Plant Cell Reports
,Plant Cell Reports (1985) 4:252-255
© Springer-Verlag 1985
Occurrence of anthocyanoplasts in cell suspension cultures of sweet potato Masayuki Nozue and Hitoshi Yasuda Department of Biology, Faculty of Science, Shinshu University, Matsumoto 390, Japan Received January 21, 1985 / Revised version received July 29, 1985 - Communicated by F. Constabel
Abstract
Intensely pigmented and spherical vesicles(anthocyanoplasts) were found in anthocyanin-containing c e l l s of sweet potato (Ipomoea batatas) suspension cultures. Anthocyanin s y ~ ~ o f i r s t occur 24-48 h a f t e r exposure to l i g h t , and then numerous small red vesicles were detected under a microscope. The f r e quency of anthocyanoplast-containing c e l l s r a p i d l y increased to f i n a l l y about 80% of the t o t a l cultured c e l l s a f t e r 5 days of i r r a d i a t i o n . F u l l y developed anthocyanoplasts reached 10-15 ~m in diameter. On the other hand, neither anthocyanin synthesis nor development of anthocyanoplasts was induced in the dark-cultured c e l l s . 2,4-D also i n h i b i t e d anthocyanin synthesis and development of these vesicles. The results suggest that anthocyanoplasts might be a site of anthocyanin synthesis and/or accumulation. Abbreviation: 2,4-D=2,4-dichlorophenoxyacetic acid Introduction
Although a number of attempts have been made to e l u c i date the s u b c e l l u l a r l o c a l i z a t i o n of secondary metabo l i t e s and t h e i r biosynthetic s i t e , the functional d i f f e r e n t i a t i o n and regulatory mechanism of the biosynthesis have not yet been c l e a r l y established. I t has been generally agreed that the main s i t e of anthocyanin accumulation is the central vacuole. However, knowledge of the r e l a t i o n between the s i t e of anthocyanin biosynthesis and the s i t e of i t s accumulation is at present fragmentary. Fritsch and Grisebach (1975) suggested that some enzymes involved in anthocyanin biosynthesis may occur on or in the tonoplast, or in the vacuolar sap. Other reports also suggested that anthocyanin and other flavonoids can be synthesized in chloroplasts (Ranjeva et a l . 1977). On the contrary, Hrazdina et a l . (1978; 1980) showed that the enzymes associated with biosynthesis occur not in the plastids and vacuoles, but in the cytosol f r a c t i o n s . I t is thought at present that the anthocyanin and other flavonoid biosynthesis occurs near the endoplasmic reticulum (ER) by cytoplasmic and ER-bound enzymes acting in close proximity to each other, and then transported by unknown transport mechanisms to the central vacuole of c e l l s , where they accumulate(Hrazdina1982). Recently, i t has been reported that intensely pigmented vesicles (anthocyanoplasts) were found w i t h i n the main vacuoles of anthocyanin-containinq c e l l s in young seedlings of red cabbage (Pecket and Small 1980). Yasuda and Shinoda (1985) have shown that the red spherical bodies, which contained anthocyanin, were
Offprint requests to: M. Nozue
found in the seedling hypocotyls of red radish. These findings led to the speculation that these vesicles were l i k e l y to be the s i t e of anthocyanin biosynthesis. We also found such pigmented vesicles ih the light-induced anthocyanin-synthesizing c e l l s of sweet potato suspension cultures. In the present report, we investigated the r e l a t i o n s h i p between development of anthocyanoplasts and anthocyanin accumulation and also discussed the possible functions of these vesi c l e s during the l i g h t - i n d u c e d anthocyanin synthesis in the c e l l suspension cultures. Materials and Methods
