover glass (1818 mm) and stood for a minute at room temperature. After removal of the suspension buffer the cover glass was put in a plastic dish (35 mm f) and 2 ml of the storage buffer (the concentration of KCl in storage buffer varied between 0.1 mM and 10 mM as the experiment demanded) was poured

Acknowledgments
We are grateful to Mr. Okumura of Okumura Farm, Mr. Shimizu of Gunma Horticultural Experiment Station and Mr. Sakamoto of Sakamoto Farm for giving us the hydrangeas. We thank to Ms. Y. Kawano and Ms. M. Ito of Sugiyama Jogakuen University for technical assistance. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports,

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Hayashi, K. and Abe, Y. (1953) Studien ber anthocyane. XXIII. Papierchromatographische bersicht der anthocyane im pflanzeneich. I. Misc. Rep. Res. Inst. Nat. Resour. 29: 18. Kondo, T., Toyama, Y., Yoshida, K., Shimizu, Y., Fujimori, E., Haraguchi, H. (1999) Cause of flower color variation of hydrangea, Hydrangea macrophylla. In Abstract papers of the 41st Symposium on the Chemistry of Natural Products 1315 October, Nagoya. 265270. Kondo, T., Yoshida, K., Nakagawa, A., Kawai, T., Tamura, H. and Goto, T. (1992) Structural basis of blue-colour development in flower petals from Commelina communis. Nature 358: 515518. Kurkdjian, A. and Guern, J. (1989) Intercellular pH: measurement and importance in cell activity. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40: 271303. Lawrence, W.J.C., Price, J.R., Robinson, G.M. and Robinson, R. (1938) CCXV. A survey of anthocyanins. V. Biochem. J. 32: 16611667. Ma, J.F., Ryan, P.R. and Delhaize, E. (2001) Aluminium tolerance in plants and the complexing role of organic acids. Trends Plant Sci. 6: 273278. Miller, A.J., Cookson, S.J., Smith, S.J. and Wells, D.M. (2001) The use of microelectrodes to investigate compartmentation and the transport of metabolized inorganic ion in plants. J. Exp. Bot. 52: 541549. Mol, J., Grotewold, E. and Koes, R. (1998) How genes paint flowers and seeds. Trends Plant Sci. 3: 212217. Nishimura, M., Hara-Nishimura, I. and Akazawa, T. (1987) Preparation of protoplast from plant tissue for organelle isolation. Methods Enzymol. 148: 27 34. Noda, K.-i., Glover, B., Linstead, P. and Martin, C. (1994) Flower colour intensity depends on specialized cell shape controlled by a Myb-related transcription factor. Nature 369: 661664. Okazaki, Y., Tazawa, M. and Iwasaki, N. (1994) Light-induced changes in cytosolic pH in leaf cells of Egeria densa: measurements with pH-sensitive microelectrodes. Plant Cell Physiol. 35: 943950. Pantoja, O. and Willmer, C.M. (1986) Pressure effects on membrane potentials of mesophyll cell protoplasts and epidermal cell protoplast of Commelina communis L. J. Exp. Bot. 37: 315320. Racusen, R.H., Kinnersley, A.M. and Galston, A.W. (1977) Osmotically induced changes in electrical properties of plant protoplast membranes. Science 198: 405407. Robinson, G.M. and Robinson, R. (1939) The colloid chemistry of leaf and flower pigments and the precursors of the anthocyanins. J. Amer. Chem. Soc. 61: 16061607. Roos, W. (2000) Ion mapping in plant cells methods and applications in signal transduction research. Planta 210: 347370. Saftner, R.A. and Raschke, K. (1981) Electrical potential in stomatal complexes. Plant Physiol. 37: 11241132. Saito, N. (1967) Light absorption of anthocyanin-containing tissue of fresh flowers by the use of the opal glass transmission method. Phytochemistry 6: 10131018. Shabala, S., Newman, I., Whittington, J. and Juswono, U. (1998) Protoplast ion fluxes: their measurement and variation with time, position and osmoticum. Planta 204: 146152. Stewart, R.N., Norris, K.H. and Asen, S. (1975) Microspectrophotometric measurement of pH and pH effect on color of petal epidermal cells. Phytochemistry 14: 937942. Takeda, K., Kariuda, M. and Itoi, H. (1985a) Blueing of sepal colour of Hydrangea macrophylla. Phytochemistry 24: 22512254. Takeda, K., Kubota, R. and Yagioka, C. (1985b) Copigments in the blueing of sepal colour of Hydrangea macrophylla. Phytochemistry 24: 12071209. Takeda, K., Yamashita, T., Takahashi, A. and Timberlake, C.F. (1990) Stable blue complexes of anthocyanin-aluminium-3-p-coumaroyl- or 3-caffeoylquinic acid involved in the blueing of Hydrangea flower. Phytochemistry 29: 10891091. Yoshida, K., Kondo, T., Okazaki, Y. and Katou, K. (1995) Cause of blue petal colour. Nature 373: 291. Yoshida, K., Mori, M., Shinkai, Y., Toyama, Y. and Kondo, T. (2002) Mechanism of blue flower color development in colored cells. In Abstract papers of the 44th Symposium on the Chemistry of Natural Products 911 October, Tokyo. 409414.

