Planta
Planta 149, 176-180(1980)
9 by Springer-Verlag 1980
Qualitative and Quantitative Aspects of Betalains Biosynthesis in Amaranthus caudatus L. var. pendula Seedlings J. Bianco-Colomas Laboratoire de Physiologic V~g~tale, Facult6 des Sciences et des Techniques, Parc Valrose, F-06034 Nice-Cedex, France
Abstract. Seedlings of Amaranthus caudatus L. var. pendula were used to study the influence of several
treatments: white light, 3,4-dihydroxyphenylalanine (DOPA), kinetin, gibberellic acid (GA3) on betalains biosynthesis. The pigments, betacyanins and betaxanthins, were separated using a Sephadex G-15 column chromatography. Qualitative as well as quantitative differences were observed according to the treatments applied. The amaranthin biosynthesis seemed to be favored in the absence of DOPA. Under the combined effect of kinetin and white light a small quantity of betanin was also synthesized. Adding exogenous DOPA led to a more diversified production which included betacyanins (amaranthin and betanin), betaxanthins (vulgaxanthin and miraxanthin), and even dopachrome. As a general rule, kinetin activated the betalains biosynthesis whereas GA3 inhibited it. The stimulating effect of white light was always much greater than that of kinetin. Key words: Amaranthus - Betalains - Cytokinin Dihydroxyphenylalamine - Gibberellin - Light (betalain synthesis).
Introduction Amaranthus seedlings have been used for many years
to study the regulation of betalains biosynthesis (Bamberger and Mayer 1960; K6hler and Conrad 1966; Mabry and Wohlpart 1968 ; Piattelli et al. 1969; Koehler 1972; Rast et al. 1972; Colomas et al. 1973; French et al. 1973). It was with this particular material that the role of tyrosine and DOPA as precursors was demonstrated (H6rhammer et al. 1964; Garay Abbreviations: GA3 =gibbereIlic acid; GA4 =gibberellin 4; B H T =
2,6-diter-butyl-4-methylphenol ; DOPA = 3,4-dihydroxyphenyl alanine; Kinetin = 6-furfurylaminopurine
0032-0935/80/0149/0176/$01.00
and Towers 1966; Miller et al. 1968; Constabel and Nassif-Makki 1971; French et al. 1974). Numerous studies were undertaken to determine the influence of physical (light, temperature, pH) and chemical (growth regulators) factors on this biosynthesis. Mabry and Wohlpart (1968) demonstrated that light is an absolute requirement for the amaranthin synthesis in A. tricolor. In other species, for example A. caudatus, the pigment accumulation takes place even in the dark, increasing markedly under the influence of white light (Rast et al. 1972; Woodhead and Swain 1974). It is a well known fact that cytokinins promote amaranthin synthesis in several species in the dark (Bamberger and Mayer 1960; K6hler and Conrad 1966; Bigot 1968; Piattelli et al. 1971; Colomas 1975; Elliott 1979) or under light conditions (Giudici de Nicola et al. 1972; Koehler 1972). GA 3 inhibits the production of amaranthin induced by cytokinins in the dark in A. caudatus (Bigot 1968). The same inhibitory effect was observed both for GA 3 and GAg with A. tricolor seedlings cultivated in the dark or in red light (Colomas et al. 1973). For Stobart et al. (1970), Kinsman et al. (1975), and Stobart and Kinsman (1977), GA 3 overcomes the light induced biosynthesis but does not affect that induced by kinetin. The above-mentioned authors, although often using species synthetizing several betalains, studied the quantative variations induced under the influence of different treatments only at the amaranthin level. We thought it would be interesting, using A. caudatus seedlings which are able to synthetize both betacyanins and betaxanthins (French et al. 1974; Giudici de Nicola et al. 1975; Colomas 1977), to examine the influence of DOPA, light, kinetin, and GA3 at the different pigment levels. This work was made possible by the separation method previously described (Colomas 1977).