Subcultures of High Anthocyanin-Producing Cell Line The high anthocyanin-producing cell l i n e used in the present experiments was obtained by the visual select i o n from callus cultures i n i t i a t e d with roots of sweet potato (Ipomoea batatas Lam. cv. K i n t o k i ) . Cell suspension c u - - u - T t u - r ~ h e cell l i n e were established by PRL-4C (Gamborg 1966) l i q u i d medium supplemented with 0.1 mg/l 2,4-D and 3% sucrose. Cell aggregates were obtained by pouring 7-day-old cultures through a stainless steel sieve with 500 pm mesh size, then through a 44 pm sieve and were taken f o r a l l subcultures. Two g wet weight of these c e l l s and cell clusters was trasferred to 300-ml Erlenmeyer flasks containing 50 ml of fresh medium every 7 days, and maintained at 25 °C in the dark on a rotary shaker at 110 cycles/min. Induction of Anthocyanin Synthesis Anthocyanin synthesis was induced by the f o l l o w i n g procedures. The seven-day-old c e l l s (1.0 g wet weight) prepared as above were trasferred to 100-ml Erlenmeyer flasks containing 10 ml of the induction medium (PRL4C medium without 2,4-D) and cultured under continuous i l l u m i n a t i o n with cool white fluorescent tubes (Toshiba Neoline FL40 SW; ca. 12,000 lux) at 25 °C on a rotary shaker at 120 cycles/min. Measurement of Cell Growth and Anthocyanin Content Cells were separated from the culture medium by f i l t r a t i o n through f i l t e r paper (Toyo No. I) with a suction funnel and then weighed. Results were expressed as fresh weight (g) per c u l t u r e . For q u a n t i t a t i v e determination of anthocyanin, c e l l s (20-40 mg fresh weight) trapped on a f i l t e r paper were extracted f o r 24 h with 5.0 ml of methanol containing 0.5% HCl (v/v) at 4 °C in the dark. A f t e r removing the insoluble materials by c e n t r i f u g a t i o n at 2,000 g f o r 20 min, the absorbance of the clear supernatant was measured
253 with a spectrophotometer. Anthocyanin content was expressed as absorbance at 530 nm per g fresh weight of c e l l s .
130
0
Counting the Pigmented Cells and Anthocyanoplast -Containing Cells At i n t e r v a l s a f t e r onset of i r r a d i a t i o n , c e l l s were counted under a microscope a f t e r maceration with Cellulase 'Onozuka' R-IO, Macerozyme R-IO (bothYakult Pharmaceutical Co., L t d . , Nishinomiya, Japan) and Pectolyase Y-23 (Seishin Pharmaceutical Co., L t d . , Tokyo, Japan) as described previously (Nishimaki and Nozue 1988). A v i s i b l e red cell (protoplast) was counted as an anthocyanin-containing cell and an i n tensely red and spherical vesicle was regarded as an anthocyanoplast. Results were expressed as percentages of these c e l l s in t o t a l cultured c e l l s . A l l experiments were repeated two or three times, and about a thousand c e l l s were observed in each experiment.
o
0
7o
C Results and Discussion
0
Light-lnduced Anthocyanin Synthesis The stimulatory e f f e c t of l i g h t on anthocyanin synthesis has been well investigated with many tissue cultures (Stickland and Sunderland 1972; Matsumoto et al. 1973; Wellmann et a l . 1976; Yamakawa et a l . 1983). In the present experiments, the 7-day-old c e l l s of the high anthocyanin-producing cell l i n e which was maintained in 2,4-D-containing medium in the dark, were transferred to the induction medium (2,4-D free) and cultured under continuous i l l u m i n a t i o n . There was no anthocyanin synthesis in the subcultures, however, anthocyanin synthesis was induced w i t h i n 48 h and i t markedly increased under f u r t h e r i r r a d i a t i o n (Fig. I ) . Anthocyanin synthesis was not induced in the darkcultured c e l l s . The percentage of anthocyanin-containing c e l l s reached more than 95% during the i l l u m i n a t i o n period. Ozeki and Komamine (1981) have established the system in which anthocyanin synthesis was triggered by t r a n s f e r r i n g c e l l s from a medium containing 2,4-D to a medium lacking auxin in carrot suspension c u l tures. However, the present data showed that continuous i l l u m i n a t i o n was an indispensable f a c t o r for anthocyanin synthesis in sweet potato suspension cultures. Development of Anthocyanoplasts in the L i g h t Induced Cells The seven-day-old c e l l s of subcultures were transferred to 2,4-D free medium and exposed to l i g h t , and then development of anthocyanoplasts in the pigmentsynthesizing c e l l s were observed under a microscope. Such pigmented vesicles were not found in the c e l l s subcultured in the dark (Fig. 2A). On the other hand, small red vesicles, approximately 