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Biological activities of abscisic acid analogs in the morphological change of the green alga Haematococcus pluvialis
JOURNAL OF FERMENTATIONAND BIOENGINEERWG Vol. 85, No. 5, 529-531. 1998
Biological Activities of Abscisic Acid Analogs in the Morphological Change of the Green Alga Haematococcus pluvialis MAKIO KOBAYASHI,‘*
YASUSHI TODOROKI,2 NOBUHIRO HIRAI,2 YOSHIRO HAJIME OHIGASHI,2 AND YASUNOBU TSUJI’
KURIMURA,’
Research Laboratory of Higashimaru Shoyu Co. Ltd., 100-3 Tominaga, Tatsuno, Hyogo 679-4193’ and Division of AppIied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502,? Japan Received 9 December 1997IAccepted
26 January 1998
The plant hormone abscisic acid (ABA) induces a morphological change from green vegetative ceils to red cyst cells, of Haematococcus pluvialis containing carotenoids, in plate culture. Long-lasting analogs of ABA, (+)-8’,8’,8’-trifluoro and (+)-8’,8’-difluoro-ABAs, which can resist metabolic inactivation, induce carotenoid production at lOO-fold lower concentration than (+)-ABA. These analogs can be used as effective regulators to produce carotenoids in H. pluvialis cells. [Key words: abscisic acid, Haematococcus pluvialis]
astaxanthin,
carotenogenesis,
Astaxanthin, 3,3’-dihydroxy-,&,%carotene-4,4’-dione, is a red ketocarotenoid used as a pigmentation source for marine fish aquaculture (1) and as an antioxidant for clinical applications (2). Immotile cyst cells of the unicellular green alga Haematococcus pluvialis produce astaxanthin in the resting stage of reduced photosynthetic activity, while green vegetative cells with two flagellae, are motile and have photosynthetic activity (3, 4). Encystment and carotenogenesis occur within 6 weeks in liquid culture, and within 4 weeks in plate culture, probably due to nutrient starvation and drought stress, respectively (5). Nutrient deficiency, such as nitrogen limitation, also induces encystment (6), and astaxanthin biosynthesis in cyst cells is also enhanced by active oxygen (7), high temperature (8), and high concentrations of salt (9, 10). Enhanced carotenogenesis in cyst cells may be a manifestation of the natural algal defense system under adverse environmental conditions (11). Abscisic acid (ABA) is best known for its function as a plant hormone in the adaptation to drought and other stresses in higher plants (12, 13). Recently, we found that in the presence of ABA, vegetative cells of H. pluvialis in plate culture formed mature red cyst cells with enhanced carotenogenesis within 2 weeks, and endogenous levels of ABA increased in parallel with the morphological change (5). Therefore, ABA could function as a plant hormone in algal morphogenesis, encystment, and carotenogenesis in H. pluvialis under drought stress, suggesting that ABA could be used as an effective regulator for the production of astaxanthin by H. pluviaiis, ABA is rapidly metabolized to inactive phaseic acid via unstable 8’-hydroxy-ABA in plants (12). Todoroki et al. (14-16) synthesized potent long-lasting analogs of ABA, (+)-8’,8’,8’-trifluoro, (+)-8’,8’-difluoro, (+)-3’fluoro and (+)-8’-methoxy-ABAs (Fig. l), which were designed to resist metabolic inactivation. Another analog, (+)-8’-fluoro-ABA, was synthesized to mimic 8’hydroxy-ABA in order to investigate the role of the 8’hydroxy group in the expression of its activity (17). In this study, we examined and compared the effects of
drought
stress,
encystment,
plant
hormone,
