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Phenylpropanoid metabolite supports cell aggregate formation in strawberry cell suspension culture
JOURNAL OF BIOSCIENCE AND BIOENGINEERING Vol. 102, No. 1, 8–13. 2006 DOI: 10.1263/jbb.102.8
© 2006, The Society for Biotechnology, Japan
Phenylpropanoid Metabolite Supports Cell Aggregate Formation in Strawberry Cell Suspension Culture Jun-ichi Edahiro1,2 and Minoru Seki1,3* Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan,1 Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan,2 and Department of Chemical Engineering, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan 3 Received 24 October 2005/Accepted 10 February 2006
Plant cells in suspension culture tend to aggregate and form large clumps. In suspension culture, large cell aggregates are frequently subjected to hydrodynamic shear stress; however, a certain degree of cell aggregation is often required for cell growth and metabolite production. Thus, controlling cell-aggregate size is desired to establish high productivity of useful products using plant cell suspension culture. In this study, we focused on the relationship between cell-aggregate formation and secondary metabolism. We found that anthocyanin concentration showed a good correlation with cell-aggregate size in the cultured strawberry cell line FAR (Fragaria ananassa R), which produces anthocyanin and other phenylpropanoid metabolites constitutively without illumination. This result suggests that there is a relationship between cell-aggregate formation and the accumulation of phenylpropanoid metabolites. To investigate the direct effect of phenylpropanoid metabolism on cell-aggregate formation, the time course of cell-aggregate size was monitored when phenylpropanoid metabolism was suppressed by a metabolic inhibitor, L-α-aminooxyβ-phenylpropionic acid (AOPP), a specific inhibitor of phenylalanine ammonia lyase which is the starting and key enzyme of the phenylpropanoid pathway. In the absence of AOPP, the average diameter of cell aggregates increased on day 8 of culture. This increase in cell-aggregate size was completely suppressed by the addition of 0.1 mM AOPP, without any reduction in cell growth rate or soluble protein content. These results indicate that cell-aggregate formation is directly supported by a secondary metabolite produced from the phenylpropanoid pathway, suggesting that cell-aggregate size can be controlled by AOPP without inhibition of primary metabolism. [Key words: plant cultured cell, Fragaria ananassa, cell aggregate, phenylpropanoid metabolism, secondary metabolism, anthocyanin, phenylalanine ammonia lyase (PAL), L-α-aminooxy-β-phenylpropionic acid (AOPP)]
Hulst et al. reported that secondary metabolite production increased with increasing cell-aggregate diameter starting at several millimeters (4). Their study showed that high mass transfer resistance caused by large particle size induced a lack of oxygen in the center of the cell aggregate, which resulted in the promotion of secondary metabolite production. In another plant cell type, Ping et al. suggested that the existence of diffusional resistance around cell aggregates due to the presence of a solid phase could hinder some intracellular substrates diffusing into the medium, which resulted in the activation of metabolic reactions (5). Because plants are multicellular organisms, a certain degree of intercellular transport of physiologically active substances is often required for the synthesis of secondary metabolites (6). In the studies described above, the formation of a certain size of cell aggregate is required for secondary metabolism. In these situations, it is desired to establish a technique for controlling cell-aggregate size, while maintaining high productivity of the target metabolite.
Recently, plant cell cultures have been studied actively as a potential source of high-value biological products. Plant cells in suspension cultures tend to aggregate and form cell clusters ranging from a few cells to several thousands of cells, often reaching a few centimeters in diameter. A large cell aggregate is frequently subjected to hydrodynamic shear stress in suspension culture (1). Therefore, smaller cell aggregates are preferred from the viewpoint of process engineering. To reduce the size of cell aggregates in suspension cultures, several methods have been examined, such as the repetitive selection of small cell aggregates (2) and the addition of cell-wall degrading enzymes to the medium (3). On the other hand, a certain degree of aggregation is often required for cellular metabolism (4). In some studies, cellaggregate formation induced secondary metabolite production, which resulted in increased productivity. For example, * Corresponding author. e-mail: [email protected] phone: +81-(0)72-254-9296 fax: +81-(0)72-254-9911 8
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Cell-aggregate size is governed by the cohesiveness of the cell wall. The cell wall is considered as a composite material made of fiber (cellulose), a matrix (lignin, hemicellulose) and fillers (water, tannins). Both lignin and tannin are secondary metabolites synthesized from the phenylpropanoid pathway, which is a typical pathway in plant secondary metabolism. As these secondary metabolites have a function to increase cellular cohesiveness, it is reasonable that large cell aggregates accumulate a large amount of secondary metabolites related to these compounds. In the case of lignin, a correlation between cell-aggregate size and lignification was reported (7). Several researchers have also reported that a certain size of cell aggregate was required for the cellular accumulation of anthocyanin (8, 9), which is a natural pigment and is synthesized from the phenylpropanoid pathway similar to lignin and tannin. However, to prove the hypothesis that the accumulation of these secondary metabolites promotes cellular cohesiveness and increases aggregate size, it is desired that a direct and definite relationship between the accumulation of these metabolites and the formation of cell aggregates by inhibiting their biosynthesis by a metabolic inhibitor be shown. Phenylpropanoid metabolites were produced from phenylalanine ammonia lyase (PAL; EC 4.3.1.5), which is the starting and key enzyme of the phenylpropanoid pathway. Therefore, it was