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Plant Physiol. (1999) 119: 1217-1232 The Decisive Step in Betaxanthin Biosynthesis Is a Spontaneous Reaction1
Abteilung Sekundärstoffwechsel, Institut für Pflanzenbiochemie, Halle (Saale), Germany
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.
Betalains, i.e. the red-violet betacyanins and yellow
betaxanthins, are water-soluble pigments of chemotaxonomic significance occurring in certain members of the plant order Caryophyllales and some
higher fungi (Steglich and Strack, 1990 The present study was undertaken to confirm the spontaneous character
of the last decisive step in betaxanthin biosynthesis. To demonstrate
the amino acid specificity and the stereoselectivity of this reaction,
nearly all proteinogenic amino acids, including some
(R)-forms were fed to hairy root cultures of yellow beet. In
addition, the effect of increasing the endogenous level of Phe on
betaxanthin formation was studied by treating the hairy root cultures
with AIP, a potent inhibitor of PAL activity. Spontaneous aldimine
formation in planta was further confirmed by feeding experiments with
young fodder beet plants and plants that do not belong to
betalain-forming taxa, i.e. broad bean (Vicia faba L.) and
pea (Pisum sativum L.).
Plant Material
Partial Syntheses of Diastereoisomeric Betaxanthins Method 1: BA from Lyophilized Red Beet Juice Lyophilized red beet juice (10 g) (Roth, Karlsruhe, Germany) was dissolved under stirring in 20 mL of water. After centrifugation for 10 min at 15,000g the supernatant was partitioned three times with 30 mL of EtOAc to remove soluble material. Aqueous NH3 solution (25%) was added to the ice-cooled solution, pH 4.8, to reach pH 11.3. After hydrolysis for 30 min at room temperature, 5 N HCl was slowly added under ice-cooling to reach pH 2.0. The mixture was immediately partitioned three times with 50 mL of EtOAc. The combined solvent fractions were concentrated in vacuo to 3 mL and then re-extracted with 3 mL of water. The yellow aqueous phase was added (100 µL each) to Eppendorf vials containing different (S)-amino acids and amines (dopamine and tyramine) (25 µmol in 100 µL of water), vortexed for 1 min, and centrifuged for 5 min at 15,000g. The supernatants were analyzed by HPLC using solvent systems 1 and 2. The yield was 0.8 to 6.0 nmol, depending on the amino acids or amine used.Method 2: BA from Betanin:Isobetanin Aqueous NH3 solution (25%) was added to a solution of betanin:isobetanin (2:1; 3 µmol in 2 mL of water) to reach pH 11.3 for hydrolysis at room temperature (30 min). After acidification to pH 2.0 with 5 N HCl under ice-cooling, the mixture was immediately partitioned three times with 5 mL of EtOAc. After evaporation of the solvent under reduced pressure the residue was solved in 3 mL of water and processed as described in method 1. This method yielded 0.2 to 0.8 nmol, or 0.6% to 2.4%, depending on the amino acids or amine used.Method 3: Direct Addition of Hydrolyzed Betanin/Isobetanin to Amino Acids Betanin:isobetanin (9:1; 800 nmol in 2 mL of water) was hydrolyzed as described in method 2 and added without acidification in 100-µL aliquots to 25 µmol (S)- and (R)-amino acids in 100 µL of water. After vortexing (1 min), the reaction mixtures were reduced to dryness in a concentrator (model 5301, Eppendorf), resuspended in 200 µL of water, and centrifuged for 5 min at 15,000g. The supernatants were analyzed as described in method 1. In this case the yield was 0.5 to 17.6 nmol, or 2% to 77%, depending on the amino acid or amine used.Isolation and Identification of the Major Betalains from Hairy Root Culture Hairy root culture material (line 5A; 20 g) was frozen in liquid N2, homogenized in a mortar, and extracted with 60 mL of 80% aqueous methanol containing 50 mM ascorbate. The extract was concentrated to 5 mL and applied on a Dowex 1 × 8 (formiate form), 50- to 100-mesh column (250 × 30 mm i.d.). The elution was performed with water (500 mL) and with a stepwise formic acid gradient (0.5, 1.0, 2.0, 3.5, and 7.0 N). The fractions (2.0 and 3.5 N) containing the main betaxanthin were concentrated and purified by semiprep HPLC (solvent system 3). Identification was performed by ESI-MS (positive ion mode and positive daughter ion scan).