these analogs on encystment and carotenogenesis of H. pluvialis. The effects were evaluated from the intracellular carotenoid/chlorophyll (Car/Chl) ratio since this ratio is a good parameter to distinguish among vegetative (green), immature cyst (brown) and mature cyst (red) cells (5, 7, 18). The Car/Chl ratios are usually about 0.5, 1.0 and 7.0 in vegetative, immature, and mature cyst cells, respectively (5, 10, 18). (+)-ABA, a natural isomer, was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Synthesis of (+)-8’,8’,8’-trifluoro, (+)-8’,8’-difluoro, (+)-8’-fluoro, (+)-3’-fluoro and (+)-8’-methoxy-ABAs were previously reported by Todoroki et al. (14-17). H. pluviafis Flotow NIES-144 was obtained from the National Institute for Environmental Studies, Tsukuba, Japan. The basal acetate medium was described previously (4). For the basal culture, 10 ml of a 4-d-old culture was inoculated into 100ml of fresh basal medium in 200 ml Erlenmeyer flasks. The flasks were then incubated mixotrophically at 20°C under 12-h light/l2-h darkillumination cycles at 20 PE md2s- I with white fluorescent lamps as described (4). The effects of the analogs on encystment and carotenogenesis of H. pfuvialis were examined as follows. A 1O-3 M aqueous solution of the analog was added to 1OOml of basal medium containing 2% (w/v) agar (5, 19) to yield final concentrations of IO-‘-10 m4M. Aliquots of 0.1 ml of the 4-d basal culture that contained 5 x lo5 vegetative cells/ml were spread on agar plates containing test compounds in triplicate as described (5). The plates were incubated for 2 weeks at 20°C under continuous illumination of 120 p*Em--2s-1 with white fluorescent lamps in a sterile cultivator in the second stage of the two-stage culture (7, 18). Cultured cells (lo-20mg fresh weight) were recovered from each plate using a spatula, and extracted with 10 ml of 90% (v/v) acetone. Intracellular pigments, carotenoid and chlorophyll in the acetone extracts were assayed as described (4, 7, 18). To examine the isomerization and decomposition of ABA and its analogs in light, aqueous solutions of (+)ABA and its analogs were added to 1 ml of basal media in 1Oml vials to yield a final concentration of 10m5 M.
* Corresponding author. 529
530
KOBAYASHI ET AL.
J . FERMENT.BIOENG ,
FIG. 1. Structures of (+)-ABA and its (+)-analogs. ABA: R,=H, R2=CH3; 8’,8’,8’-trifluoro-ABA: R,=H, Rz=CFj; 8’,8’difluoro-ABA: R, =H. R,=CHF,: 8’-fluoro-ABA: R, =H. R,= CHZF; 8-methox;-ABk: i, =H, -‘R2=CH20CH3; 3’_&or&AiA: R,=F, Rz=CH4.
The media were incubated under the same conditions as those used for plate culture of cells, and aliquots were collected after 7 d. The collected media were kept at 5°C until HPLC analysis. Concentrations of (+)-ABA and its analogs in the media were analyzed by HPLC (PLC5D, Eyela, Tokyo) using a Chromatorex ODS column (4.5 i.d. x lSOmm, Fuji Silysia Chemical Ltd., Kasugai, Aichi). The media were eluted with 50% (v/v) methanol in 0.1% (v/v) aqueous acetic acid at a flow rate of 1.O ml/min, and UV-absorbing materials were detected at 254 nm. Peaks in the chromatograms were identified by comparing their retention times with those of authentic standards, and the concentrations of the compounds were calculated from calibration curves of the authentic samples obtained by plotting peak areas and amounts. All analyses were performed in triplicate, and the means were calculated. Retention times of (+)-ABA, its analogs, and their 2-E isomers, are as follows: ABA, 7.2 min; 2-E-ABA, 5.8 min; 8’,8’,8’-trifluoro-ABA, 8.4 min; 2-E-8’,8’,8’-trifluoro-ABA, 7.5 min; 8’,8’-difluoro-ABA, 7.7 min; 2-E-8’,8’-difluoro-ABA, 6.4 min; 8’-fluoro-ABA, 6.7 min; 2-E-8’-fluoro-ABA, 5.3 min; 3’-fluoro-ABA, 8.5 min; 2-E-3’-fluoro-ABA, 6.6 min; 8’-methoxy-ABA, 7.7 min; 2E-8’-methoxy-ABA, 6.7 min. The effects of (+)-ABA and its analogs on the Car/ Chl ratio are shown in Fig. 2. The Car/Chl ratio of cells incubated for 2 weeks without (+)-ABA was about 2.0. (+)-ABA at 3 x lop7 M increased the ratio to 2.8, and