assumed that L-α-aminooxy-β-phenylpropionic acid (AOPP), a specific inhibitor of PAL, would prove useful in this case. In this study, we investigated the correlation between secondary metabolites synthesized from the phenylpropanoid pathway and cell-aggregate size in cultured strawberry cells producing anthocyanin constitutively. By applying AOPP, a specific inhibitor of PAL, we evaluated the direct effect of secondary metabolism on cell-aggregate formation without affecting primary metabolism. MATERIALS AND METHODS Plant materials and culture conditions Cultured strawberry cells, designated FAR, which were derived from the petioles of Fragaria ananassa cv. Shikinari, were used in this study. FAR cells can produce anthocyanin constitutively without light irradiation (10). A suspension culture of FAR was subcultured every 14 d at 25°C in Linsmaier–Skoog medium (11) (abbreviated as LS medium) containing 1 mg/l 2,4-dichlorophenoxyacetic acid, 0.1 mg/l benzylaminopurine, and 0.4 mg/l thiamine–HCl. Cell fractionation by aggregate size Cells harvested from an 8-d-cultured cell suspension were partitioned into two fractions by sieving through nylon mesh as described below. The cell suspension was filtered through a nylon mesh (opening, 95 µm) and the filtrate was refiltered through a nylon mesh with a smaller opening size (37 µm). The first cell fraction (designated L-cells) contained large cell aggregates that were retained on the rough mesh (95 µm). The other fraction had smaller cell aggregates (S-cells) which passed through the rough mesh (95 µm) and were retained on the fine mesh (37 µm). As a control, the cell suspension was directly filtered through the fine mesh (37 µm). Following this filtration, cells retained on the mesh were harvested and designated nonfractionated cells. For each cell fraction, anthocyanin content per dry cell weight was measured by extraction and the average diameter of cell aggregates was measured using an image analysis technique (details are described in the following section). Every experiment was repeated three times and the average values and standard
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errors were calculated. A significance test (One-way ANOVA) was performed using Sigma stat software (Rockware, Golden, CO, USA). Cell culture in medium supplemented with AOPP AOPP, purchased from Wako Pure Chemical Industries (Osaka), was used as a specific inhibitor of the biosynthesis of phenylpropanoid metabolites. Fresh FAR cells (2 g), which were harvested by sieving through a nylon mesh (opening, 37 µm), were inoculated to 100 ml of the LS medium supplemented with 0.1 mM AOPP. For the control culture, cells were inoculated to the LS medium containing no AOPP. For each culture, the dry cell weight and intracellular contents of anthocyanin, tannin and soluble proteins were measured. The average cell-aggregate diameter was also measured. Every experiment was repeated twice and the average values and standard errors were calculated. Analyses of cell samples Cell culture samples were harvested every 2 d for analyses by the suction-filtering of 2 ml of the culture samples using no. 4A filter paper (Advantec, Tokyo). The procedures for measuring cell and anthocyanin concentrations were described in our previous report (12). Tannins To prepare a tannin assay plate, a gel plate containing 0.4% agarose and 1.0 mg/l bovine serum albumin fraction V (Sigma, St. Louis, MO, USA) was prepared on a 9-cm diameter plastic Petri dish. A 4-mm-diameter well was bored at the center of the tannin assay plate. To extract tannins, 4 ml of 70% acetone (acetone:water=70: 30, v/v) was added to 100 mg of the suction-filtered cells and the mixture was allowed to stand overnight at 4°C. The supernatant was collected by centrifugation and was dried under vacuum. The residue was redissolved in 20 µl of distilled water and placed in the well of the plate. After sitting for 3 d at room temperature, a white circle formed around the well. The area of the circle was measured as the amount of tannin in the sample (13). Tannic acid (Wako) was used as a reference. Lignins To estimate lignin deposition onto the cell wall, cell samples are stained with phloroglucinol (Wako), and observed under a microscope (14). Soluble proteins To remove phenolic compounds having an inhibitory effect on the protein assay, 100 mg of the suction-filtered cells was extracted 3 times with 0.5 ml of 80% ethanol (ethanol: water= 80:20, v/v) at 80°C for 20 min. The cell residue was again extracted with 0.5 ml of 0.3 N KOH at room temperature. The extract was assayed using the Bio-Rad protein assay method (Bio-Rad, Hercules, CA, USA) and the soluble protein contents were calculated from a standard curve of bovine serum albumin (BSA) solution. Cell-aggregate size Cell-aggregate size was measured by an image analysis technique. Harvested cells filtered through a nylon mesh (opening 37 µm) were dispersed in 1 ml of distilled water, and a 40-µl aliquot was spread on a slide glass. Cell images were captured by an observation system consisting of an IX7 microscope (Olympus Optical, Tokyo), and a DXC-151 CCD camera (Sony, Tokyo). From the captured images, the cross-sectional area of each cell aggregate was determined using NIH Image (ver. 1.62; National Institutes of Health, Bethesda, MD, USA), and was converted to the equivalent diameter of a spherical object. For each test, at least 1000 aggregates were measured and the average diameter and standard errors were calculated.
RESULTS AND DISCUSSION Large cell aggregates of FAR cells accumulate high level of anthocyanin As a typical secondary metabolite produced from the phenylpropanoid pathway, the natural pigment anthocyanin has been intensively studied. At first, we investigated the relationship between anthocyanin accu-
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FIG. 1. Cellular anthocyanin concentration of size-fractionated FAR cells on day 8. White bar represents smaller cells that passed through the nylon mesh (S-cells), black bar represents larger cells retained on the nylon mesh (L-cells) and gray bar represents nonfractionated cells. L-Cells accumulate a larger amount of anthocyanin than other cell fractions. *Significantly different from nonfractionated cells (p < 0.002). **Significantly different from nonfractionated cells (p<0.001).