3 ~m in d i ameter, were f i r s t detected in the anthocyanincontaining c e l l s between 72-96 h a f t e r onset of i r r a d i a t i o n (Fig. 2B). The number of red small vesicles in each i r r a d i a t e d cell s i g n i f i c a n t l y increased at the stage of maximum pigment content (Fig. 2C), and subsequently decreased to only one or a few vesicles. F u l l y developed anthocyanoplasts reached 10-15 ~m in diameter a f t e r 17 days (Fig. 2D). At t h i s stage, anthocyanin in the central vacuoles disappeared in a few c e l l s , but remained w i t h i n the vesicles (Fig. 2E). Frequency of these c e l l s increased at the late c u l t u r e period, and most anthocyanoplasts remained in the discolored c e l l s a f t e r pigment accumulation had reached a maximum. This f i n d i n g suggested that the f u l l y developed anthocyanoplast is one of the sites of anthocyanin accumulation in the suspension cultures of sweet potato. However, anthocyanin content progres-
""
0 01 2 3 4 5 6 7 Days after onset of irradiation
Fig. I. Light-induced anthocyanin synthesis in cell suspension cultures of sweet potato. Irradiated c e l l s ( o ) ; Dark-cultured c e l l s ( e ) .
s i v e l y increased a f t e r the disappearance ofanthocyanoplasts in the seedlings of red cabbage (Pecket and Small 1980) and red radish (Yasuda et al. unpublished). The difference between the present observations and those of seedlings may be due to the f a c t that we used cultured c e l l s and they used i n t a c t plant tissues to induce anthocyanin synthesis. In red cabbage seedlings (Pecket and Small 1980), anthocyanoplasts of light-grown seedlings were cons i s t e n t l y larger than those of dark-grown seedlings, which accords with greater a b i l i t y of the former to synthesize pigment. Few pigmented vesicles and l i t t l e anthocyanin accumulation were found in the darkcultured c e l l s in t h i s experiment (Fig. 2F). These results indicated that l i g h t exposure is an important f a c t o r in development of anthocyanoplasts. Relationship bewteen Anthocyanoplasts and Pigment Content during Light I r r a d i a t i o n Fig. 3 shows the r e l a t i o n s h i p between occurrence of anthocyanoplasts and anthocyanin synthesis in the irradiated cells. Anthocyanin synthesis began to f i r s t occur 24-48 h a f t e r i r r a d i a t i o n . Once the maximum level of pigmented c e l l s was reached, the increase in anthocyanin content stopped (Fig. 3A). On the other hand, the frequency of anthocyanoplastcontaining c e l l s r a p i d l y increased 72 h a f t e r onset of i r r a d i a t i o n and then almost l i n e a r l y f o r another 48 h. F i n a l l y the c e l l s containing vesicles reached about 80%. These r e s u l t s indicated a close r e l a t i o n between occurrence of anthocyanoplasts and pigment synthesis in the i r r a d i a t e d c e l l s . Effect of 2,4-D on Light-lnduced Anthocyanin Synthesis and Occurrence of Anthocyanoplasts
254
!:|"
---
I
I
I
I
6
8
10
12
100
l"
0 0
2
4
1
Oays after onset of irradiation
Fig. 3. Changes in cell g~'owth, anthocyanin content, frequency of pigmented ceqls and anthocyanoplast-containing c e l l s in the l i g h t induced c e l l s of sweet potato suspension cultures. A: Cell growth ( o ) ; anthocyanin content (m) and anthocyanin-containing c e l l s (•). B: anthocyanoplast-containing cells ( A ) . Fig. 2. Development of anthocyanoplasts in cell suspension cultures of sweet potato. A: Seven-day-old cells in subcultures by PRL-4C medium supplemented with 0.1 mg/l 2,4-D in the dark. B: Three-day-old c e l l s a f t e r transferred to 2,4-D free medium and exposed to l i g h t . Small red vesicles were detected (arrow). Anthocyanin was s l i g h t l y accumulated in the vacuoles. C: Numerous small pigmented vesicles (arrow) in the anthocyanin-containing c e l l s a f t e r 6 days. D: Fully developed anthocyanoplasts (arrow) a f t e r 17 days. High amount of anthocyanin was accumulated in the vacuoles. E: Pigmented vesicles (arrow) and discolored vacuoles a f t e r 17 days. F: Dark-cultured cells a f t e r 10 days. Bars indicate 10 #m.