Feeding Experiments Amino acids (Gly, [S]-Ala, [S]-Ser, [S]-Thr, [S]-Leu, [S]-Ile, [S]-Val, [S]-Gln, [S]-Asn, [S]-Glu, [S]-Asp, [S]-Lys, [S]-Arg, [S]-Orn, [S]-Met, [S]-Trp, [S]-Phe, [S]-His, [S]-Pro, [S]-Hyp, and [S]-4-thiaprolin) were dissolved in water and fed by sterile filtration to hairy root cultures of yellow beet at d 7 after subcultivation (final amino acid concentration, 2 mM). Cultures with water added served as a control. After 24 h the hairy roots were harvested, extracted, and analyzed by HPLC. The competition experiments (addition of [S]-Phe and [R]-Phe separately and combined) and the feeding of (R)-amino acids were performed in the same way. In saturation experiments (S)-Ala, (R)-Ala, and (S)-Thr were fed at d 7 under the same conditions with increasing final concentrations (2, 5, 10, 20, and 50 mM). In addition to the (NH4)2SO4 concentration (1 mM) of the nutrition solution, (NH4)2SO4 was fed at d 7 under the same conditions to reach final concentrations of 3, 6, 11, 21, and 51 mM. Furthermore, 5 mM (S)-Leu was fed from d 4 to 8, and the hairy roots were harvested 24 h after each application. To study the competition in the uptake of (R)-Phe in the presence of (S)-Phe (final concentration: 2 mM each) at d 7, 20 µL (0.74 MBq) of 3H-labeled (S)-Phe ([S]-[2,6-3H2]Phe, specific activity 200 TBq mmol 1, TRK 552, Amersham) and 50 µL (0.185 MBq) of 14C-labeled
(R)-Phe
([R]-[1-14C]Phe, specific
activity: 200 GBq mmol1, ARC 1116, Biotrend,
Cologne, Germany) were applied. To monitor uptake and for the
calculation of the 3H-to-4C ratio in the nutrition
solution, two 50 µL-aliquots were used 0, 1, 2, 4, 8, 12, and 24 h after addition for analysis by liquid scintillation counting (Ultima
Gold XR, Packard Instruments, Meriden, CT) for 2 min with LS 6000 TA
(Beckman).
Quantification of Betalains After harvesting hairy roots, suspension-cultured cells, or hypocotyls, the material was washed briefly with distilled water, blotted dry between filter paper, frozen in liquid N2, and homogenized in a mortar. The betalains were extracted with 80% aqueous methanol containing 50 mM ascorbate at a tissue:solvent ratio of 1 g 3 mL1. After centrifugation at 15,000g
for 10 min at 4°C, the supernatants were removed. Two aliquots of 20 µL were diluted to 1 mL with water and the absorbance was measured at
475 nm (for betaxanthins) and 540 nm (for betacyanins) with a
photometer (Shimadzu, Columbia, MD). For quantification of the
compounds, the mean molar extinction coefficient for betaxanthins
(48 × 106 cm2
mol1; Girod and Zryd, 1991b ) and for betanin
(62 × 106 cm2
mol1; Wyler et al., 1959) were used. After HPLC
analysis of the extracts the peak areas of individual compounds were
compared with those of standard compounds. The HPLC separations were
performed (solvent system 1) using an autosampler (20-µL injections).
BA was quantified by HPLC using a purified standard (molar extinction
coefficient, 25 × 106 cm2 mol1; Girod
and Zryd, 1991b).