mulation and cell-aggregate size. The average diameters of cell aggregates were 390 µm, 200 µm and 336 µm for L-cells, S-cells and nonfractionated cells, respectively. Because the S-cells contained elongated or tabular-shaped cell aggregates rather than spherical ones, it was reasonable that the average diameter of the S-cells was larger than the opening of the mesh (95 µm). The relationship between cell-aggregate size and anthocyanin accumulation is shown in Fig. 1. The anthocyanin concentration of the L-cells was 95% higher (p<0.001) than that of the S-cells and 20% higher (p<0.002) than that of the nonfractionated cells, showing that the large cell aggregates of FAR cells accumulated a high level of anthocyanin. In other studies, it was also reported that a certain size of cell-aggregate (approximately several hundred micrometers) showed the highest anthocyanin accumulation, using grape, carrot and ohelo cells (8, 9, 15). These results indicate a certain relationship between anthocyanin production and cell-aggregate formation. Additionally, Madhusudhan and Ravishankar reported that anthocyanin content decreased when the diameter of cell aggregates exceeded 1 mm (9). This was possibly because cells at the core of cell aggregates could not receive light, which is an important factor in the biosynthesis of anthocyanins and flavonoids (16). Moreover, when the diameter exceeded 3 mm, the oxygen supply to the aggregate core was restricted by both cell respiration and mass transfer resistance (15), which resulted in the inhibition of anthocyanin biosynthesis (16). In this study, the FAR cells used can produce anthocyanin constitutively without light irradiation. Additionally, although it was confirmed that the anthocyanin productivity of FAR cells was decreased when the cells were cultured at a low oxygen concentration (data not shown), the oxygen supply would not be a limiting factor for anthocyanin production FAR in cell aggregates because they do not grow above 500 µm in diameter (data not shown). These experimental results suggest that FAR cells are suitable for investigating the rela-
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tionship between anthocyanin production and aggregate formation in cultured plant cells independently of the external environment, that is, oxygen supply and photoirradiation. AOPP inhibits synthesis of secondary metabolites without reducing cell growth FAR cells accumulate not only anthocyanin but also other phenolic compounds that are considered to be products of the phenylpropanoid pathway up to 10% of the dry cell weight. As described in the previous section, Fig. 1 shows that there is a relationship between cell-aggregate size and cellular anthocyanin concentration. However, in this case, there is a possibility that cellaggregate formation is directly controlled not by anthocyanin but by another phenylpropanoid metabolite which is produced synchronously with anthocyanin. Therefore, we focused on lignin and tannin, two phenolic compounds that are synthesized via the phenylpropanoid pathway and are accumulated in the plant cell wall. Kuboi and Yamada (7) reported that large size cell aggregates accumulated a large amount of lignin, which is deposited in the secondary cell wall and functions as mechanical support for the plant body. However, in the present study, the phloroglucinol test showed that FAR cells did not accumulate lignin in their cell wall. Next, we focused on the functions of tannin for supporting cell-aggregate formation. Condensed tannin (polyleucoanthocyanidin) is a strongly hydrophilic phenolic polymer, which is synthesized from the direct precursor of anthocyanin. It is contained in the cell wall as a filler and binds strongly to polysaccharide which is a main component of the plant cell wall. Tannin has been reported to have a lignin-like action. Bean seeds containing condensed tannin become hard when they are stored at high temperature and humidity for a long time because of the condensation of tannin with cell-wall polysaccharide (17). In several plant species that inhabit arid regions, tannin functions to inhibit the formation of cracks in the cell wall (18). However, the effect of tannin on cell-aggregate formation has not yet been investigated in cultured plant cells. AOPP, an analogue of L-phenylalanine, strongly binds to PAL and inhibits the deamination of L-phenylalanine in a competitive manner (19). However, AOPP has little affinity to L-phenylalanine-tRNA synthetase. This suggests that it does not inhibit the synthesis of the protein (20). We consider that AOPP is suitable for determining the influence of phenylpropanoid metabolites on the formation of cell aggregates because it can totally inhibit the production of phenylpropanoid metabolites without adversely effecting primary metabolism. For this purpose, the biosynthesis of phenylpropanoid metabolites in FAR cells was totally inhibited by AOPP and the cellular concentrations of tannin and anthocyanin were measured. As indicators of primary metabolism, the cell growth and cellular concentration of soluble proteins were also measured. In the culture without AOPP, the cellular concentrations of anthocyanin (Fig. 2) and tannin (Fig. 3) remained higher than the initial concentration (day 0) and significantly increased from days 8 to 10. When AOPP was added to the medium, the concentrations of both decreased significantly and remained at a low level during throughout the culture period. On the other hand, the addition of AOPP had no detectable influence on the time course of cell concentration
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FIG. 2. Time course of cellular concentration of anthocyanin. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition significantly inhibited the anthocyanin production.
FIG. 3. Time course of cellular concentration of tannin. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition significantly inhibited tannin production.
(Fig. 4) and cellular concentration of soluble protein (Fig. 5). These results indicate that FAR cells could produce proteins and proliferate in the presence of AOPP. From the results described above, we confirmed that the addition of AOPP strongly inhibited the biosynthesis of the phenylpropanoid metabolites without adversely effecting primary metabolism. These results suggest that the addition of AOPP into the culture medium of FAR cells is a suitable experimental system for evaluating the direct influence of phenylpropanoid metabolites on cell-aggregate formation. AOPP decreased cell-aggregate size Figure 6 shows the time course of the average cell-aggregate diameter. For the first 4 d of culture, the diameter did not change significantly, in both cultures with and without AOPP. From day 4 to day 8, the diameter increased in the culture without AOPP. On the other hand, in the culture with AOPP, the diameter remained constant throughout the same culture period. From days 8 to 10, the diameter decreased in both cultures with and without AOPP.
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FIG. 4. Time course of cell concentration. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition did not affect cell concentration.
FIG. 5. Time course of cellular concentration of soluble proteins. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition did not inhibit protein production.
FIG. 6. Time course of average aggregate diameter. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition decreased cell-aggregate diameter from days 8 to 10.