J, Bianco-Colomas: Betalain Biosynthesis in
Amaranthus
177
Material and Methods
Results
Seeds (10,000) of A. eaudatus L. var. pendula were allowed to germinate for 24 h on moistened filter paper in Petri dishes in the dark at 26 ~ C. The seedlings were then kept in water or transferred into kinetin, DOPA, or GAs solutions. For the next 96 h they were exposed to light, fluorescent light giving 12.5 W m -2 at seedlings level, or maintained in the dark, in both cases at 23 ~ C. The concentrations chosen were 10-5M for kinetin (Bigot 1968), 5.10 6M for GA3 (Colomas et al. 1973), and 2.5.10 aM for DOPA (Colomas 1977). Seedlings, five days old, were homogenized at 5~ C in a mortar in presence of BHT. The homogenate was centrifuged at 18,000 g for 10 min and the supernatant adjusted to pH 3 with 1 M HC1. After a second centrifugation, the supernatant was adjusted to pH Z3 with 1 M citric acid. The crude extracts were submitted to Sephadex G-15 column chromatography as previously described (Colomas 1977). The column was eluted with an acetate buffer (M, pH4.5) at a flow rate of 6 ml per hour. The collected fractions (1 mi) were studied using a Varian spectrophotometer (605 M). For each fraction the absorption maximum was determined and the absorbance measured. Each experiment was repeated at least five times. In some cases incubation experiments were performed using crude extracts obtained from seedlings maintained for five days in the dark on distilled water. One ml crude extract without BHT added and one ml aqueous DOPA solution at 5.10- 3 M were introduced in glass vials of 24 ml capacity stoppered with cotton plugs. The vials were shaken (100 rev min 1) and exposed to light at 23 ~ C. The experiments lasted either 30 or 120 min.
1. Qualitative Changes ( T a b l e ) Seedlings Grown without DOPA. T h e t a b l e s h o w s t h a t s e e d l i n g s g r o w n o n d i s t i l l e d w a t e r in t h e d a r k o r in the light synthetize only one betacyanin (fractions 14 t o 22). T h i s p i g m e n t w a s f o r m e r l y i d e n t i f i e d as a m a r a n t h i n ( C o l o m a s 1977). T h e a d d i t i o n o f G A 3 caused the disappearance of this pigment in seedlings cultivated in the dark. A d d i n g k i n e t i n t o s e e d l i n g s e x p o s e d t o l i g h t ind u c e d t h e s y n t h e s i s o f a n o t h e r b e t a c y a n i n f o u n d in t h e f r a c t i o n s f r o m 2 4 t o 28. T h i s p i g m e n t h a s f o r m e r ly b e e n i d e n t i f i e d as b e t a n i n ( C o l o m a s 1977). W h e n both kinetin and GA 3 were applied, the qualitative results were very similar to those obtained with kinetin only. Seedlings Grown with DOPA. W h a t e v e r t h e t r e a t m e n t a p p l i e d (light, k i n e t i n , G A 3 ) , a m a r a n t h i n a n d b e t a n i n w e r e a l w a y s p r e s e n t i n t h e f r a c t i o n s f r o m 14 t o 22 a n d 24 t o 28, r e s p e c t i v e l y . In addition, the DOPA treatment induced the form a t i o n o f b e t a x a n t h i n s , f r a c t i o n s 32 t o 39, w h i c h h a v e p r e v i o u s l y b e e n i d e n t i f i e d as v u l g a x a n t h i n a n d
Table 1. Separation by Sephadex gel G-15 chromatography of the pigments synthesized in the different experimental conditions Fractions 14 22 2m,x 540 n m amaranthin
17 22 2max475 n m dopachrome
24 28 )~max 540 nm betanin
32-39 2m,x 475 nm betaxanthins
Water
Darkness Light
+ +
nd" nd
nd nd
nd nd
Kinetin
Darkness Light
+ +
nd nd
nd +
nd nd
GA3
Darkness Light
nd +
nd nd
nd nd
nd nd
Kinetin + GA 3
Darkness Light
+ +
nd nd
nd +
nd nd
DOPA
Darkness Light
+ +
+ +
+ +
+ +
DOPA + Kinetin
Darkness Light
+ +
+ +
+ +
+ +
DOPA +GA3
Darkness Light
+ +
+ +
+ +
nd +
DOPA +Kinetin + GA 3
Darkness
+
+
+
nd
Light
+
+
+
+
and = n o t detectable
J. Bianco-Colomas: Betalain Biosynthesis in Amaranthus
178
miraxanthin (Colomas 1977). They did n,ot appear when GA3 was applied to seedlings grown in darkness, with or without kinetin. Whatever treatment the seedlings received, the presence of another substance showing an absorption maximum at 475 nm was detected in the fractions from 17 to 22. A compound exhibiting the same characteristics (2 max and elution volume) was detected in the crude extract after incubation for 30 rain with DOPA at 23 ~ C. When the incubation lasted a longer time, 120 rain, the coloration became progressively darker and finally black. Since dopachrome, one of the intermediaries of melanogenesis, is orange colored and shows an absorption maximum at 475 rim, we propose to assimilate to it the compound found in the fractions from 17 to 22.