Enzymic cyclo-Dopa Preparation Mushroom tyrosinase (800 µL, 1 mg mL1,
Sigma) was added to 10 mL of Dopa (10 mM in 0.02 M KPi buffer, pH 6.8) and shaken for 8 min at room
temperature. The reaction mixture was then treated with 10 mL of 0.2 M ascorbate. Proteins were precipitated by addition of 20 mL of methanol after 5 min, and the mixture was centrifuged at
15,000g for 5 min. The supernatant was concentrated to 6 mL and purified by preparative HPLC. The yield was 40 µmol (40%), and
the  max was 286 nm (HPLC-PDA) (the
max was 285 nm in 20% HCl [Wyler and
Chiovini, 1968]).
Spontaneous Condensation of cyclo-Dopa with BA as a Function of pH Ten microliters of BA (5 nmol) in water was added to a mixture of 80 µL of citrate/phosphate buffer (0.1 M citrate, 0.2 M NaPi buffer, 10 mM ascorbate, pH range 3.0-6.5) and 10 µL of cyclo-Dopa (40 nmol, 160 mM ascorbate) in a 100-µL cuvette (final ascorbate concentration, 25 mM). The increase in A540 was measured in intervals of 30 s for 10 min with a photometer (Beckman) at room temperature. The actual pH in the reaction mixture was measured directly after the experiment. The same assay at pH 6.0 was used to test the effect of protein extracts from a hairy root culture of yellow beet on betanidin formation by replacing 20 µL of the buffer with 20 µL of the protein extract.Preparation of Protein Extracts Protein extracts from the hairy root culture of yellow beets were prepared according to the methods (including [NH4]2SO4 precipitation) of Steiner et al. (1996), De-Eknamkul et al. (1997), and
Terradas and Wyler (1991) (60% acetone precipitation).
Photometric Assay for Vulgaxanthin I Formation Ten microliters of 100 mM (S)-Gln in water (final [S]-Gln concentration, 10 mM) was added to a mixture of 80 µL of citrate/phosphate buffer (0.1 M citrate, 0.2 M NaPi-buffer, and 10 mM ascorbate, pH 6.0) and 10 µL of BA (5 nmol) in water in a 100 µL-cuvette. The extinction at 475 nm was measured in intervals of 30 s for 10 min with a photometer (Beckman) at room temperature. The actual pH in the reaction mixture was measured directly after the experiment. The same assay was used to test the effect of protein extracts from hairy roots on the vulgaxanthin I formation by replacing 20 µL of the buffer with 20 µL of the protein extract.HPLC Assay for Vulgaxanthin I Formation A mixture of 50 µL 0.1 M KPi-buffer, pH 6.5, containing 50 mM ascorbate, 20 µL of 10 mM (S)-Gln, and 20 µL of protein extract from hairy roots was preincubated at 30°C for 5 min. The reaction was started by the addition of 10 µL of BA (5 nmol) in water. After 60 min at 30°C, 100 µL of methanol was added, centrifuged at 15,000g for 5 min, and the supernatant (50 µL) was analyzed by HPLC (solvent system 1).Amino Acid Analyses The plant material (1 g fresh weight) was frozen in liquid N2 and homogenized in a mortar. The amino acids were extracted with ethanol at a solvent:tissue ratio of 4 mL g1. After centrifugation at 15,000g
for 10 min at 4°C, the supernatants were removed and the pellets were
re-extracted with 3 mL of 80% aqueous ethanol. Water (3 mL) was added
to the combined supernatants, and the mixture was partitioned with 5 mL
of CHCl3 until the CHCl3 fractions were colorless. The aqueous upper phases were concentrated to
dryness in a rotary evaporator, dissolved in water, and aliquots were
used for amino acid analysis (ABI 420A, Applied Biosystems, Foster
City, CA) with the inclusion of (S)-Gln and
(S)-Asn in the amino acid standards. For Dopa analysis,
extraction was carried out in the presence of ascorbate (100 mM) and the extract was analyzed by HPLC as
described previously (Steiner et al., 1996).