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EDAHIRO AND SEKI
Firstly, we would like to emphasize that cell-aggregate diameter increased from days 4 to 8 in the culture without AOPP and that this change was completely inhibited by AOPP addition. These results show for the first time that the inhibition of secondary metabolite production directly induced a decrease in cell-aggregate diameter and indicate that phenylpropanoid metabolites support cell-aggregate formation during this culture period. Additionally, these results also suggest that cell-aggregate size can be controlled by conrolling of phenylpropanoid metabolism with AOPP. As described in the Introduction section, smaller cell aggregates are preferred in suspension culture because of low hydrodynamic stress. This method may be a promising technique for controlling cell-aggregate size in production processes of useful products by cultured plant cells. Secondly, we would like to discuss in detail the relationship between cell-aggregate size and cellular accumulations of anthocyanin, tannin and other phenylpropanoid metabolites. The decrease in cell-aggregate diameter by AOPP from days 4 to 8 of culture (Fig. 6) shows that some phenylpropanoid metabolites support cell-aggregate formation as described previously. However, in the culture without AOPP, the cellular concentrations of anthocyanin (Fig. 2) and tannin (Fig. 3) rapidly increased from days 8 to 10 following the increase in cell-aggregate diameter (from days 4 to 8, in Fig. 6). Additionally, cell-aggregate diameter rapidly decreased from days 8 to 10 (Fig. 6) although cells accumulated high levels of anthocyanin and tannin (Figs. 2 and 3). These results suggest that these metabolites do not support aggregate formation directly. Contrary to the initial hypothesis, the production of these metabolites (anthocyanin and tannin) is promoted as a result of the increase in cell-aggregate size induced by the phenylpropanoid metabolite which supports cell-aggregate formation. On the basis of this mechanism, we will now discuss the effect of AOPP on cell-aggregate size. There was no difference in the size of cell aggregates in culture with and without AOPP up to day 4 of culture (Fig. 6). This result suggests that cells did not accumulate phenylpropanoid metabolites which support cell-aggregate formation for the first 4 d. Additionally, cellaggregate diameter decreased from days 8 to 10 in both cultures with and without AOPP (Fig. 6). Because this change was induced regardless of the presence of AOPP, it was suggested that a certain change was induced in a cell wall component other than a phenylpropanoid metabolite, which resulted in dissociation of the cell aggregate. Kuboi and Yamada (7), and Hanagata et al. (21) investigated the mechanisms of both the formation and dissociation of cell aggregates. They also reported that large cell aggregates released many small aggregates and became smaller at the end of the culture period even when they accumulated a large amount of lignin (7). From the results in this study, we found that phenylpropanoid metabolites support cell-aggregate formation in FAR cells. We examined the relationship between the time course of cell-aggregate size and the cellular accumulation of wellknown phenylpropanoid metabolites, namely, anthocyanin, tannin and lignin. However, the results suggest that these metabolites do not directly affect cell-aggregate formation. The metabolite in question is still unknown. On the other
hand, the results also suggested that the productions of anthocyanin and tannin were induced as the result of increase in cell-aggregate size, contrary to the initial hypothesis. Additionally, for the production of useful products by plant cell suspension, it is important to control cell-aggregate size to prevent cell damage from hydrodynamic stress. We suggest that the use of AOPP, the metabolic inhibitor of the phenylpropanoid pathway, is a promising method to control cellaggregate size. REFERENCES 1. Moreira, J. L., Cruz, P. E., Santana, P. C., Aunins, J. G., and Carrondo, M. J. T.: Formation and disruption of animalcell aggregates in stirred vessels — mechanisms and kineticstudies. Chem. Eng. Sci., 50, 2747–2764 (1995). 2. Dixon, R. A.: Isolation and maintenance of callus and cell suspension cultures, p. 1–20. In Dixon, R. A. (ed.), Plant cell culture. IRL Press, Oxford (1995). 3. King, P. J., Cox, B. J., Fowler, M. W., and Street, H. E.: Metabolic events in synchronized cell cultured of Acer pseudoplatanus L. Planta, 117, 109–122 (1974). 4. Hulst, A. C., Meyer, M. M. T., Breteler, H., and Tramper, J.: Effect of aggregate size in cell-cultures of Tagetes-patula on thiophene production and cell-growth. Appl. Microbiol. Biotechnol., 30, 18–25 (1989). 5. Ping, H. J., Chang, H. W., and Kwong, T. H.: Diffusion-enhanced bioreactions: a hypothetical mechanism for plant cell aggregation. Bull. Math. Biol., 55, 869–889 (1993). 6. Lindsey, K. and Yeoman, M. M.: The relationship between growth rate, differentiation and alkaloid accumulation in cell cultures. J. Exp. Bot., 34, 1055–1065 (1983). 7. Kuboi, T. and Yamada, Y.: Changing cell aggregations and lignification in tobacco suspension cultures. Plant Cell Physiol., 19, 437–443 (1978). 8. Nagamori, E., Hiraoka, K., Honda, H., and Kobayashi, T.: Enhancement of anthocyanin production from grape (Vitis vinifera) callus in a viscous additive-supplemented medium. Biochem. Eng. J., 9, 59–65 (2001). 9. Madhusudhan, R. and Ravishankar, G. A.: Gradient of anthocyanin in cell aggregates of Daucus carota in suspension cultures. Biotechnol. Lett., 18, 1253–1256 (1996). 10. Nakamura, M., Takeuchi, Y., Miyanaga, K., Seki, M., and Furusaki, S.: High anthocyanin accumulation in the dark by strawberry (Fragaria ananassa) callus. Biotechnol. Lett., 21, 695–699 (1999). 11. Linsmaier, E. M. and Skoog, F.: Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18, 100– 127 (1965). 12. Edahiro, J., Nakamura, M., Seki, M., and Furusaki, S.: Enhanced accumulation of anthocyanin in cultured strawberry cells by repetitive feeding of L-phenylalanine into the medium. J. Bioeng. Biosci., 99, 43–47 (2005). 13. Hagerman, A. E.: Radial diffusion method for determining tannin in plant extracts. J. Chem. Ecol., 13, 437–450 (1987). 14. Matsushita, G. and Nakao, J.: Studies on the mechanism of lignin color reaction (XIII). Mokuzai gakkaishi, 25, 588–594 (1979). 15. Pépin, M. F., Smith, M. A. L., and Reid, J. F.: Application of imaging tools to plant cell culture: relationship between plant cell aggregation and flavonoid production. In Vitro Cell. Dev. Biol.-Plant, 35, 290–295 (1999). 16. Zhong, J. J., Yoshida, M., Fujiyama, K., Seki, T., and Yoshida, T.: Enhancement of anthocyanin production by Perilla-frutescens cells in a stirred bioreactor with internal light irradiation. J. Ferment. Bioeng., 75, 299–303 (1993). 17. Stanley, D. W.: A possible role for condensed tannins in bean
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hardening. Food Res. Int., 25, 187–192 (1992). 18. Pizzi, A. and Cameron, F. A.: Flavonoid tannins-structural wood components for drought-resistance mechanisms of plants. Wood Sci. Technol., 20, 119–124 (1986). 19. Hanson, K. R.: Phenylalanine ammonia-lyase: mirror-image packing of D- and L-phenylalanine and D- and L-transition state analogs into the active site. Arch. Biochem. Biophys., 211, 575–588 (1981).
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20. Leubnermetzger, G. and Amrhein, N.: Phenylalanine analogs — potent inhibitors of phenylalanine ammonia-lyase are weak inhibitors of phenylalanine-transfer-RNA synthetases. Z. Naturforsch. C, 49, 781–790 (1994). 21. Hanagata, N., Ito, A., Uehara, H., Asari, F., Takeuchi, T., and Karube, I.: Behavior of cell aggregate of Carthamustinctorius L. cultured-cells and correlation with red pigment formation. J. Biotechnol., 30, 259–269 (1993).
