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(Last modification: 04. Mar. 2009)

 

Flavonoids (Anthocyanins and Flavonols) in Catharanthus roseus

 

 

Recent reviews

  • Mustafa, N. R., Verpoorte, R., 2007. Phenolic compounds in Catharanthus roseus. Phytochemistry Reviews 6, 243-258.

  • Piovan, A., Filippini, R., 2007. Anthocyanins in Catharanthus roseus in vivo and in vitro: A review. Phytochemistry Reviews 6, 235-242.

Flavonoids in C. roseus

  • Anthocyanins:
    Forsyth and Simmonds, 1957; Carew and Krueger, 1976; Knobloch et al., 1982; Milo et al., 1985; Piovan et al., 1998; Filippini et al., 2003

  • Flavonols:
    Forsyth and Simmonds, 1957; Nishibe et al., 1996; Brun et al., 1999


 

Flavonoids in Cell Cultures

 

     One of the interesting questions is whether cell cultures (callus or suspension) can synthesize the same anthocyanins as intact plants. Although the experiments could not be carried out with the sophistication as possible today, already the first early experiments showed that the formation of the backbones characteristic for C. roseus (petunidin, malvidin, hirsutidin) was possible in callus cultures, and that it was induced by light (Carew and Krueger, 1976). Comparable experiments with dark-grown suspension cultures showed that the cultures turned red after dilution into a 8% sucrose solution and irradiation, and the compounds were the same as in the flowers, although the ratio was changed a bit: in petals, hirsutidin was the prominent compound, while the cultures contained mostly malvidin and petunidin (Knobloch et al., 1982). Look at the figure above: It seems that there is a difference in the expression of the O-methyltransferases neccessary for the various methylation steps. Later work demonstrated that the anthocyanic profiles of flowers from both field-grown and in vitro regenerated plants were qualitatively similar (Piovan et al., 1998).
     Some interesting finding with the cell suspension cultures: a closer inspection of the induced cell cultures revealed that only about 5% of the cells turned red, indicating that the cells were very heterogeneous in their response (Knobloch et al., 1982). This was confirmed in other experiments, and these also suggested a delay of anthocyanin formation under certain conditions: in the lag phase of growth (the period directly after dilution of the culture into fresh medium) and in the cell division phase (the period after the initial lag phase of growth); the majority of the anthocyanins was accumulated after these stages (i.e. in the phase when the increase of the culture mass mainly reflects cell enlargement by water uptake, not increase of cell numbers) (Hall and Yeoman, 1986b). Additional studies confirmed that only a certain percentage of the cells (about 10%) was productive in anthocyanin formation (Hall and Yeoman, 1986a; 1987), and this was also shown in even more detailed later studies (Filippini et al., 2003).
The mechanism of partial differentation with respect to capacity of flavonoid biosynthesis remained unclear.

    Other experiments investigated the effects of various chemicals. One of the studies (Godoy-Hernández and Loyola-Vargas, 1997) found drastic increases in total anthocyanins (1476% !) after application of 20 mM acetylsalicylic acid (Aspirin), but it seems a bit difficult to see a physiological relevance at such high concentration of the chemical. The same might be argued for experiments with nicotinamide concentrations of 8.2 mM (Berglund et al., 1993). Another study showed that gibberellic acid (GA3) led to an anthocyanin increase (Ohlsson and Berglund, 2001).

 


 

What do we know from our work about the enzymes in flavonoid biosynthesis in C. roseus?

  • Hotze et al. (1995) cloned and characterized the cytochrome P450 cinnamic acid 4-hydroxlase (C4H) and reproduced the cloning of the previously described P450 reductase (Meijer et al., 1993) that is necessary as electron donor in the P450 reaction. Both enzymes were always present in the cell cultures, also when the cells were kept in the dark. The main purpose of that work, however, was to establish a relatively simple method for functional P450 expression in E. coli: the construction of enzymatically active fusion proteins containing a cytochrome P450 fused to the P450 reductase: more...