the Car/Chl ratio increased in a concentration-dependent manner between lop7 and 10e4M, confirming that (+)ABA enhances carotenoid biosynthesis in cyst cells (5). The effects of the analogs on the Car/Chl ratio were varied. The long-lasting analogs of ABA, (+)-8’,8’,8’-trifluoro and ( f)-8’,8’-difluoro-ABAs, were effective at lOO-fold lower concentrations, 10-6M, than (+)-ABA which was effective at lo-4M. Their Car/Chl ratio, 7.5, at 1OW M was higher than that of (+)-ABA, 6.9, at 10e4M. The high activities of (+)-8’,8’,8’-trifluoro and (+)-8’,8’difluoro-ABAs were expected, since the activities of these metabolism-resistant analogs increased in long-term tests such as the rice seedling assay (15). These analogs can be used as effective regulators for the production of astaxanthin by H. pluvialis cells. (+)-8’-Methoxy-ABA showed an activity similar to that of (i-)-ABA at lo-GM, and higher activity at lop5 M. This activity, however, was not higher than was expected. The 8’-methoxy group may not contribute to the resistance to metabolic inactivation in H. pluvialis, the activity of (+)-8’-methoxy-ABA indicated that the presence of an 8’-methoxy group did not decrease the
-8
-7
-6
-5
-4
-3
Log M FIG. 2. Effects of (+)-ABA and its (+)-analogs on encystment and carotenogenesis in vegetative cells of H. pluviulis in plate culture. Incubation period: 2 weeks. Symbols: 0, (+)-ABA; 0, 8’,8’,8’trifluoro-ABA; A , 8’,8’-difluoro-ABA; A, 8’-fluoro-ABA; n , 8’methoxy-ABA; 0, 3’-fluoro-ABA. Symbols represent t_ SE of the mean. Car/Chl ratio, Carotenoid/chlorophyll ratio.
activity either. Tolerance of activities against 8’-modification of ABA has also been shown in other assays using 8’-alkyl-ABAs (20). (+)-8’-Fluoro and (+)-3’-fluoro-ABAs were active even at 10e7 M, while (+)-ABA was not active at this concentration. However, their activities at lo-5 M were lower than those of (+)-ABA at the same concentration although these analogs had activities similar to or higher than (+)-ABA in other assays (16, 17). ABA, which has the 2-Z side chain, isomerizes to its inactive 2-E isomer, and is partially decomposed by UV irradiation (21), suggesting that these analogs isomerized or decomposed during the test. (+)-ABA and its analogs at lop5 M in basal media were incubated for 7 d under the same conditions as those used for plate culture of cells in order to examine isomerization and decomposition of the compounds in light. Concentrations of ABA and its analogs decreased to 5.2-5.8 x 10.‘jM after 7 d, and concentrations of its 2-E isomers increased to 3.2-4.2X 10m6M. Total concentrations of ABA and its analogs including its 2-E isomers were between 8.5 and 9.7 x 1O-6 M after 7 d. Unidentified compounds were detected on HPLC analysis, but their amounts were small. This result indicated that both the concentrations of isomers and total concentrations of ABA analogs after 7 d were similar to those of (+)-ABA, suggesting that decreases in the activities of (+)-8’-Auoro-and (+)-3’-fluoro-ABA by isomerization and decomposition during the test probably did not occur. The low activities at 10e5 M may indicate toxicity of the monofluoro analogs at high concentrations. If this is so, the toxicity could be caused by formation of hydrogen bonds of the monofluoro groups with hydrogendonating groups such as amino or hydroxyl groups in cellular components (22, 23). This system with H. pluvialis can be used in the bioassay of ABA for the following reasons: (i) induction of encystment of vegetative cells of H. pluvialis, and subse-
NOTES
VOL. 85, 1998
quent enhancement of carotenoid biosynthesis in cysts, which are characteristic activities of ABA; (ii) ABA showed good correlation between activity and concentration; and (iii) the red coloration of colonies in plate cultures was correlated with ABA activity. REFERENCES