HPLC Analytical and semipreparative HPLC was performed with a system from Waters (Milford, MA). The liquid chromatograph was equipped with a 5-µm Nucleosil C18 column (250 × 4 mm i.d., Macherey-Nagel, Düren, Germany), and the following solvent and gradient systems were used. Solvent system 1: A, 1.5% ortho-phosphoric acid in water; B, 80% acetonitrile in water; linear gradient from 100% A to 70% A in (A plus B) within 40 min. The flow rate was 1 mL min1. Solvent system 2: A, 50 mM NaH2PO4, and 2.5 mM
triethylamine, adjusted to pH 4.2 with H3PO4 and B,
40% acetonitrile in water (buffered-ion pairing system and a step-wise
gradient, according to the method of Trezzini and Zryd [1991]).
Solvent system 3: A, 1% formic acid in water; B, 80% acetonitrile in
water; gradient as in solvent system 1.
ESI-MS Positive ESI-MS was performed (TSQ 7000, Finnigan, Bremen, Germany; electrospray voltage 4.5 kV, N2 as sheath gas) using a syringe pump (Harvard Apparatus, South Natick, MA) operating at a flow rate of 5 µL min1.
Diastereoisomeric Betaxanthins Can Easily Be Prepared and Separated by HPLC Betaxanthin standards necessary for the identification of the metabolites of amino acid feedings to hairy root cultures and plants have been prepared in three different ways. The simplest and most rapid procedure (method 1) is the hydrolysis of commercial lyophilized red beet juice (containing racemic betanin) by aqueous ammonia solution, extraction of the liberated racemic BA after acidification (optimal at pH 1.0-2.0), and its addition to different amino acids directly yielding the diastereoisomeric betaxanthins ([2S/S]- and [2S/R]-forms). Using the solvent system and the stepwise gradient system (Trezzini and Zryd, 1991) (solvent system 2) and an
optimized linear gradient system (solvent system 1), 21 of 25 isomer
pairs could be separated. However, due to the racemic nature of the
starting material, an isomer assignment of the separated peaks was not
possible. Starting the partial synthesis with purified betanin:isobetanin mixtures (2:1) (method 2), the betaxanthin isomers
were obtained in the same ratio, the larger peaks of the isomer pairs
corresponded to the (2S/S)-forms (Table
I). With this method some
(R)-amino acids were also transformed to the corresponding
betaxanthins and analyzed by HPLC (Table
II). In addition, a modified version
(method 3) of the known synthetic procedure (Trezzini and Zryd, 1991)
was used to synthesize betaxanthins in higher amounts; but for use as
standards a subsequent purification was necessary.
Betalain Accumulation Coincides with Rapid Growth of Yellow Beet Hairy Root Cultures In liquid culture, both hairy root culture lines 5A and 7 showed the most intensive fresh weight increase between d 7 and 9, which was paralleled by a steep increase in miraxanthin content (Fig. 1). HPLC analysis of an extract (Fig. 2) showed that the betalain mixture consisted predominantly of betaxanthins, a major form and a minor form, together with a lower portion (<30%) of different betacyanins. Whereas nothing was known about the identity of the betacyanins of the hairy root culture, the two betaxanthins were recently identified as vulgaxanthin I ([S]-Gln-betaxanthin) and portulacaxanthin II ([S]-Tyr-betaxanthin) (Hempel and Böhm, 1997).
The identity of the betaxanthin (max = 468 nm)
eluting at 12.0 min was confirmed as vulgaxanthin I, which is typical for the genus Beta, but the major betaxanthin
(Rt = 24.7 min; max = 457 nm) did not match synthetically prepared
(S)-Tyr-betaxanthin (Rt = 25.2 min;
max = 469 nm). Therefore, hairy root material was extracted and the major betaxanthin was purified by conventional anion-exchange chromatography on a Dowex 1 × 8 column (Strack et
al., 1993) and by semipreparative HPLC (data not shown). The purified
betaxanthin was analyzed by ESI-MS.