  • Kaltenbach et al. (1999) described the molecular characterization of flavonoid 3',5'-hydroxylase (F3'5'H, a cytochrome P450) and chalcone synthase, a detailed functional analysis of the F3'5'H and the tissue-specific expression in plants: more... Just for the context discussed here: the data showed that F3'5'H and flavanone hydroxylase (FHT) are present in dark-grown cell suspension cultures; and they were further induced by irradiation. Note: the FHT is not discussed here, but in the discussion of the F3'5'H (more...).
    An important information from those investigations: The bottle-neck in flavonoid synthesis  in cell cultures was chalcone synthase: it was completely absent in dark-grown cultures, and had to be induced by irradiation.

  • Cacace et al. (2003) reported the molecular and functional analysis of a a new type of O-methyltransferase OMT that performs two sequential methylations at the 3'- and 5'-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol). The resulting methylation pattern is characteristic for the anthocyanins with malvidin and hirsutidin backbone and the syringetin derivative in C. roseus (see Figure above). Like F3'5'H and flavanone hydroxylase, the OMT showed a quite high expression in dark-grown cell cultures.

  • Schröder et al. (2004) described an O-methyltransferase that was not expected in C. roseus: We were looking for the 7-OMT postulated in the biosynthesis of hirsutidin (see top of page for the structure), but the enzyme characterized here, accepting 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols as substrates, performed a methylation in the 4'-position of the B-ring. The resulting flavonoids with vicinal O-methyl groups at the B-ring have not yet been described from C. roseus, and the role of this OMT in vivo is still an enigma. Interestingly, the expression of this OMT ("OMT6") was very low in dark-grown cultures, but it was strongly induced by light. The significance remains unclear as well.

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References

 

Flavonoids in C. roseus

  • Brun, G., Dijoux, M. G., David, B., Mariotte, A. M., 1999. A new flavonol glycoside from Catharanthus roseus. Phytochemistry 50, 167-169.

  • Carew, D. P., Krueger, R. J., 1976. Anthocyanidins of Catharanthus roseus callus cultures. Phytochemistry 15, 442-

  • Filippini, R., Caniato, R., Piovan, A., Cappelletti, E. M., 2003. Production of anthocyanins by Catharanthus roseus. Fitoterapia 74, 62-67.

  • Forsyth, W. G. C., Simmonds, N. W., 1957. Anthocyanidins of Lochnera rosea. Nature 180, 247-

  • Knobloch, K.-H., Bast, G., Berlin, J., 1982. Medium- and light-induced formation of serpentine and anthocyanins in cell suspension cultures of Catharanthus roseus. Phytochemistry 21, 591-594.

  • Milo, J., Levy, A., Akavia, N., Ashri, A., Palevitch, D., 1985. Inheritance of corolla color and anthocyanin pigments in periwinkle (Catharanthus roseus [L.] G. Don). Zeitschrift für Pflanzenzüchtung 95, 352-360.

  • Nishibe, S., Takenaka, T., Fujikawa, T., Yasukawa, K., Takido, M., Morimitsu, Y., Hirota, A., Kawamura, T., Noro, Y., 1996. Bioactive phenolic compounds from Catharanthus roseus and Vinca minor. Natural Medicines (Tokyo) 50, 378-383.

  • Piovan, A., Filippini, R., Favretto, D., 1998. Characterization of the anthocyanins of Catharanthus roseus (L.) G. Don in vivo and in vitro by electrospray ionization ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 12, 361-367.
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Flavonoids and enzymes in cell cultures