I t is well known that higher concentrations of auxins generally i n h i b i t the formation of anthocyanin and other phenolic compounds in the various cell cultures (Constabel et a l . 1971; Davies 1972; Westcott and Henshaw 1976). Therefore, we examined the effect of 2,4-D in the induction medium on the light-induced anthocyanin synthesis and occurrence of anthocyanoplasts. The seven-day-old subcultured c e l l s in the Table I .
dark were transferred to fresh medium supplemented with O, 0.1 and 0.3 mg/l 2,4-D and then exposed to light. Cell growth was markedly promoted, but both anthocyanin content and frequency of anthocyanoplastcontaining cells were inhibited by addition of 2,4-D in the medium (Table I ) . These results suggested that the occurrence and development of pigmented vesicles depend on the a b i l i t y to produce anthocyanin in the light-induced c e l l s . Pecket and Small (1980) suggested that anthocyanoplasts are the s i t e of anthocyanin biosynthesis from observations on the l i g h t and dark-grown red cabbage seedlings. Our present observations also indicated that appearance of anthocyanoplasts was apparently related to the pigment formation. However, the present data did not show any d i r e c t evidence as to whether anthocyanoplasts were the s i t e of biosynthesis of anthocyanin i n c e l l suspension cultures of sweet potato. Further experiments were necessary to answer the question by using the isolated anthocyanoplasts. Acknowledgement
This work was supported in part by the grant from Association for the Advancement of Science of Nagano Prefecture, Japan.
Effect of 2,4-D in the induction medium on cell growth, pigmented c e l l s , anthocyanopla~ts and anthocyanin content in the light-induced c e l l s of sweet potato suspension cultures
Concentration of 2,4-D (mg/l) 0 0.1 0,3
Cell growth (g FW/culture) 0.89 + 0.06 b 1.55 + 0.04 2.34 + 0.02
Pigmentedc e l l s (%) 98.4 92.5 62.1
Anthocyanoplasts (%) 79.2 26.8 5.6
Anthocyanin content (A530/g FW) 314.0 + I0.0 b 143.2 + 4.3 46.8 + 2.3
alrradiated c e l l s were harvested 10 days a f t e r transfer to the induction medium, bData were the mean ~ SE.
255 References
Constabel F, Shyluk JP, Gamborg OL (1971) Planta 96: 306-316 Davies ME (1972) Planta 104:50-56 Fritsch H, Grisebach H (1975) Phytochemistry 14:24372442 Gamborg OL (1966) Can J Biochem 44:791-799 Hrazdina G (1982) In: Harborne JB, Mabry TJ (eds) The Flavonoids Advances in Research, Chapman and Hall London, New York. pp 135-188 Hrazdina G, Alscher-Herman R, Kish, VM (1980) Phytochemistry 19:1355-1359 Hrazdina G, Wagner GJ, Siegelman HW (1978) Phytochemistry 17:53-56
Matsumoto T, Nishida K, Noguchi M, Tamaki E (1973) Agric Biol Chem 37:561-567 Nishimaki T, Nozue M (1985) Plant Cell Rep(submitted) Ozeki Y, Komamine A (1981) Physiol Plant 53:570-577 Pecket RC, Small CJ (1980) Phytochemistry 19:25712576 Ranjeva R, A l i b e r t G, Boudet AM (1977) Plant Sci Lett 10:235-242 Stickland S, Sunderland N (1972) Ann Bot 36:443-457 Wellmann E, Hrazdina G, Grisebach H (1976) Phytochemistry 15:913-915 Westcott RJ, Henshaw GG (1976) Planta 131:67-73 Yamakawa T, Kato S, Ishida K, Kodama T, Minoda Y (1983) Agric Biol Chem 47:2185-2191 Yasuda H, Shinoda H (1985) Cytologia (in press)
,Plant Cell Reports (1985) 4:252-255
© Springer-Verlag 1985
Occurrence of anthocyanoplasts in cell suspension cultures of sweet potato Masayuki Nozue and Hitoshi Yasuda Department of Biology, Faculty of Science, Shinshu University, Matsumoto 390, Japan Received January 21, 1985 / Revised version received July 29, 1985 - Communicated by F. Constabel
Abstract