Microalgae aquaculture feeds. J. Appl. Phycol., 4, 233-245 (1992). 2. Miki, W.: Biological functions and activities of animal carotenoids. Pure Appl. Chem., 63, 141-146 (1991). 3. Boussiba, S. and Vonshak, A.: Astaxanthin accumulation in the green alga Haematococcus pluvialis. Plant Cell Physiol., 32, 1077-1082 (1991). 4. Kobayashi, M., Kakizono, T., and Nagai, S.: Astaxanthin production by a green alga, Haematococcus phi& accompanied with morphological changes in acetate media. J. Ferment. Bioeng., 71, 335-339 (1991). 5. Kobayashi, M., Hirai, N., Kurimura, Y., Ohigashi, H., and Tsuji, Y.: Abscisic acid-dependent algal morphogenesis in the unicellular green alga Haematococcus pluvialis. Plant Growth Regul., 22, 79-85 (1997). 6. Kakizono, T., Kobayashi, M., and Nagai, S.: Effect of carbon/ nitrogen ratio on encystment accompanied with astaxanthin formation in a green alga, Haematococcus pluvialis. J. Ferment. Bioeng., 74, 403-405 (1992). 7. Kobayashi, M., Kakizono, T., and Nagai, S.: Enhanced carotenoid biosynthesis by oxidative stress in acetate-induced cyst cells of a green unicellular alga, Haematococcus pluvialis. Appl. Environ. Microbial., 59, 867-873 (1993). 8. Tjahjono, A. E., Hayama, Y., Kakizono, T., Terada, Y., 1. Benemann,
Nishio,
J. R.:
N., and Nagai,
S.: Hyper-accumulation
of astaxanthin
in a green alga Haematococcus pluvialis at elevated temperatures. Biotechnol. Lett., 16. 133-138 (1994). 9. Harker, M., Tsavalos, A. J., and Young; A. J.: Autotrophic growth and carotenoid production of Haematococcus pluvialis in a 30 liter air-lift photobioreactor. J. Ferment. Bioeng., 82, 113-118 (1996). 10. Kobayashi, M., Kurimura, Y., and Tsuji, Y.: Light-independent, astaxanthin production by the green microalga Hae-
531
matococcus phviaiis under salt stress. Biotechnol. Lett., 19, 507-509 (1997). 11. Kobayashi, M., Kakizono, T., Nishio, N., Nagai, S., Kurimura, Y., and Tsuji, Y.: Antioxidant role of astaxanthin in the green alga Haematococcus pluvialis. Appl. Microbial. Biotechnol., 48, 351-356 (1997). 12. Addicott, F. T.: Abscisic acid, Praeger, New York (1983). 13. Zeevaart, J. A. D. and Creelman, R. A.: Metabolism and physiology of abscisic acid. Annu. Rev. Plant Physiol. Plant Mol. Biol., 39, 439-473 (1988). 14. Todoroki, Y., Hirai, N., and Koshimizu, K.: 8’- and 9’-Methoxy-
abscisic acids as antimetabolic analogs of abscisic acid. Biosci. Biotech. Biochem., 58, 707-715 (1994). 15. Todoroki, Y., Hirai, N., and Koshimizu, K.: 8’,8’-Difluoroand 8’,8’,8’-trifluoroabscisic acids as highly potent, long-lasting analogues of abscisic acid. Phytochemistry, 38, 561-568 (1995). 16. Todoroki, Y., Hirai, N., and Ohigashi, H.: Synthesis, biological activity and metabolism of (s)-( +)J’-fluoroabscisic acid. Tetrahedron, 51, 691 l-6926 (1995). 17. Todoroki, Y., Hirai, N., and Koshimizu, K.: Synthesis and biological activity of I-deoxy-l’-fluoro- and 8-fluoroabscisic acids. Phytochemistry, 40, 633-641 (1995). 18. Kobayashi, M., Kurimura, Y., Kakizono, T., Nishio, N., and Tsuji, Y.: Morphological changes in the life cycle of the green alga Haematococcus pluvialis. J. Ferment. Bioeng., 84, 94-97 (1997). 19. Marslilek, B., Zahradm’ckovB, H., and Hronkovai, M.: Extracellular production of abscisic acid by soil algae under salt, acid or drought stress. 2. Naturforsch., 47c, 701-704 (1992). 20. Nakano, S., Todoroki, Y., Hirai, N., and Ohigashi, H.: Synthesis and biological
activitv
of 7’-. 8’-. and 9’-alkvl analoeues
of abscisic acid. Biosci. Biotech. Biochem., 59, 1699-1706 (1995). 21. Planeher, B.: Note on the isomerization of abscisic acid by irradiation with UV light. Gartenbauwissenschaft. 44. 184-191 (1979).