  • Berglund, T., Ohlsson, A. B., Rydström, J., 1993. Nicotinamide increases glutathione and anthocyanin in tissue culture of Catharanthus roseus. Journal of Plant Physiology 141, 596-600.
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  • Cacace, S., Schröder, G., Wehinger, E., Strack, D., Schmidt, J., Schröder, J., 2003. A flavonol O-methyltransferase from Catharanthus roseus performing two sequential methylations. Phytochemistry 62, 127-137.
        Protein extracts from dark-grown cell suspension cultures of Catharanthus roseus (Madagascar periwinkle) contained several O-methyltransferase activities, including the 16-hydroxytabersonine O-methyltransferase (16HT-OMT) in indole alkaloid biosynthesis. This enzyme was enriched through several purification steps, including affinity chromatography on adenosine agarose. SDS-PAGE of the purified protein preparation revealed a protein band at the size expected for plant OMTs (38-43 kDa). Mass spectrometry indicated two dominant protein species of similar mass in this band, and sequences of tryptic peptides showed similarities to known OMTs. Homology-based RT-PCR identified cDNAs for four new OMTs. Two of these cDNAs (CrOMT2 and CrOMT4) encoded the proteins dominant in the preparation enriched for 16HT-OMT. The proteins were closely related (73% identity), but both shared only 48-53% identity with the closest relatives found in the public databases. The enzyme functions were investigated with purified recombinant proteins after cDNA expression in E. coli. Unexpectedly, both proteins had no detectable 16HT-OMT activity, and CrOMT4 was inactive with all substrates investigated. CrOMT2 was identified as a flavonoid OMT that was expressed in dark-grown cell cultures and copurified with 16HT-OMT. It represented a new type of OMT that performs two sequential methylations at the 3'- and 5'-positions of the B-ring in myricetin (flavonol) and dihydromyricetin (dihydroflavonol). The resulting methylation pattern is characteristic for C. roseus flavonol glycosides and anthocyanins, and it is proposed that CrOMT2 is involved in their biosynthesis.
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  • Carew, D. P., Krueger, R. J., 1976. Anthocyanidins of Catharanthus roseus callus cultures. Phytochemistry 15, 442
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  • Filippini, R., Caniato, R., Piovan, A., Cappelletti, E. M., 2003. Production of anthocyanins by Catharanthus roseus. Fitoterapia 74, 62-67.
    Return to text

  • Godoy-Hernández, G., Loyola-Vargas, V. M., 1997. Effect of acetylsalicylic acid on secondary metabolism of Catharanthus roseus tumor suspension cultures. Plant Cell Reports 16, 287-290.
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  • Hall, R. D., Yeoman, M. M., 1986a. Factors determining anthocyanin yield in cell cultures of Catharanthus roseus (L.) G. Don. New Phytologist 103, 33-43.
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  • Hall, R. D., Yeoman, M. M., 1986b. Temporal and spatial heterogeneity in the accumulation of anthocyanins in cell cultures of Catharanthus roseus (L.) G.Don. Journal of Experimental Botany 37, 48-60.
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  • Hall, R. D., Yeoman, M. M., 1987. Intercellular and intercultural heterogeneity in secondary metabolite accumulation in cultures of Catharanthus roseus following cell line selection. Journal of Experimental Botany 38, 1391-1398.
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  • Hotze, M., Schröder, G., Schröder, J., 1995. Cinnamate 4-hydroxylase from Catharanthus roseus, and a strategy for the functional expression of plant cytochrome P450 proteins as translational fusions with P450 reductase in Escherichia coli. FEBS Letters 374, 345-350.
         A PCR-based approach was used to isolate cDNAs for cinnamate 4-hydroxylase (C4H, CYP73) from Catharanthus roseus cell cultures. The protein shared 75.9% identity with C4H from other plants, and the transcription was induced under various stress conditions. The cloned protein was used to investigate the functional expression of plant P450/P450-reductase fusions in E. coli. Fusions containing a modified N-terminal membrane anchor were located in the membrane and possessed C4H activity without solubilization or addition of other factors. The results indicate that the fusion protein strategy provides a useful tool to analyze the activities encoded in the rapidly increasing number of plant P-450 sequences of uncertain or unknown function. We also discuss critical elements of the strategy: the choice of the E. coli host strain, the N-terminal membrane anchor, and the conditions for protein expression.
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    Return to text or go to description of P450/P450 reductase fusions: more...