© 2006, The Society for Biotechnology, Japan
Phenylpropanoid Metabolite Supports Cell Aggregate Formation in Strawberry Cell Suspension Culture Jun-ichi Edahiro1,2 and Minoru Seki1,3* Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan,1 Research Center of Advanced Bionics, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan,2 and Department of Chemical Engineering, Graduate School of Engineering, Osaka Prefecture University, 1-1 Gakuen-cho, Sakai, Osaka 599-8531, Japan 3 Received 24 October 2005/Accepted 10 February 2006
Plant cells in suspension culture tend to aggregate and form large clumps. In suspension culture, large cell aggregates are frequently subjected to hydrodynamic shear stress; however, a certain degree of cell aggregation is often required for cell growth and metabolite production. Thus, controlling cell-aggregate size is desired to establish high productivity of useful products using plant cell suspension culture. In this study, we focused on the relationship between cell-aggregate formation and secondary metabolism. We found that anthocyanin concentration showed a good correlation with cell-aggregate size in the cultured strawberry cell line FAR (Fragaria ananassa R), which produces anthocyanin and other phenylpropanoid metabolites constitutively without illumination. This result suggests that there is a relationship between cell-aggregate formation and the accumulation of phenylpropanoid metabolites. To investigate the direct effect of phenylpropanoid metabolism on cell-aggregate formation, the time course of cell-aggregate size was monitored when phenylpropanoid metabolism was suppressed by a metabolic inhibitor, L-α-aminooxyβ-phenylpropionic acid (AOPP), a specific inhibitor of phenylalanine ammonia lyase which is the starting and key enzyme of the phenylpropanoid pathway. In the absence of AOPP, the average diameter of cell aggregates increased on day 8 of culture. This increase in cell-aggregate size was completely suppressed by the addition of 0.1 mM AOPP, without any reduction in cell growth rate or soluble protein content. These results indicate that cell-aggregate formation is directly supported by a secondary metabolite produced from the phenylpropanoid pathway, suggesting that cell-aggregate size can be controlled by AOPP without inhibition of primary metabolism. [Key words: plant cultured cell, Fragaria ananassa, cell aggregate, phenylpropanoid metabolism, secondary metabolism, anthocyanin, phenylalanine ammonia lyase (PAL), L-α-aminooxy-β-phenylpropionic acid (AOPP)]
Hulst et al. reported that secondary metabolite production increased with increasing cell-aggregate diameter starting at several millimeters (4). Their study showed that high mass transfer resistance caused by large particle size induced a lack of oxygen in the center of the cell aggregate, which resulted in the promotion of secondary metabolite production. In another plant cell type, Ping et al. suggested that the existence of diffusional resistance around cell aggregates due to the presence of a solid phase could hinder some intracellular substrates diffusing into the medium, which resulted in the activation of metabolic reactions (5). Because plants are multicellular organisms, a certain degree of intercellular transport of physiologically active substances is often required for the synthesis of secondary metabolites (6). In the studies described above, the formation of a certain size of cell aggregate is required for secondary metabolism. In these situations, it is desired to establish a technique for controlling cell-aggregate size, while maintaining high productivity of the target metabolite.
Recently, plant cell cultures have been studied actively as a potential source of high-value biological products. Plant cells in suspension cultures tend to aggregate and form cell clusters ranging from a few cells to several thousands of cells, often reaching a few centimeters in diameter. A large cell aggregate is frequently subjected to hydrodynamic shear stress in suspension culture (1). Therefore, smaller cell aggregates are preferred from the viewpoint of process engineering. To reduce the size of cell aggregates in suspension cultures, several methods have been examined, such as the repetitive selection of small cell aggregates (2) and the addition of cell-wall degrading enzymes to the medium (3). On the other hand, a certain degree of aggregation is often required for cellular metabolism (4). In some studies, cellaggregate formation induced secondary metabolite production, which resulted in increased productivity. For example, * Corresponding author. e-mail: [email protected] phone: +81-(0)72-254-9296 fax: +81-(0)72-254-9911 8
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Cell-aggregate size is governed by the cohesiveness of the cell wall. The cell wall is considered as a composite material made of fiber (cellulose), a matrix (lignin, hemicellulose) and fillers (water, tannins). Both lignin and tannin are secondary metabolites synthesized from the phenylpropanoid pathway, which is a typical pathway in plant secondary metabolism. As these secondary metabolites have a function to increase cellular cohesiveness, it is reasonable that large cell aggregates accumulate a large amount of secondary metabolites related to these compounds. In the case of lignin, a correlation between cell-aggregate size and lignification was reported (7). Several researchers have also reported that a certain size of cell aggregate was required for the cellular accumulation of anthocyanin (8, 9), which is a natural pigment and is synthesized from the phenylpropanoid pathway similar to lignin and tannin. However, to prove the hypothesis that the accumulation of these secondary metabolites promotes cellular cohesiveness and increases aggregate size, it is desired that a direct and definite relationship between the accumulation of these metabolites and the formation of cell aggregates by inhibiting their biosynthesis by a metabolic inhibitor be shown. Phenylpropanoid metabolites were produced from phenylalanine ammonia lyase (PAL; EC 4.3.1.5), which is the starting and key enzyme of the phenylpropanoid pathway. Therefore, it was assumed that L-α-aminooxy-β-phenylpropionic acid (AOPP), a specific inhibitor of PAL, would