Aldimine Formation in Hairy Root Cultures after Feeding of Amino Acids Shows Neither Amino Acid Specificity Nor Stereoselectivity The results of (S)-amino acid feeding to a hairy root culture (Table III) showed that all amino acids were accepted in the formation of the corresponding betaxanthins, but to a different extent. Also, (S)-4-thiaprolin, a synthetic amino acid, led to formation of the respective betaxanthin. In parallel feedings of the (S)- and (R)-isomers of different amino acids, both stereoisomers were incorporated into the corresponding betaxanthins to the same extent (Table IV). Simultaneous application of (S)- and (R)-Phe to hairy root cultures unexpectedly yielded a (S)-Phe-betaxanthin/(R)-Phe-betaxanthin ratio of 10:1. This unexpected result could be clarified by uptake studies using (S)-[2,6-3H2]Phe/(R)-[1-14C]Phe mixtures. The 3H-to-14C ratio of the compounds decreased in the nutrition solution within the feeding time (24 h) from 4.4 to 0.75 (Fig. 3).
Exogenously Applied Amino Acids Compete with the Endogenous Dopamine in Betaxanthin Formation Feeding of (S)-Thr, an amino acid of high solubility, with final concentrations up to 50 mM in the nutrition solution to hairy root culture at d 7, led to an increased (S)-Thr-betaxanthin formation (optimum, 10 mM (S)-Thr), with simultaneous decreased BA and miraxanthin V levels compared with the control (Fig. 4). To suppress the miraxanthin V formation more efficiently, high amounts of (S)-Leu (5 mM) were given daily to the hairy root culture between d 4 and 8, and hairy roots were harvested 24 h after each addition. The strong increase of the miraxanthin V and BA content seen in the controls was totally suppressed, with a simultaneous increase in the (S)-Leu-betaxanthin level (Fig. 5).
Endogenously Increased Phe Level Leads to the Formation of (S)-Phe-Betaxanthin In another experiment, betaxanthin formation was affected indirectly without amino acid feeding. The addition of AIP, a strong inhibitor of PAL (EC 4.3.1.5), to a hairy root culture led to an increase of the endogenous (S)-Phe level and, subsequently, (S)-Phe-betaxanthin, which was missing in the control culture (Fig. 6), was detectable.
Feeding of Amino Acids to Fodder Beet Seedlings Confirms the Hairy Root Culture Results To show that the results of the amino acid feeding experiments with hairy root cultures are transferable to whole plants, amino acids were fed to de-rooted fodder beet plants via the hypocotyls. (S)- and (R)-Phe were taken up and incorporated into the corresponding betaxanthins in the same way as in the hairy root culture (Fig. 7). The application of AIP (50 µM) to the same system for 24 h also led to the increase of the (S)-Phe level and to the formation of (S)-Phe-betaxanthin, although to a smaller extent than in the hairy root culture experiment. As AIP itself is an amino acid and could result in the formation of a derived betaxanthin, the AIP-betaxanthin was synthesized as the standard, but no AIP-betaxanthin was found in the extract after AIP feeding, obviously due to the low concentration applied. Furthermore, feeding of cyclo-Dopa (2 mM) in the presence of ascorbate for stabilization, a red coloration of the hypocotyls was observed after only 60 min. HPLC analysis of the extract proved that betanidin had been formed and was accompanied by low amounts of betanin (Fig. 8).
BA Feeding Leads to Betaxanthin Formation in Plants That Do Not Belong to the Caryophyllales BA isolated from fodder beet hypocotyls and purified by preparative HPLC (data not shown) was fed in phosphate-buffered solution, pH 6.8, for 24 h to 2-week-old de-rooted broad bean and pea seedlings via the hypocotyls. Although the uptake was low, HPLC analysis of the hypocotyl extracts of both plants showed the presence of betaxanthins, identified by their characteristic online UV/Vis spectra. The major betaxanthin from the broad bean experiment (Fig. 9) was readily identified as dopaxanthin (max = 470 nm) by comparison with a synthetic
standard compound. Amino acid analysis of hypocotyl extracts of broad
bean seedlings revealed that Dopa was present at the highest
concentration of all amino acids determined (Table
V).