  • Kaltenbach, M., Schröder, G., Schmelzer, E., Lutz, V., Schröder, J., 1999. Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties, and cell-type specific expression in plants. Plant Journal 19, 183-193.
           We investigated the P450 dependent flavonoid hydroxylase from the ornamental plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68-78% identity with other CYP75s, and genomic blots suggested one or two genes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3',5'-hydroxylase, but 3'-hydroxylated products were also detected. The substrate specificity was investigated with the C. roseus enzyme and a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 reductase. Both enzymes accepted flavanones as well as flavones, dihydroflavonols, and flavonols, and both performed 3'- as well as 3'5'-hydroxylation. Kinetics with C. roseus cultures on the level of enzyme activity, protein, and RNA showed that the F3'5'H was present in dark-grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4-hydroxylase and flavanone 3ß-hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3'5'H substrates. Immunohistochemical localization of F3'5'H had not been performed before. A comparison of CHS and F3'5'H in cotyledons and flower buds from C. roseus identified CHS expression preferentially in the epidermis, while F3'5'H was only detected in the phloem. The cell-type specific expression suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.
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  • Knobloch, K.-H., Bast, G., Berlin, J., 1982. Medium- and light-induced formation of serpentine and anthocyanins in cell suspension cultures of Catharanthus roseus. Phytochemistry 21, 591-594.
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  • Meijer, A. H., Cardoso, M. I. L., Voskuilen, J. Th., De Waal, A., Verpoorte, R., Hoge, J. H. C., 1993. Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants. Plant Journal 4, 47-60.
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  • Ohlsson, A. B., Berglund, T., 2001. Gibberellic acid-induced changes in glutathione metabolism and anthocyanin content in plant tissue. Plant Cell, Tissue and Organ Culture 64, 77-80.
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  • Piovan, A., Filippini, R., Favretto, D., 1998. Characterization of the anthocyanins of Catharanthus roseus (L.) G. Don in vivo and in vitro by electrospray ionization ion trap mass spectrometry. Rapid Communications in Mass Spectrometry 12, 361-367.
    Return to text

  • Schröder, G., Wehinger, E., Lukacin, R., Wellmann, F., Seefelder, W., Schwab, W., Schröder, J., 2004. Flavonoid methylation: a novel 4'-O-methyltransferase from Catharanthus roseus, and evidence that partially methylated flavanones are substrates of four different flavonoid dioxygenases. Phytochemistry 65, 1085-1094.
        Catharanthus roseus (Madagascar periwinkle) flavonoids have a simple methylation pattern. Characteristic are B-ring 5' and 3' methylations and a methylation in the position 7 of the A-ring. The first two can be explained by a previously identified unusual O-methyltransferase (CrOMT2) that performs two sequential methylations. We used a homology based RT-PCR strategy to search for cDNAs encoding the enzyme for the A-ring 7 position. Full-length cDNAs for three proteins were characterized (CrOMT5, CrOMT6, CrOMT7). The deduced polypeptides shared 59-66% identity among each other, with CrOMT2, and with CrOMT4 (a previously characterized protein of unknown function). The five proteins formed a cluster separate from all other OMTs in a relationship tree. Analysis of the genes showed that all C. roseus OMTs had a single intron in a conserved position, and a survey of OMT genes in other plants revealed that this intron was highly conserved in evolution. The three cDNAs were cloned for expression of His-tagged recombinant proteins. CrOMT5 was insoluble, but CrOMT6 and CrOMT7 could be purified by affinity chromatography. CrOMT7 was inactive with all compounds tested. The only substrates found for CrOMT6 were 3'-O-methyl-eriodictyol (homoeriodictyol) and the corresponding flavones and flavonols. The mass spectrometric analysis showed that the enzyme was not the expected 7OMT, but a B-ring 4'OMT. OMTs with this specificity had not been described before, and 3',4'-dimethylated flavonoids had not been found so far in C. roseus, but they are well-known from other plants. The identification of this enzyme activity raised the question whether methylation could be a part of the mechanisms channeling flavonoid biosynthesis. We investigated four purified recombinant 2-oxoglutarate-dependent flavonoid dioxygenases: flavanone 3ß-hydroxylase, flavone synthase, flavonol synthase, and anthocyanidin synthase. 3'-O-methyl-eriodictyol was a substrate for all four enzymes. The activities were only slightly lower than with the standard substrate naringenin, and in some cases much higher than with eriodictyol. Methylation in the A-ring, however, strongly reduced or abolished the activities with all four enzymes. The results suggested that B-ring 3' methylation is no hindrance for flavonoid dioxygenases. These results characterized a new type of flavonoid O-methyltransferase, and also provided new insights into the catalytic capacities of key dioxygenases in flavonoid biosynthesis.
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