prove useful in this case. In this study, we investigated the correlation between secondary metabolites synthesized from the phenylpropanoid pathway and cell-aggregate size in cultured strawberry cells producing anthocyanin constitutively. By applying AOPP, a specific inhibitor of PAL, we evaluated the direct effect of secondary metabolism on cell-aggregate formation without affecting primary metabolism. MATERIALS AND METHODS Plant materials and culture conditions Cultured strawberry cells, designated FAR, which were derived from the petioles of Fragaria ananassa cv. Shikinari, were used in this study. FAR cells can produce anthocyanin constitutively without light irradiation (10). A suspension culture of FAR was subcultured every 14 d at 25°C in Linsmaier–Skoog medium (11) (abbreviated as LS medium) containing 1 mg/l 2,4-dichlorophenoxyacetic acid, 0.1 mg/l benzylaminopurine, and 0.4 mg/l thiamine–HCl. Cell fractionation by aggregate size Cells harvested from an 8-d-cultured cell suspension were partitioned into two fractions by sieving through nylon mesh as described below. The cell suspension was filtered through a nylon mesh (opening, 95 µm) and the filtrate was refiltered through a nylon mesh with a smaller opening size (37 µm). The first cell fraction (designated L-cells) contained large cell aggregates that were retained on the rough mesh (95 µm). The other fraction had smaller cell aggregates (S-cells) which passed through the rough mesh (95 µm) and were retained on the fine mesh (37 µm). As a control, the cell suspension was directly filtered through the fine mesh (37 µm). Following this filtration, cells retained on the mesh were harvested and designated nonfractionated cells. For each cell fraction, anthocyanin content per dry cell weight was measured by extraction and the average diameter of cell aggregates was measured using an image analysis technique (details are described in the following section). Every experiment was repeated three times and the average values and standard
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errors were calculated. A significance test (One-way ANOVA) was performed using Sigma stat software (Rockware, Golden, CO, USA). Cell culture in medium supplemented with AOPP AOPP, purchased from Wako Pure Chemical Industries (Osaka), was used as a specific inhibitor of the biosynthesis of phenylpropanoid metabolites. Fresh FAR cells (2 g), which were harvested by sieving through a nylon mesh (opening, 37 µm), were inoculated to 100 ml of the LS medium supplemented with 0.1 mM AOPP. For the control culture, cells were inoculated to the LS medium containing no AOPP. For each culture, the dry cell weight and intracellular contents of anthocyanin, tannin and soluble proteins were measured. The average cell-aggregate diameter was also measured. Every experiment was repeated twice and the average values and standard errors were calculated. Analyses of cell samples Cell culture samples were harvested every 2 d for analyses by the suction-filtering of 2 ml of the culture samples using no. 4A filter paper (Advantec, Tokyo). The procedures for measuring cell and anthocyanin concentrations were described in our previous report (12). Tannins To prepare a tannin assay plate, a gel plate containing 0.4% agarose and 1.0 mg/l bovine serum albumin fraction V (Sigma, St. Louis, MO, USA) was prepared on a 9-cm diameter plastic Petri dish. A 4-mm-diameter well was bored at the center of the tannin assay plate. To extract tannins, 4 ml of 70% acetone (acetone:water=70: 30, v/v) was added to 100 mg of the suction-filtered cells and the mixture was allowed to stand overnight at 4°C. The supernatant was collected by centrifugation and was dried under vacuum. The residue was redissolved in 20 µl of distilled water and placed in the well of the plate. After sitting for 3 d at room temperature, a white circle formed around the well. The area of the circle was measured as the amount of tannin in the sample (13). Tannic acid (Wako) was used as a reference. Lignins To estimate lignin deposition onto the cell wall, cell samples are stained with phloroglucinol (Wako), and observed under a microscope (14). Soluble proteins To remove phenolic compounds having an inhibitory effect on the protein assay, 100 mg of the suction-filtered cells was extracted 3 times with 0.5 ml of 80% ethanol (ethanol: water= 80:20, v/v) at 80°C for 20 min. The cell residue was again extracted with 0.5 ml of 0.3 N KOH at room temperature. The extract was assayed using the Bio-Rad protein assay method (Bio-Rad, Hercules, CA, USA) and the soluble protein contents were calculated from a standard curve of bovine serum albumin (BSA) solution. Cell-aggregate size Cell-aggregate size was measured by an image analysis technique. Harvested cells filtered through a nylon mesh (opening 37 µm) were dispersed in 1 ml of distilled water, and a 40-µl aliquot was spread on a slide glass. Cell images were captured by an observation system consisting of an IX7 microscope (Olympus Optical, Tokyo), and a DXC-151 CCD camera (Sony, Tokyo). From the captured images, the cross-sectional area of each cell aggregate was determined using NIH Image (ver. 1.62; National Institutes of Health, Bethesda, MD, USA), and was converted to the equivalent diameter of a spherical object. For each test, at least 1000 aggregates were measured and the average diameter and standard errors were calculated.
RESULTS AND DISCUSSION Large cell aggregates of FAR cells accumulate high level of anthocyanin As a typical secondary metabolite produced from the phenylpropanoid pathway, the natural pigment anthocyanin has been intensively studied. At first, we investigated the relationship between anthocyanin accu-
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EDAHIRO AND SEKI
FIG. 1. Cellular anthocyanin concentration of size-fractionated FAR cells on day 8. White bar represents smaller cells that passed through the nylon mesh (S-cells), black bar represents larger cells retained on the nylon mesh (L-cells) and gray bar represents nonfractionated cells. L-Cells accumulate a larger amount of anthocyanin than other cell fractions. *Significantly different from nonfractionated cells (p < 0.002). **Significantly different from nonfractionated cells (p<0.001).