Intensely pigmented and spherical vesicles(anthocyanoplasts) were found in anthocyanin-containing c e l l s of sweet potato (Ipomoea batatas) suspension cultures. Anthocyanin s y ~ ~ o f i r s t occur 24-48 h a f t e r exposure to l i g h t , and then numerous small red vesicles were detected under a microscope. The f r e quency of anthocyanoplast-containing c e l l s r a p i d l y increased to f i n a l l y about 80% of the t o t a l cultured c e l l s a f t e r 5 days of i r r a d i a t i o n . F u l l y developed anthocyanoplasts reached 10-15 ~m in diameter. On the other hand, neither anthocyanin synthesis nor development of anthocyanoplasts was induced in the dark-cultured c e l l s . 2,4-D also i n h i b i t e d anthocyanin synthesis and development of these vesicles. The results suggest that anthocyanoplasts might be a site of anthocyanin synthesis and/or accumulation. Abbreviation: 2,4-D=2,4-dichlorophenoxyacetic acid Introduction
Although a number of attempts have been made to e l u c i date the s u b c e l l u l a r l o c a l i z a t i o n of secondary metabo l i t e s and t h e i r biosynthetic s i t e , the functional d i f f e r e n t i a t i o n and regulatory mechanism of the biosynthesis have not yet been c l e a r l y established. I t has been generally agreed that the main s i t e of anthocyanin accumulation is the central vacuole. However, knowledge of the r e l a t i o n between the s i t e of anthocyanin biosynthesis and the s i t e of i t s accumulation is at present fragmentary. Fritsch and Grisebach (1975) suggested that some enzymes involved in anthocyanin biosynthesis may occur on or in the tonoplast, or in the vacuolar sap. Other reports also suggested that anthocyanin and other flavonoids can be synthesized in chloroplasts (Ranjeva et a l . 1977). On the contrary, Hrazdina et a l . (1978; 1980) showed that the enzymes associated with biosynthesis occur not in the plastids and vacuoles, but in the cytosol f r a c t i o n s . I t is thought at present that the anthocyanin and other flavonoid biosynthesis occurs near the endoplasmic reticulum (ER) by cytoplasmic and ER-bound enzymes acting in close proximity to each other, and then transported by unknown transport mechanisms to the central vacuole of c e l l s , where they accumulate(Hrazdina1982). Recently, i t has been reported that intensely pigmented vesicles (anthocyanoplasts) were found w i t h i n the main vacuoles of anthocyanin-containinq c e l l s in young seedlings of red cabbage (Pecket and Small 1980). Yasuda and Shinoda (1985) have shown that the red spherical bodies, which contained anthocyanin, were
Offprint requests to: M. Nozue
found in the seedling hypocotyls of red radish. These findings led to the speculation that these vesicles were l i k e l y to be the s i t e of anthocyanin biosynthesis. We also found such pigmented vesicles ih the light-induced anthocyanin-synthesizing c e l l s of sweet potato suspension cultures. In the present report, we investigated the r e l a t i o n s h i p between development of anthocyanoplasts and anthocyanin accumulation and also discussed the possible functions of these vesi c l e s during the l i g h t - i n d u c e d anthocyanin synthesis in the c e l l suspension cultures. Materials and Methods
Subcultures of High Anthocyanin-Producing Cell Line The high anthocyanin-producing cell l i n e used in the present experiments was obtained by the visual select i o n from callus cultures i n i t i a t e d with roots of sweet potato (Ipomoea batatas Lam. cv. K i n t o k i ) . Cell suspension c u - - u - T t u - r ~ h e cell l i n e were established by PRL-4C (Gamborg 1966) l i q u i d medium supplemented with 0.1 mg/l 2,4-D and 3% sucrose. Cell aggregates were obtained by pouring 7-day-old cultures through a stainless steel sieve with 500 pm mesh size, then through a 44 pm sieve and were taken f o r a l l subcultures. Two g wet weight of these c e l l s and cell clusters was trasferred to 300-ml Erlenmeyer flasks containing 50 ml of fresh medium every 7 days, and maintained at 25 °C in the dark on a rotary shaker at 110 cycles/min. Induction of Anthocyanin Synthesis Anthocyanin synthesis was induced by the f o l l o w i