Protein Extracts Do Not Catalyze the Formation of Vulgaxanthin I and Betanidin Despite the evidence for spontaneity in the condensation reaction, enzyme extracts were prepared to study the possible catalysis of the condensation reaction. De-Eknamkul et al. (1997) found enzyme-catalyzed
condensation of dopamine with the iridoid aldehyde secologanin,
including aldimine formation. Protein extracts from hairy root culture
prepared according to their procedure and similarly, the other extracts
(acetone powder,
[NH4]2SO4
precipitation), did not catalyze the formation of vulgaxanthin I
or betanidin (measured photometrically or by HPLC). Whereas the
spontaneous condensation of cyclo-Dopa with BA could be
kinetically measured at 540 nm (Table
VI), the monitoring of vulgaxanthin I
formation at 475 nm failed. Table VI shows the increasing rates of the
spontaneous formation of betanidin with the decreasing pH values. In
the physiologically relevant pH range above 6.0, the spontaneous
reaction was negligible. This was the prerequisite for all of the
enzymatic attempts at betaxanthin formation.
General Features of Betalain Biosynthesis In contrast to the well-characterized genes and enzymes involved in anthocyanin biosynthesis (Heller and Forkmann, 1993), there are only
two enzymes known to be involved in the biosynthesis of the basic
skeleton of the betalains: tyrosinase, which is responsible for the
formation of Dopa and cyclo-Dopa (Mueller et al., 1996; Steiner et al., 1996, 1999) and Dopa dioxygenase, which catalyzes the
Dopa extradiol cleavage, leading to the formation of the chromophor BA
(Girod and Zryd, 1991a; Terradas and Wyler, 1991; Hinz et al., 1997;
Mueller et al., 1997a, 1997b) (for a recent review, see Roberts and
Strack, 1999). Two further steps need to be clarified: (a) the
possible glucosylation of cyclo-Dopa before its
condensation with BA as an alternative to the glucosylation of
betanidin (Heuer and Strack, 1992; Heuer et al., 1996; Vogt et al.,
1997), and (b) the condensation reaction between BA and amino acids
(including cyclo-Dopa) and amines (i.e. aldimine formation).
This condensation reaction was studied in betaxanthin and
betacyanin biosynthesis in vivo and in vitro to verify the earlier
suggestion that this step proceeds nonenzymatically, i.e. spontaneously
(Trezzini, 1990; Trezzini and Zryd, 1990; Hempel and Böhm, 1997).
If the condensation reaction was catalyzed by an enzyme, amino acid
specificity and stereoselectivity for the natural (S)-forms
of amino acids should be found.
Partial Syntheses and Separation of Betaxanthins As a prerequisite for the analysis of the products expected from the amino acid feedings, a simple method for the synthesis of stereoisomeric betaxanthins and their analytical separation had to be elaborated. Only the separation of one isomeric betaxanthin pair ([2S/11S]-indicaxanthin/[2S/11R]-indicaxanthin) was known (Terradas and Wyler, 1991). A method for the extraction of BA from acidified solutions (Döpp et al., 1982) was adapted to the isolation of BA from hydrolyzed betanin:isobetanin mixtures. This BA
had to be added to amino acids and amines to get the diastereoisomeric betaxanthins suitable as standards. As the isomer ratio of the betaxanthins obtained was the same as in the starting material (betanin/isobetanin), a significant racemization of the BA under the
hydrolysis conditions did not occur. Trezzini and Zryd (1991) used a
buffered-ion pairing solvent system and a stepwise gradient (solvent
system 2) to separate semisynthetic betaxanthins by HPLC.