22. Penglis, A. A. E.: Fluorinated carbohydrates. Adv. Carbohydr. Chem. Biochem., 38. 195-285 (1981). 23. Welch, J. T.: Ad;an&es in the ireparation of biologically active organofluorine (1987).
compounds.
Tetrahedron,
43,
3123-3197
Biological Activities of Abscisic Acid Analogs in the Morphological Change of the Green Alga Haematococcus pluvialis MAKIO KOBAYASHI,‘*
YASUSHI TODOROKI,2 NOBUHIRO HIRAI,2 YOSHIRO HAJIME OHIGASHI,2 AND YASUNOBU TSUJI’
KURIMURA,’
Research Laboratory of Higashimaru Shoyu Co. Ltd., 100-3 Tominaga, Tatsuno, Hyogo 679-4193’ and Division of AppIied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwake-cho, Sakyo-ku, Kyoto 606-8502,? Japan Received 9 December 1997IAccepted
26 January 1998
The plant hormone abscisic acid (ABA) induces a morphological change from green vegetative ceils to red cyst cells, of Haematococcus pluvialis containing carotenoids, in plate culture. Long-lasting analogs of ABA, (+)-8’,8’,8’-trifluoro and (+)-8’,8’-difluoro-ABAs, which can resist metabolic inactivation, induce carotenoid production at lOO-fold lower concentration than (+)-ABA. These analogs can be used as effective regulators to produce carotenoids in H. pluvialis cells. [Key words: abscisic acid, Haematococcus pluvialis]
astaxanthin,
carotenogenesis,
Astaxanthin, 3,3’-dihydroxy-,&,%carotene-4,4’-dione, is a red ketocarotenoid used as a pigmentation source for marine fish aquaculture (1) and as an antioxidant for clinical applications (2). Immotile cyst cells of the unicellular green alga Haematococcus pluvialis produce astaxanthin in the resting stage of reduced photosynthetic activity, while green vegetative cells with two flagellae, are motile and have photosynthetic activity (3, 4). Encystment and carotenogenesis occur within 6 weeks in liquid culture, and within 4 weeks in plate culture, probably due to nutrient starvation and drought stress, respectively (5). Nutrient deficiency, such as nitrogen limitation, also induces encystment (6), and astaxanthin biosynthesis in cyst cells is also enhanced by active oxygen (7), high temperature (8), and high concentrations of salt (9, 10). Enhanced carotenogenesis in cyst cells may be a manifestation of the natural algal defense system under adverse environmental conditions (11). Abscisic acid (ABA) is best known for its function as a plant hormone in the adaptation to drought and other stresses in higher plants (12, 13). Recently, we found that in the presence of ABA, vegetative cells of H. pluvialis in plate culture formed mature red cyst cells with enhanced carotenogenesis within 2 weeks, and endogenous levels of ABA increased in parallel with the morphological change (5). Therefore, ABA could function as a plant hormone in algal morphogenesis, encystment, and carotenogenesis in H. pluvialis under drought stress, suggesting that ABA could be used as an effective regulator for the production of astaxanthin by H. pluviaiis, ABA is rapidly metabolized to inactive phaseic acid via unstable 8’-hydroxy-ABA in plants (12). Todoroki et al. (14-16) synthesized potent long-lasting analogs of ABA, (+)-8’,8’,8’-trifluoro, (+)-8’,8’-difluoro, (+)-3’fluoro and (+)-8’-methoxy-ABAs (Fig. l), which were designed to resist metabolic inactivation. Another analog, (+)-8’-fluoro-ABA, was synthesized to mimic 8’hydroxy-ABA in order to investigate the role of the 8’hydroxy group in the expression of its activity (17). In this study, we examined and compared the effects of
drought
stress,
encystment,
plant
hormone,