n g procedures. The seven-day-old c e l l s (1.0 g wet weight) prepared as above were trasferred to 100-ml Erlenmeyer flasks containing 10 ml of the induction medium (PRL4C medium without 2,4-D) and cultured under continuous i l l u m i n a t i o n with cool white fluorescent tubes (Toshiba Neoline FL40 SW; ca. 12,000 lux) at 25 °C on a rotary shaker at 120 cycles/min. Measurement of Cell Growth and Anthocyanin Content Cells were separated from the culture medium by f i l t r a t i o n through f i l t e r paper (Toyo No. I) with a suction funnel and then weighed. Results were expressed as fresh weight (g) per c u l t u r e . For q u a n t i t a t i v e determination of anthocyanin, c e l l s (20-40 mg fresh weight) trapped on a f i l t e r paper were extracted f o r 24 h with 5.0 ml of methanol containing 0.5% HCl (v/v) at 4 °C in the dark. A f t e r removing the insoluble materials by c e n t r i f u g a t i o n at 2,000 g f o r 20 min, the absorbance of the clear supernatant was measured
253 with a spectrophotometer. Anthocyanin content was expressed as absorbance at 530 nm per g fresh weight of c e l l s .
130
0
Counting the Pigmented Cells and Anthocyanoplast -Containing Cells At i n t e r v a l s a f t e r onset of i r r a d i a t i o n , c e l l s were counted under a microscope a f t e r maceration with Cellulase 'Onozuka' R-IO, Macerozyme R-IO (bothYakult Pharmaceutical Co., L t d . , Nishinomiya, Japan) and Pectolyase Y-23 (Seishin Pharmaceutical Co., L t d . , Tokyo, Japan) as described previously (Nishimaki and Nozue 1988). A v i s i b l e red cell (protoplast) was counted as an anthocyanin-containing cell and an i n tensely red and spherical vesicle was regarded as an anthocyanoplast. Results were expressed as percentages of these c e l l s in t o t a l cultured c e l l s . A l l experiments were repeated two or three times, and about a thousand c e l l s were observed in each experiment.
o
0
7o
C Results and Discussion
0
Light-lnduced Anthocyanin Synthesis The stimulatory e f f e c t of l i g h t on anthocyanin synthesis has been well investigated with many tissue cultures (Stickland and Sunderland 1972; Matsumoto et al. 1973; Wellmann et a l . 1976; Yamakawa et a l . 1983). In the present experiments, the 7-day-old c e l l s of the high anthocyanin-producing cell l i n e which was maintained in 2,4-D-containing medium in the dark, were transferred to the induction medium (2,4-D free) and cultured under continuous i l l u m i n a t i o n . There was no anthocyanin synthesis in the subcultures, however, anthocyanin synthesis was induced w i t h i n 48 h and i t markedly increased under f u r t h e r i r r a d i a t i o n (Fig. I ) . Anthocyanin synthesis was not induced in the darkcultured c e l l s . The percentage of anthocyanin-containing c e l l s reached more than 95% during the i l l u m i n a t i o n period. Ozeki and Komamine (1981) have established the system in which anthocyanin synthesis was triggered by t r a n s f e r r i n g c e l l s from a medium containing 2,4-D to a medium lacking auxin in carrot suspension c u l tures. However, the present data showed that continuous i l l u m i n a t i o n was an indispensable f a c t o r for anthocyanin synthesis in sweet potato suspension cultures. Development of Anthocyanoplasts in the L i g h t Induced Cells The seven-day-old c e l l s of subcultures were transferred to 2,4-D free medium and exposed to l i g h t , and then development of anthocyanoplasts in the pigmentsynthesizing c e l l s were observed under a microscope. Such pigmented vesicles were not found in the c e l l s subcultured in the dark (Fig. 2A). On the other hand, small red vesicles, approximately 3 ~m in d i ameter, were f i r s t detected in the anthocyanincontaining c e l l s between 72-96 h a f t e r onset of i r r a d i a t i o n (Fig. 2B). The number of red small vesicles in each i r r a d i a t e d cell s