Characterization of the Hairy Root Culture from Yellow Beet and Identification of the Major Betalains During the logarithmic phase of the fresh-weight increase of hairy roots (d 7-9) a parallel rise in the betaxanthin content occurred (Fig. 1). Both hairy root lines (5A and 7) produced a betalain mixture consisting of a minor and a major betaxanthin, together with a low portion (<30%) of different betacyanins (Fig. 2). In contrast to the results of Hempel and Böhm (1997), the major betaxanthin has been
identified as dopamine-betaxanthin by ESI-MS, as well as by comparison
(Rt 24.7 = min; max = 457 nm) and co-chromatography in HPLC with synthetic
dopamine-betaxanthin. This betaxanthin, first isolated as miraxanthin V
(max = 458.5 nm in the presence of HCl)
from flowers of Mirabilis jalapa (Piattelli et al., 1965),
is already known as a constitutive component of the betaxanthins of
callus cultures of B. vulgaris (Girod and Zryd, 1991b).
Furthermore, in accordance with previous results (Hempel and
Böhm, 1997), the identity of the minor betaxanthin (max = 468 nm) eluting at 12 min was confirmed as
(S)-Gln-betaxanthin (vulgaxanthin I) by comparison and
co-chromatography with the synthetic vulgaxanthin I
standard. The occurrence of eight betalains in the roots of the yellow
beets from which the hairy root cultures were derived has been
reported, including vulgaxanthin I and vulgaxanthin II (Savolainen and
Kuusi, 1978).
Feeding of Amino Acids to Hairy Root Culture of Yellow Beets and
Other Cell Cultures
Feeding Experiments with Fodder Beet Plants HPLC of hypocotyl extracts from fodder beet plants showed a betaxanthin and BA pattern very similar to that of the hairy root culture of yellow beets. Therefore, we used these plants to prove whether or not the condensation reaction in amino acid feedings proceeds in the same way as in hairy root cultures. (S)- and (R)-Phe were taken up by de-rooted fodder beet plants and were incorporated into the corresponding betaxanthins in the same way and to the same extent as in the hairy root culture without any stereoselectivity (Fig. 7). The application of AIP (50 µM) to the same plant material for 24 h also led to an increase in the (S)-Phe-level and to the formation of (S)-Phe-betaxanthin, although to a smaller extent than in the hairy root culture experiment (data not shown).BA Feeding to Broad Bean and Pea Plants To find additional evidence for the spontaneous character of the condensation reaction, BA was fed to broad bean and pea seedlings, which do not belong to the betalain-synthesizing Caryophyllales. The analyses of both extracts after BA feeding showed the presence of betaxanthins, in contrast to extracts of buffer-treated plants. The major betaxanthin from the broad bean experiment (Fig. 9) was identified as dopaxanthin (max = 470 nm) on
the basis of the Rt and co-injection with a
synthesized standard compound. Amino acid analysis and Dopa
determination of hypocotyl extracts by HPLC revealed that the Dopa
concentration was higher than that of all other amino acids (Table V).
Because the Asn concentration was also high, the preferred formation of
dopaxanthin was an unexpected outcome, and may indicate a different
localization of different amino acids, leading to a more facilitated
access of BA to Dopa than to Asn. The pattern of distribution and
concentration of amino acids in the vacuole is similar to that in the
cytoplasm, but quite different from that in the chloroplast (Mimura et
al., 1990), the site of synthesis of many amino acids in higher plants. Because it is reasonable to assume that broad bean hypocotyls (which do
not synthesize betalains) do not have an enzyme catalyzing the
condensation reaction, the dopaxanthin formation observed must have
resulted from a nonenzymic spontaneous process.
Spontaneous versus Enzymatic Condensation Reactions The formation of the aldimine bond in the betaxanthin biosynthesis proceeds in two steps: the nucleophilic addition of the amino group at the aldehyde group leads to an intermediate from which water is eliminated, forming the aldimine bond (Fig. 11). The reaction of an amine with an aldehyde is an enzymatically important catalyzed step in benzylisoquinoline biosynthesis (dopamine with 4-hydroxyphenylacetaldehyde or 3,4-dihydroxyphenylacetaldehyde), which leads, however, to the cyclized intermediates norcoclaurine and norlaudanosoline, respectively (Rueffer and Zenk, 1987). Similarly, the
condensation of dopamine with the iridoid aldehyde secologanin was
found to be catalyzed by cell-free extracts of Alangium
lamarckii (De-Eknamkul et al., 1997). This cyclization proceeded,
in contrast to the betaxanthin formation, directly to
tetrahydroisoquinoline derivatives (R- and
S-form), which spontaneously cyclized further in lactam
formation. The first reaction steps can also proceed nonenzymic at pH
5.0 (Itoh et al., 1995).
