The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Abstract

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris "Golden Beet") showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris "Altamo") plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.

Figure 1

Time course of growth (fresh weight increase) and betaxanthin content (miraxanthin V and vulgaxanthin I) in hairy root culture of yellow beet.

Figure 2

HPLC elution profiles of betalains in hairy root culture of yellow beets. Top, betaxanthins (A₄₇₅); Bottom, betacyanins (A₅₄₀). Full scales are different in top (0.50 absorbance units) and bottom (0.05 absorbance units). Peak numbers correspond to the biosynthetic scheme in Figure 10.

Figure 3

Time course of uptake (24 h) of tritium-labeled (S)-Phe and ¹⁴C-labeled (R)-Phe (alone and combined, in the presence of 2 mm (S)- and (R)-Phe) by hairy root culture of yellow beets (at d 7) and ³H-to-¹⁴C ratio in the nutrient solution. Each value is the average of duplicate samples.

Figure 4

Feeding of (S)-Thr in increasing concentrations (final: 2–50 mm) to hairy root culture of yellow beet (at d 7) for 24 h and betalain analysis by HPLC.

Figure 5

Repeated daily feeding of (S)-Leu (5 mm) to hairy root culture of yellow beets from d 4 and 8 and betalain analysis by HPLC 24 h after each application. Dotted lines, Control; solid lines, (S)-Leu feeding.

Figure 6

Repeated daily feeding of AIP (16 μm) to hairy root culture of yellow beets between d 4 and 7 and betalain analysis by HPLC at d 8. A, Control; B, AIP feeding. Full scales of A₄₇₅ = 0.6 absorbance units. The inset in B is the PDA spectrum of the newly formed (S)-Phe-betaxanthin. Peak numbers correspond to the biosynthetic scheme in Figure 10.

Figure 7

Feeding of (S)-Phe and (R)-Phe (10 mL at 10 mm) to 10 23-d-old de-rooted fodder beet plants via the hypocotyls for 48 h and betalain analysis of hypocotyl extracts by HPLC. A, Control; B, (S)-Phe feeding; C, (R)-Phe feeding. Full scales of A₄₀₅ = 0.24 absorbance units. Peak numbers correspond to the biosynthetic scheme in Figure 10.

Figure 8

Feeding of cyclo-Dopa to five 23-d-old de-rooted fodder beet plants via hypocotyls for 1.5 h and betalain analysis of hypocotyl extracts by HPLC. A, Control (80 mm ascorbate); B, cyclo-Dopa feeding (2 mm, in the presence of 80 mm ascorbate). Scales of A₄₀₅ (full scale = 0.12 absorbance units) and A₅₄₀ (full scale = 0.5 absorbance units) are the same in A and B. Peak numbers correspond to the biosynthetic scheme in Figure 10. 3′, Isobetanidin (the [2S/15R]-isomer of betanidin).

Figure 9

Feeding of BA (1.4 mL of 0.285 mm in 0.1 m KPi buffer, pH 6.8) to two 14-d-old de-rooted broad bean plants via the hypocotyls for 24 h and betalain analysis of hypocotyl extracts by HPLC. A, Control (0.1 m KPi buffer, pH 6.8); B, BA feeding (0.285 mm in 0.1 m KPi buffer, pH 6.8). Scales of A₄₇₅ (full scale = 0.07 absorbance units) are the same in A and B.

Figure 10

Scheme of betalain biosynthesis in hairy root culture. Enzymatically catalyzed steps: E IA, hydroxylating activity of tyrosinase; E IB, oxidizing activity of tyrosinase; E II, Dopa dioxygenase; E III, Dopa decarboxylase; E IV, glucosyltransferase. Spontaneous steps: V, cyclization reactions; and VI, condensation reactions (aldimine formation). Compound numbers of this scheme correspond to peak numbers in Figures 2, 6, 7, and 9.

Figure 11

Scheme illustrating the formation of the aldimine bond in betaxanthin biosynthesis. In the first step the amino group of amino acids is added to the aldehyde moiety of the BA and the intermediate then eliminates water, resulting in the aldimine bond.