mulation and cell-aggregate size. The average diameters of cell aggregates were 390 µm, 200 µm and 336 µm for L-cells, S-cells and nonfractionated cells, respectively. Because the S-cells contained elongated or tabular-shaped cell aggregates rather than spherical ones, it was reasonable that the average diameter of the S-cells was larger than the opening of the mesh (95 µm). The relationship between cell-aggregate size and anthocyanin accumulation is shown in Fig. 1. The anthocyanin concentration of the L-cells was 95% higher (p<0.001) than that of the S-cells and 20% higher (p<0.002) than that of the nonfractionated cells, showing that the large cell aggregates of FAR cells accumulated a high level of anthocyanin. In other studies, it was also reported that a certain size of cell-aggregate (approximately several hundred micrometers) showed the highest anthocyanin accumulation, using grape, carrot and ohelo cells (8, 9, 15). These results indicate a certain relationship between anthocyanin production and cell-aggregate formation. Additionally, Madhusudhan and Ravishankar reported that anthocyanin content decreased when the diameter of cell aggregates exceeded 1 mm (9). This was possibly because cells at the core of cell aggregates could not receive light, which is an important factor in the biosynthesis of anthocyanins and flavonoids (16). Moreover, when the diameter exceeded 3 mm, the oxygen supply to the aggregate core was restricted by both cell respiration and mass transfer resistance (15), which resulted in the inhibition of anthocyanin biosynthesis (16). In this study, the FAR cells used can produce anthocyanin constitutively without light irradiation. Additionally, although it was confirmed that the anthocyanin productivity of FAR cells was decreased when the cells were cultured at a low oxygen concentration (data not shown), the oxygen supply would not be a limiting factor for anthocyanin production FAR in cell aggregates because they do not grow above 500 µm in diameter (data not shown). These experimental results suggest that FAR cells are suitable for investigating the rela-
J. BIOSCI. BIOENG.,
tionship between anthocyanin production and aggregate formation in cultured plant cells independently of the external environment, that is, oxygen supply and photoirradiation. AOPP inhibits synthesis of secondary metabolites without reducing cell growth FAR cells accumulate not only anthocyanin but also other phenolic compounds that are considered to be products of the phenylpropanoid pathway up to 10% of the dry cell weight. As described in the previous section, Fig. 1 shows that there is a relationship between cell-aggregate size and cellular anthocyanin concentration. However, in this case, there is a possibility that cellaggregate formation is directly controlled not by anthocyanin but by another phenylpropanoid metabolite which is produced synchronously with anthocyanin. Therefore, we focused on lignin and tannin, two phenolic compounds that are synthesized via the phenylpropanoid pathway and are accumulated in the plant cell wall. Kuboi and Yamada (7) reported that large size cell aggregates accumulated a large amount of lignin, which is deposited in the secondary cell wall and functions as mechanical support for the plant body. However, in the present study, the phloroglucinol test showed that FAR cells did not accumulate lignin in their cell wall. Next, we focused on the functions of tannin for supporting cell-aggregate formation. Condensed tannin (polyleucoanthocyanidin) is a strongly hydrophilic phenolic polymer, which is synthesized from the direct precursor of anthocyanin. It is contained in the cell wall as a filler and binds strongly to polysaccharide which is a main component of the plant cell wall. Tannin has been reported to have a lignin-like action. Bean seeds containing condensed tannin become hard when they are stored at high temperature and humidity for a long time because of the condensation of tannin with cell-wall polysaccharide (17). In several plant species that inhabit arid regions, tannin functions to inhibit the formation of cracks in the cell wall (18). However, the effect of tannin on cell-aggregate formation has not yet been investigated in cultured plant cells. AOPP, an analogue of L-phenylalanine, strongly binds to PAL and inhibits the deamination of L-phenylalanine in a competitive manner (19). However, AOPP has little affinity to L-phenylalanine-tRNA synthetase. This suggests that it does not inhibit the synthesis of the protein (20). We consider that AOPP is suitable for determining the influence of phenylpropanoid metabolites on the formation of cell aggregates because it can totally inhibit the production of phenylpropanoid metabolites without adversely effecting primary metabolism. For this purpose, the biosynthesis of phenylpropanoid metabolites in FAR cells was totally inhibited by AOPP and the cellular concentrations of tannin and anthocyanin were measured. As indicators of primary metabolism, the cell growth and cellular concentration of soluble proteins were also measured. In the culture without AOPP, the cellular concentrations of anthocyanin (Fig. 2) and tannin (Fig. 3) remained higher than the initial concentration (day 0) and significantly increased from days 8 to 10. When AOPP was added to the medium, the concentrations of both decreased significantly and remained at a low level during throughout the culture period. On the other hand, the addition of AOPP had no detectable influence on the time course of cell concentration
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FIG. 2. Time course of cellular concentration of anthocyanin. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition significantly inhibited the anthocyanin production.
FIG. 3. Time course of cellular concentration of tannin. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition significantly inhibited tannin production.
(Fig. 4) and cellular concentration of soluble protein (Fig. 5). These results indicate that FAR cells could produce proteins and proliferate in the presence of AOPP. From the results described above, we confirmed that the addition of AOPP strongly inhibited the biosynthesis of the phenylpropanoid metabolites without adversely effecting primary metabolism. These results suggest that the addition of AOPP into the culture medium of FAR cells is a suitable experimental system for evaluating the direct influence of phenylpropanoid metabolites on cell-aggregate formation. AOPP decreased cell-aggregate size Figure 6 shows the time course of the average cell-aggregate diameter. For the first 4 d of culture, the diameter did not change significantly, in both cultures with and without AOPP. From day 4 to day 8, the diameter increased in the culture without AOPP. On the other hand, in the culture with AOPP, the diameter remained constant throughout the same culture period. From days 8 to 10, the diameter decreased in both cultures with and without AOPP.
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FIG. 4. Time course of cell concentration. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition did not affect cell concentration.
FIG. 5. Time course of cellular concentration of soluble proteins. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition did not inhibit protein production.
FIG. 6. Time course of average aggregate diameter. Circles represent the culture without AOPP and triangles represent the culture supplemented with AOPP at a concentration of 0.1 mM. AOPP addition decreased cell-aggregate diameter from days 8 to 10.