these analogs on encystment and carotenogenesis of H. pluvialis. The effects were evaluated from the intracellular carotenoid/chlorophyll (Car/Chl) ratio since this ratio is a good parameter to distinguish among vegetative (green), immature cyst (brown) and mature cyst (red) cells (5, 7, 18). The Car/Chl ratios are usually about 0.5, 1.0 and 7.0 in vegetative, immature, and mature cyst cells, respectively (5, 10, 18). (+)-ABA, a natural isomer, was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Synthesis of (+)-8’,8’,8’-trifluoro, (+)-8’,8’-difluoro, (+)-8’-fluoro, (+)-3’-fluoro and (+)-8’-methoxy-ABAs were previously reported by Todoroki et al. (14-17). H. pluviafis Flotow NIES-144 was obtained from the National Institute for Environmental Studies, Tsukuba, Japan. The basal acetate medium was described previously (4). For the basal culture, 10 ml of a 4-d-old culture was inoculated into 100ml of fresh basal medium in 200 ml Erlenmeyer flasks. The flasks were then incubated mixotrophically at 20°C under 12-h light/l2-h darkillumination cycles at 20 PE md2s- I with white fluorescent lamps as described (4). The effects of the analogs on encystment and carotenogenesis of H. pfuvialis were examined as follows. A 1O-3 M aqueous solution of the analog was added to 1OOml of basal medium containing 2% (w/v) agar (5, 19) to yield final concentrations of IO-‘-10 m4M. Aliquots of 0.1 ml of the 4-d basal culture that contained 5 x lo5 vegetative cells/ml were spread on agar plates containing test compounds in triplicate as described (5). The plates were incubated for 2 weeks at 20°C under continuous illumination of 120 p*Em--2s-1 with white fluorescent lamps in a sterile cultivator in the second stage of the two-stage culture (7, 18). Cultured cells (lo-20mg fresh weight) were recovered from each plate using a spatula, and extracted with 10 ml of 90% (v/v) acetone. Intracellular pigments, carotenoid and chlorophyll in the acetone extracts were assayed as described (4, 7, 18). To examine the isomerization and decomposition of ABA and its analogs in light, aqueous solutions of (+)ABA and its analogs were added to 1 ml of basal media in 1Oml vials to yield a final concentration of 10m5 M.
* Corresponding author. 529
530
KOBAYASHI ET AL.
J . FERMENT.BIOENG ,
FIG. 1. Structures of (+)-ABA and its (+)-analogs. ABA: R,=H, R2=CH3; 8’,8’,8’-trifluoro-ABA: R,=H, Rz=CFj; 8’,8’difluoro-ABA: R, =H. R,=CHF,: 8’-fluoro-ABA: R, =H. R,= CHZF; 8-methox;-ABk: i, =H, -‘R2=CH20CH3; 3’_&or&AiA: R,=F, Rz=CH4.
The media were incubated under the same conditions as those used for plate culture of cells, and aliquots were collected after 7 d. The collected media were kept at 5°C until HPLC analysis. Concentrations of (+)-ABA and its analogs in the media were analyzed by HPLC (PLC5D, Eyela, Tokyo) using a Chromatorex ODS column (4.5 i.d. x lSOmm, Fuji Silysia Chemical Ltd., Kasugai, Aichi). The media were eluted with 50% (v/v) methanol in 0.1% (v/v) aqueous acetic acid at a flow rate of 1.O ml/min, and UV-absorbing materials were detected at 254 nm. Peaks in the chromatograms were identified by comparing their retention times with those of authentic standards, and the concentrations of the compounds were calculated from calibration curves of the authentic samples obtained by plotting peak areas and amounts. All analyses were performed in triplicate, and the means were calculated. Retention times of (+)-ABA, its analogs, and their 2-E isomers, are as follows: ABA, 7.2 min; 2-E-ABA, 5.8 min; 8’,8’,8’-trifluoro-ABA, 8.4 min; 2-E-8’,8’,8’-trifluoro-ABA, 7.5 min; 8’,8’-difluoro-ABA, 7.7 min; 2-E-8’,8’-difluoro-ABA, 6.4 min; 8’-fluoro-ABA, 6.7 min; 2-E-8’-fluoro-ABA, 5.3 min; 3’-fluoro-ABA, 8.5 min; 2-E-3’-fluoro-ABA, 6.6 min; 8’-methoxy-ABA, 7.7 min; 2E-8’-methoxy-ABA, 6.7 min. The effects of (+)-ABA and its analogs on the Car/ Chl ratio are shown in Fig. 2. The Car/Chl ratio of cells incubated for 2 weeks without (+)-ABA was about 2.0. (+)-ABA at 3 x lop7 M increased the ratio to 2.8, and the Car/Chl ratio increased in a concentration-dependent manner between lop7 and 