Culture and Technology, Japan (COE Research No. 07CE2004 to T.K., 12460052 to T.K. and on Priority Areas No. 12045234 to T.K.), NIBB Cooperative Research Program (90201 and 00158 to K.Y. and T.K.), Hayashi Memorial Foundation for Female Natural Scientists (to K.Y.) and Yamada Science Foundation (to K.Y.).

References
Abe, S., Takeda, J. and Senda, M. (1983) Resting membrane potential and action potential of Nitella expansa protoplast. Plant Cell Physiol. 21: 537 546. Abe, S. and Takeda, J. (1986) The membrane potential of enzymatically isolated Nitella expansa protoplasts as compared with their intact cells. J. Exp. Bot. 37: 238252. Allen, R.C. (1943) Influence of aluminum on the flower color of Hydrangea macrophylla DC. Boyce Thompson Institute 13: 221242. Asen, S., Norris, K.H. and Stewart, R.N. (1971a) Effect of pH and concentration of the anthocyanin-flavonol co-pigment complex on the color of Better Times Roses. J. Amer. Soc. Hort. Sci. 96: 770773. Asen, S. and Siegelman, H.W. (1957) Effect of aluminum on absorption spectra of the anthocyanin and flavonols from sepals of Hydrangea macrophylla var. Merveille. Proc. Amer. Soc. Hort. Sci. 70: 478481. Asen, S., Siegelman, H.W. and Stuart, N.W. (1957) Anthocyanin and other phenolic compounds in red and blue sepals of Hydrangea Macrophylla var. Merveille. Proc. Amer. Soc. Hort. Sci. 69: 561569. Asen, S., Stewart, R.N. and Norris, K.H. (1971b) Co-pigmentation effect of quercetin glycosides on absorption characteristics of cyanidin glycosides and color of red wing azalea. Phytochemistry 10: 171175. Asen, S., Stewart, R.N. and Norris, K.H. (1975) Anthocyanin, flavonol copigments and pH responsible for larkspur flower color. Phytochemistry 14: 26772682. Asen, S., Stewart, N.R. and Norris, K.H. (1977) Anthocyanin and pH involved in the color of Heavenly blue morning glory. Phytochemistry 16: 1118 1119. Bouteau, F., Dellis, O., Bousquet, U. and Rona, J.P. (1999). Evidence of multiple sugar uptake across the plasma membrane of laticifer protoplasts from Hevea. Bioelectrochem. Bioenergetics 48: 135139. Bouteau, F., Perino, C., Cornel, D. and Rona, J.P. (1993) Sugar absorption and potassium channels in protoplasts of Hevea brasiliensis laticiferous vessels. Bioelectrochem. Bioenergetics 31: 215228. Brouillard, R. (1988) Flavonoids and flower colour. In The Flavonoids: Advances in Research since 1980. Edited by Harborne, J.B. pp. 525538. Chapman and Hall, London. Brouillard, R. and Dangles, O. (1994) Flavonoids and flower colour. In The Flavonoids: Advances in Research since 1986. Edited by Harborne, J.B. pp. 565588. Chapman & Hall, London. Chenery, E.M. (1937) The problem of the blue hydrangea. J. R. Hort. Soc. 62: 604320. Cornel, D., Gringon, C., Rona, J.P. and Heller, R. (1983) Measurement of intracellular potassium activity in protoplasts of Acer pseudoplatanus: origin of their electropositivity. Physiol. Plant. 57: 203209. Duijn, B.V. and Hemimovaara-Dijkstra, S. (1994) Intracellular microelectrode membrane potential measurements in tobacco cell-suspension protoplasts and barley aleurone protoplasts: interpretation and artifacts. Biochim. Biophys. Acta 1193: 7784. Felle, H. (1987) Proton transport and pH control in Sinapis alba root hairs: a study carried out with double barrelled pH micro-electrodes. J. Exp. Bot. 38: 340354. Felle, H. (1993) Ion-selective microelectrodes: their use and importance in modern cell biology. Bot. Acta 106: 512. Felle, H. and Bertl, A. (1986) The fabrication of H+-selective liquid-membrane micro-electrode for use in plant cell. J. Exp. Bot. 37: 14161428. Goto, T. (1987) Structure, stability and color variation of natural anthocyanins. Prog. Chem. Org. Nat. Products 52: 113158. Goto, T. and Kondo, T. (1991) Structure and molecular stacking of anthocyanins flower color variation. Angew. Chem. Int. Ed. Engl. 30: 1733.

(Received September 21, 2002; Accepted December 24, 2002)