i g n i f i c a n t l y increased at the stage of maximum pigment content (Fig. 2C), and subsequently decreased to only one or a few vesicles. F u l l y developed anthocyanoplasts reached 10-15 ~m in diameter a f t e r 17 days (Fig. 2D). At t h i s stage, anthocyanin in the central vacuoles disappeared in a few c e l l s , but remained w i t h i n the vesicles (Fig. 2E). Frequency of these c e l l s increased at the late c u l t u r e period, and most anthocyanoplasts remained in the discolored c e l l s a f t e r pigment accumulation had reached a maximum. This f i n d i n g suggested that the f u l l y developed anthocyanoplast is one of the sites of anthocyanin accumulation in the suspension cultures of sweet potato. However, anthocyanin content progres-
""
0 01 2 3 4 5 6 7 Days after onset of irradiation
Fig. I. Light-induced anthocyanin synthesis in cell suspension cultures of sweet potato. Irradiated c e l l s ( o ) ; Dark-cultured c e l l s ( e ) .
s i v e l y increased a f t e r the disappearance ofanthocyanoplasts in the seedlings of red cabbage (Pecket and Small 1980) and red radish (Yasuda et al. unpublished). The difference between the present observations and those of seedlings may be due to the f a c t that we used cultured c e l l s and they used i n t a c t plant tissues to induce anthocyanin synthesis. In red cabbage seedlings (Pecket and Small 1980), anthocyanoplasts of light-grown seedlings were cons i s t e n t l y larger than those of dark-grown seedlings, which accords with greater a b i l i t y of the former to synthesize pigment. Few pigmented vesicles and l i t t l e anthocyanin accumulation were found in the darkcultured c e l l s in t h i s experiment (Fig. 2F). These results indicated that l i g h t exposure is an important f a c t o r in development of anthocyanoplasts. Relationship bewteen Anthocyanoplasts and Pigment Content during Light I r r a d i a t i o n Fig. 3 shows the r e l a t i o n s h i p between occurrence of anthocyanoplasts and anthocyanin synthesis in the irradiated cells. Anthocyanin synthesis began to f i r s t occur 24-48 h a f t e r i r r a d i a t i o n . Once the maximum level of pigmented c e l l s was reached, the increase in anthocyanin content stopped (Fig. 3A). On the other hand, the frequency of anthocyanoplastcontaining c e l l s r a p i d l y increased 72 h a f t e r onset of i r r a d i a t i o n and then almost l i n e a r l y f o r another 48 h. F i n a l l y the c e l l s containing vesicles reached about 80%. These r e s u l t s indicated a close r e l a t i o n between occurrence of anthocyanoplasts and pigment synthesis in the i r r a d i a t e d c e l l s . Effect of 2,4-D on Light-lnduced Anthocyanin Synthesis and Occurrence of Anthocyanoplasts
254
!:|"
---
I
I
I
I
6
8
10
12
100
l"
0 0
2
4
1
Oays after onset of irradiation
Fig. 3. Changes in cell g~'owth, anthocyanin content, frequency of pigmented ceqls and anthocyanoplast-containing c e l l s in the l i g h t induced c e l l s of sweet potato suspension cultures. A: Cell growth ( o ) ; anthocyanin content (m) and anthocyanin-containing c e l l s (•). B: anthocyanoplast-containing cells ( A ) . Fig. 2. Development of anthocyanoplasts in cell suspension cultures of sweet potato. A: Seven-day-old cells in subcultures by PRL-4C medium supplemented with 0.1 mg/l 2,4-D in the dark. B: Three-day-old c e l l s a f t e r transferred to 2,4-D free medium and exposed to l i g h t . Small red vesicles were detected (arrow). Anthocyanin was s l i g h t l y accumulated in the vacuoles. C: Numerous small pigmented vesicles (arrow) in the anthocyanin-containing c e l l s a f t e r 6 days. D: Fully developed anthocyanoplasts (arrow) a f t e r 17 days. High amount of anthocyanin was accumulated in the vacuoles. E: Pigmented vesicles (arrow) and discolored vacuoles a f t e r 17 days. F: Dark-cultured cells a f t e r 10 days. Bars indicate 10 #m.