10e4M, confirming that (+)ABA enhances carotenoid biosynthesis in cyst cells (5). The effects of the analogs on the Car/Chl ratio were varied. The long-lasting analogs of ABA, (+)-8’,8’,8’-trifluoro and ( f)-8’,8’-difluoro-ABAs, were effective at lOO-fold lower concentrations, 10-6M, than (+)-ABA which was effective at lo-4M. Their Car/Chl ratio, 7.5, at 1OW M was higher than that of (+)-ABA, 6.9, at 10e4M. The high activities of (+)-8’,8’,8’-trifluoro and (+)-8’,8’difluoro-ABAs were expected, since the activities of these metabolism-resistant analogs increased in long-term tests such as the rice seedling assay (15). These analogs can be used as effective regulators for the production of astaxanthin by H. pluvialis cells. (+)-8’-Methoxy-ABA showed an activity similar to that of (i-)-ABA at lo-GM, and higher activity at lop5 M. This activity, however, was not higher than was expected. The 8’-methoxy group may not contribute to the resistance to metabolic inactivation in H. pluvialis, the activity of (+)-8’-methoxy-ABA indicated that the presence of an 8’-methoxy group did not decrease the
-8
-7
-6
-5
-4
-3
Log M FIG. 2. Effects of (+)-ABA and its (+)-analogs on encystment and carotenogenesis in vegetative cells of H. pluviulis in plate culture. Incubation period: 2 weeks. Symbols: 0, (+)-ABA; 0, 8’,8’,8’trifluoro-ABA; A , 8’,8’-difluoro-ABA; A, 8’-fluoro-ABA; n , 8’methoxy-ABA; 0, 3’-fluoro-ABA. Symbols represent t_ SE of the mean. Car/Chl ratio, Carotenoid/chlorophyll ratio.
activity either. Tolerance of activities against 8’-modification of ABA has also been shown in other assays using 8’-alkyl-ABAs (20). (+)-8’-Fluoro and (+)-3’-fluoro-ABAs were active even at 10e7 M, while (+)-ABA was not active at this concentration. However, their activities at lo-5 M were lower than those of (+)-ABA at the same concentration although these analogs had activities similar to or higher than (+)-ABA in other assays (16, 17). ABA, which has the 2-Z side chain, isomerizes to its inactive 2-E isomer, and is partially decomposed by UV irradiation (21), suggesting that these analogs isomerized or decomposed during the test. (+)-ABA and its analogs at lop5 M in basal media were incubated for 7 d under the same conditions as those used for plate culture of cells in order to examine isomerization and decomposition of the compounds in light. Concentrations of ABA and its analogs decreased to 5.2-5.8 x 10.‘jM after 7 d, and concentrations of its 2-E isomers increased to 3.2-4.2X 10m6M. Total concentrations of ABA and its analogs including its 2-E isomers were between 8.5 and 9.7 x 1O-6 M after 7 d. Unidentified compounds were detected on HPLC analysis, but their amounts were small. This result indicated that both the concentrations of isomers and total concentrations of ABA analogs after 7 d were similar to those of (+)-ABA, suggesting that decreases in the activities of (+)-8’-Auoro-and (+)-3’-fluoro-ABA by isomerization and decomposition during the test probably did not occur. The low activities at 10e5 M may indicate toxicity of the monofluoro analogs at high concentrations. If this is so, the toxicity could be caused by formation of hydrogen bonds of the monofluoro groups with hydrogendonating groups such as amino or hydroxyl groups in cellular components (22, 23). This system with H. pluvialis can be used in the bioassay of ABA for the following reasons: (i) induction of encystment of vegetative cells of H. pluvialis, and subse-
NOTES
VOL. 85, 1998
quent enhancement of carotenoid biosynthesis in cysts, which are characteristic activities of ABA; (ii) ABA showed good correlation between activity and concentration; and (iii) the red coloration of colonies in plate cultures was correlated with ABA activity. REFERENCES
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