Considering previously published studies and the results of our experiments, we are convinced that the condensation process of the BA with amino acids (including cyclo-Dopa) or amines in plants is a spontaneous rather than an enzyme-catalyzed reaction. This assumption is substantiated by the following lines of evidence: (a) experiments failed to detect protein-catalyzed betaxanthin formation; (b) the formation of betaxanthins after amino acid feeding to a hairy root culture of yellow beets showed neither an amino acid specificity nor a stereoselectivity; (c) the betaxanthin formation was also observed with unnatural precursors ([S]-4-thiaprolin); (d) (S)-Phe-betaxanthin formation was detected after inhibition of PAL by AIP due to an increase of the endogenous (S)-Phe level; (e) the results of betaxanthin formation in hairy root cultures of yellow beets were reproduced with intact plants (fodder beets). Furthermore, the formation of a betacyanin, betanidin, has been demonstrated by feeding cyclo-Dopa to these plants; and (f) application of BA to plants that do not form betaxanthin led to betaxanthin formation.
Received October 22, 1998;
accepted January 2, 1999.
Abbreviations: AIP, 2-aminoindan 2-phosphonic acid. BA, betalamic acid. Dopa, 3-(3,4-dihydroxyphenyl)-Ala. ESI-MS, electrospray ionization MS. EtOAc, ethyl acetate. PAL, Phe ammonia-lyase. PDA, photodiode array detection. Rt, retention time.
We thank Barbara Kolbe (Institut für Pflanzenbiochemie [IPB], Halle, Germany) for skillful technical assistance in plant cell cultivation; Dr. Hartmut Böhm (Deutsches Institut für Ernährungsforschung, Bergholz-Rehbrücke, Germany) for providing hairy root cultures of yellow beets; Dr. Inna Kuzovkina (K.A. Timiryasev Institute of Plant Physiology, Russian Academy of Sciences, Moscow) for establishing a hairy root culture of red beets; Dr. Alfred Baumert (IPB) for (S)-4-thiaprolin synthesis; Dr. Nicolaus Amrhein (Eidgenössische Technische Hochschule Zürich, Switzerland) for providing AIP and proposing the AIP-feeding experiments; Dr. Jürgen Schmidt (IPB) for MS measurements; and Dr. Michael Kiess (Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany) for amino acid analyses. We are also grateful to Christine Kaufmann and Annett Kohlberg (IPB) for the figures and photographs. Finally, we thank Ulrike Steiner (IPB) for valuable discussions and Martina Rauscher (IPB) for critical reading of the manuscript.
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  F. Gandia-Herrero, J. Escribano, and F. Garcia-Carmona Betaxanthins as Substrates for Tyrosinase. An Approach to the Role of Tyrosinase in the Biosynthetic Pathway of Betalains Plant Physiology, May 1, 2005; 138(1): 421 - 432. [Abstract] [Full Text] [PDF]
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L. Christinet, F. X. Burdet, M. Zaiko, U. Hinz, and J.-P. Zryd Characterization and Functional Identification of a Novel Plant 4,5-Extradiol Dioxygenase Involved in Betalain Pigment Biosynthesis in Portulaca grandiflora Plant Physiology, January 1, 2004; 134(1): 265 - 274. [Abstract] [Full Text] [PDF]
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Abstract
Introduction
, Isobetanidin (the
[2S/15R]-isomer of betanidin).
-methylaminobutyraldehyde to N-methyl pyrrolinium
cation, and its coupling with acetoacetic acid giving hygrine (Endo et
al., 1988
-alanyldopamine quinone methide leads to papiliochrome II, a yellow wing pigment of butterflies (Saul and Sugumaran, 1991