I t is well known that higher concentrations of auxins generally i n h i b i t the formation of anthocyanin and other phenolic compounds in the various cell cultures (Constabel et a l . 1971; Davies 1972; Westcott and Henshaw 1976). Therefore, we examined the effect of 2,4-D in the induction medium on the light-induced anthocyanin synthesis and occurrence of anthocyanoplasts. The seven-day-old subcultured c e l l s in the Table I .
dark were transferred to fresh medium supplemented with O, 0.1 and 0.3 mg/l 2,4-D and then exposed to light. Cell growth was markedly promoted, but both anthocyanin content and frequency of anthocyanoplastcontaining cells were inhibited by addition of 2,4-D in the medium (Table I ) . These results suggested that the occurrence and development of pigmented vesicles depend on the a b i l i t y to produce anthocyanin in the light-induced c e l l s . Pecket and Small (1980) suggested that anthocyanoplasts are the s i t e of anthocyanin biosynthesis from observations on the l i g h t and dark-grown red cabbage seedlings. Our present observations also indicated that appearance of anthocyanoplasts was apparently related to the pigment formation. However, the present data did not show any d i r e c t evidence as to whether anthocyanoplasts were the s i t e of biosynthesis of anthocyanin i n c e l l suspension cultures of sweet potato. Further experiments were necessary to answer the question by using the isolated anthocyanoplasts. Acknowledgement
This work was supported in part by the grant from Association for the Advancement of Science of Nagano Prefecture, Japan.
Effect of 2,4-D in the induction medium on cell growth, pigmented c e l l s , anthocyanopla~ts and anthocyanin content in the light-induced c e l l s of sweet potato suspension cultures
Concentration of 2,4-D (mg/l) 0 0.1 0,3
Cell growth (g FW/culture) 0.89 + 0.06 b 1.55 + 0.04 2.34 + 0.02
Pigmentedc e l l s (%) 98.4 92.5 62.1
Anthocyanoplasts (%) 79.2 26.8 5.6
Anthocyanin content (A530/g FW) 314.0 + I0.0 b 143.2 + 4.3 46.8 + 2.3
alrradiated c e l l s were harvested 10 days a f t e r transfer to the induction medium, bData were the mean ~ SE.
255 References
Constabel F, Shyluk JP, Gamborg OL (1971) Planta 96: 306-316 Davies ME (1972) Planta 104:50-56 Fritsch H, Grisebach H (1975) Phytochemistry 14:24372442 Gamborg OL (1966) Can J Biochem 44:791-799 Hrazdina G (1982) In: Harborne JB, Mabry TJ (eds) The Flavonoids Advances in Research, Chapman and Hall London, New York. pp 135-188 Hrazdina G, Alscher-Herman R, Kish, VM (1980) Phytochemistry 19:1355-1359 Hrazdina G, Wagner GJ, Siegelman HW (1978) Phytochemistry 17:53-56
Matsumoto T, Nishida K, Noguchi M, Tamaki E (1973) Agric Biol Chem 37:561-567 Nishimaki T, Nozue M (1985) Plant Cell Rep(submitted) Ozeki Y, Komamine A (1981) Physiol Plant 53:570-577 Pecket RC, Small CJ (1980) Phytochemistry 19:25712576 Ranjeva R, A l i b e r t G, Boudet AM (1977) Plant Sci Lett 10:235-242 Stickland S, Sunderland N (1972) Ann Bot 36:443-457 Wellmann E, Hrazdina G, Grisebach H (1976) Phytochemistry 15:913-915 Westcott RJ, Henshaw GG (1976) Planta 131:67-73 Yamakawa T, Kato S, Ishida K, Kodama T, Minoda Y (1983) Agric Biol Chem 47:2185-2191 Yasuda H, Shinoda H (1985) Cytologia (in press)