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EDAHIRO AND SEKI
Firstly, we would like to emphasize that cell-aggregate diameter increased from days 4 to 8 in the culture without AOPP and that this change was completely inhibited by AOPP addition. These results show for the first time that the inhibition of secondary metabolite production directly induced a decrease in cell-aggregate diameter and indicate that phenylpropanoid metabolites support cell-aggregate formation during this culture period. Additionally, these results also suggest that cell-aggregate size can be controlled by conrolling of phenylpropanoid metabolism with AOPP. As described in the Introduction section, smaller cell aggregates are preferred in suspension culture because of low hydrodynamic stress. This method may be a promising technique for controlling cell-aggregate size in production processes of useful products by cultured plant cells. Secondly, we would like to discuss in detail the relationship between cell-aggregate size and cellular accumulations of anthocyanin, tannin and other phenylpropanoid metabolites. The decrease in cell-aggregate diameter by AOPP from days 4 to 8 of culture (Fig. 6) shows that some phenylpropanoid metabolites support cell-aggregate formation as described previously. However, in the culture without AOPP, the cellular concentrations of anthocyanin (Fig. 2) and tannin (Fig. 3) rapidly increased from days 8 to 10 following the increase in cell-aggregate diameter (from days 4 to 8, in Fig. 6). Additionally, cell-aggregate diameter rapidly decreased from days 8 to 10 (Fig. 6) although cells accumulated high levels of anthocyanin and tannin (Figs. 2 and 3). These results suggest that these metabolites do not support aggregate formation directly. Contrary to the initial hypothesis, the production of these metabolites (anthocyanin and tannin) is promoted as a result of the increase in cell-aggregate size induced by the phenylpropanoid metabolite which supports cell-aggregate formation. On the basis of this mechanism, we will now discuss the effect of AOPP on cell-aggregate size. There was no difference in the size of cell aggregates in culture with and without AOPP up to day 4 of culture (Fig. 6). This result suggests that cells did not accumulate phenylpropanoid metabolites which support cell-aggregate formation for the first 4 d. Additionally, cellaggregate diameter decreased from days 8 to 10 in both cultures with and without AOPP (Fig. 6). Because this change was induced regardless of the presence of AOPP, it was suggested that a certain change was induced in a cell wall component other than a phenylpropanoid metabolite, which resulted in dissociation of the cell aggregate. Kuboi and Yamada (7), and Hanagata et al. (21) investigated the mechanisms of both the formation and dissociation of cell aggregates. They also reported that large cell aggregates released many small aggregates and became smaller at the end of the culture period even when they accumulated a large amount of lignin (7). From the results in this study, we found that phenylpropanoid metabolites support cell-aggregate formation in FAR cells. We examined the relationship between the time course of cell-aggregate size and the cellular accumulation of wellknown phenylpropanoid metabolites, namely, anthocyanin, tannin and lignin. However, the results suggest that these metabolites do not directly affect cell-aggregate formation. The metabolite in question is still unknown. On the other
hand, the results also suggested that the productions of anthocyanin and tannin were induced as the result of increase in cell-aggregate size, contrary to the initial hypothesis. Additionally, for the production of useful products by plant cell suspension, it is important to control cell-aggregate size to prevent cell damage from hydrodynamic stress. We suggest that the use of AOPP, the metabolic inhibitor of the phenylpropanoid pathway, is a promising method to control cellaggregate size. REFERENCES 1. Moreira, J. L., Cruz, P. E., Santana, P. C., Aunins, J. G., and Carrondo, M. J. T.: Formation and disruption of animalcell aggregates in stirred vessels — mechanisms and kineticstudies. Chem. Eng. Sci., 50, 2747–2764 (1995). 2. Dixon, R. A.: Isolation and maintenance of callus and cell suspension cultures, p. 1–20. In Dixon, R. A. (ed.), Plant cell culture. IRL Press, Oxford (1995). 3. King, P. J., Cox, B. J., Fowler, M. W., and Street, H. E.: Metabolic events in synchronized cell cultured of Acer pseudoplatanus L. Planta, 117, 109–122 (1974). 4. Hulst, A. C., Meyer, M. M. T., Breteler, H., and Tramper, J.: Effect of aggregate size in cell-cultures of Tagetes-patula on thiophene production and cell-growth. Appl. Microbiol. Biotechnol., 30, 18–25 (1989). 5. Ping, H. J., Chang, H. W., and Kwong, T. H.: Diffusion-enhanced bioreactions: a hypothetical mechanism for plant cell aggregation. Bull. Math. Biol., 55, 869–889 (1993). 6. Lindsey, K. and Yeoman, M. M.: The relationship between growth rate, differentiation and alkaloid accumulation in cell cultures. J. Exp. Bot., 34, 1055–1065 (1983). 7. Kuboi, T. and Yamada, Y.: Changing cell aggregations and lignification in tobacco suspension cultures. Plant Cell Physiol., 19, 437–443 (1978). 8. Nagamori, E., Hiraoka, K., Honda, H., and Kobayashi, T.: Enhancement of anthocyanin production from grape (Vitis vinifera) callus in a viscous additive-supplemented medium. Biochem. Eng. J., 9, 59–65 (2001). 9. Madhusudhan, R. and Ravishankar, G. A.: Gradient of anthocyanin in cell aggregates of Daucus carota in suspension cultures. Biotechnol. Lett., 18, 1253–1256 (1996). 10. Nakamura, M., Takeuchi, Y., Miyanaga, K., Seki, M., and Furusaki, S.: High anthocyanin accumulation in the dark by strawberry (Fragaria ananassa) callus. Biotechnol. Lett., 21, 695–699 (1999). 11. Linsmaier, E. M. and Skoog, F.: Organic growth factor requirements of tobacco tissue cultures. Physiol. Plant., 18, 100– 127 (1965). 12. Edahiro, J., Nakamura, M., Seki, M., and Furusaki, S.: Enhanced accumulation of anthocyanin in cultured strawberry cells by repetitive feeding of L-phenylalanine into the medium. J. Bioeng. Biosci., 99, 43–47 (2005). 13. Hagerman, A. E.: Radial diffusion method for determining tannin in plant extracts. J. Chem. Ecol., 13, 437–450 (1987). 14. Matsushita, G. and Nakao, J.: Studies on the mechanism of lignin color reaction (XIII). Mokuzai gakkaishi, 25, 588–594 (1979). 15. Pépin, M. F., Smith, M. A. L., and Reid, J. F.: Application of imaging tools to plant cell culture: relationship between plant cell aggregation and flavonoid production. In Vitro Cell. Dev. Biol.-Plant, 35, 290–295 (1999). 16. Zhong, J. J., Yoshida, M., Fujiyama, K., Seki, T., and Yoshida, T.: Enhancement of anthocyanin production by Perilla-frutescens cells in a stirred bioreactor with internal light irradiation. J. Ferment. Bioeng., 75, 299–303 (1993). 17. Stanley, D. W.: A possible role for condensed tannins in bean
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hardening. Food Res. Int., 25, 187–192 (1992). 18. Pizzi, A. and Cameron, F. A.: Flavonoid tannins-structural wood components for drought-resistance mechanisms of plants. Wood Sci. Technol., 20, 119–124 (1986). 19. Hanson, K. R.: Phenylalanine ammonia-lyase: mirror-image packing of D- and L-phenylalanine and D- and L-transition state analogs into the active site. Arch. Biochem. Biophys., 211, 575–588 (1981).
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20. Leubnermetzger, G. and Amrhein, N.: Phenylalanine analogs — potent inhibitors of phenylalanine ammonia-lyase are weak inhibitors of phenylalanine-transfer-RNA synthetases. Z. Naturforsch. C, 49, 781–790 (1994). 21. Hanagata, N., Ito, A., Uehara, H., Asari, F., Takeuchi, T., and Karube, I.: Behavior of cell aggregate of Carthamustinctorius L. cultured-cells and correlation with red pigment formation. J. Biotechnol., 30, 259–269 (1993).