2. Quantitative Changes Seedlings Grown without DOPA. In the dark, on distilled water, the amaranthin synthesis was very low. Under the influence of light it increased markedly (Fig. 1A). Whereas GA3 completely inhibited the amaranthin synthesis in the dark, it induced a significant decrease in the light (Fig. 1 A). In seedlings maintained in darkness as well in those exposed to light, kinetin clearly increased the amaranthin production. Betanin appeared in small quantities under light conditions only (Fig. 1 A). GA3 inhibited significantly the kinetin induced synthesis. On the other hand, the production of betanin in very low quantities was only very slightly affected by GA 3 (Fig. 1 B).
Seedlings Grown with DOPA. In darkness, without growth regulators, the amaranthin synthesis was greatly increased under the influence of DOPA (Fig. 2A). Betanin and betaxanthins were found in small quantities, respectively, in the fractions from 24 to 28 and 32 to 39. The quantity of dopachrome found in fractions 17 to 22 was somewhat less than that of amaranthin. Under light conditions, the synthesis of all these pigments was very markedly stimulated (Fig. 2B).
% 1,o
A 9 dw, D
Z~
E E
~)"%
0 ~o 0(-
~
i
l
i
t
0 dw, L
~A
A K,L X GA3, L 9 K,D
k
0.5 i_ 0 o~
< o 14
o
16
18
20
Fraction
22
24
26
28
number
A
E 1.0
B
/~z~
L~
* K,D
I1~
A
[3 K G A 3 , L
t-' m 0.5 n .D
Planta 149, 176-180(1980)
9 by Springer-Verlag 1980
Qualitative and Quantitative Aspects of Betalains Biosynthesis in Amaranthus caudatus L. var. pendula Seedlings J. Bianco-Colomas Laboratoire de Physiologic V~g~tale, Facult6 des Sciences et des Techniques, Parc Valrose, F-06034 Nice-Cedex, France
Abstract. Seedlings of Amaranthus caudatus L. var. pendula were used to study the influence of several
treatments: white light, 3,4-dihydroxyphenylalanine (DOPA), kinetin, gibberellic acid (GA3) on betalains biosynthesis. The pigments, betacyanins and betaxanthins, were separated using a Sephadex G-15 column chromatography. Qualitative as well as quantitative differences were observed according to the treatments applied. The amaranthin biosynthesis seemed to be favored in the absence of DOPA. Under the combined effect of kinetin and white light a small quantity of betanin was also synthesized. Adding exogenous DOPA led to a more diversified production which included betacyanins (amaranthin and betanin), betaxanthins (vulgaxanthin and miraxanthin), and even dopachrome. As a general rule, kinetin activated the betalains biosynthesis whereas GA3 inhibited it. The stimulating effect of white light was always much greater than that of kinetin. Key words: Amaranthus - Betalains - Cytokinin Dihydroxyphenylalamine - Gibberellin - Light (betalain synthesis).
Introduction Amaranthus seedlings have been used for many years
to study the regulation of betalains biosynthesis (Bamberger and Mayer 1960; K6hler and Conrad 1966; Mabry and Wohlpart 1968 ; Piattelli et al. 1969; Koehler 1972; Rast et al. 1972; Colomas et al. 1973; French et al. 1973). It was with this particular material that the role of tyrosine and DOPA as precursors was demonstrated (H6rhammer et al. 1964; Garay Abbreviations: GA3 =gibbereIlic acid; GA4 =gibberellin 4; B H T =
2,6-diter-butyl-4-methylphenol ; DOPA = 3,4-dihydroxyphenyl alanine; Kinetin = 6-furfurylaminopurine
0032-0935/80/0149/0176/$01.00
and Towers 1966; Miller et al. 1968; Constabel and Nassif-Makki 1971; French et al. 1974). Numerous studies were undertaken to determine the influence of physical (light, temperature, pH) and chemical (growth regulators) factors on this biosynthesis. Mabry and Wohlpart (1968) demonstrated that light is an absolute requirement for the amaranthin synthesis in A. tricolor. In other species, for example A. caudatus, the pigment accumulation takes place even in the dark, increasing markedly under the influence of white light (Rast et al. 1972; Woodhead and Swain 1974). It is a well known fact that cytokinins promote amaranthin synthesis in several species in the dark (Bamberger and Mayer 1960; K6hler and Conrad 1966; Bigot 1968; Piattelli et al. 1971; Colomas 1975; Elliott 1979) or under light conditions (Giudici de Nicola et al. 1972; Koehler 1972). GA 3 inhibits the production of amaranthin induced by cytokinins in the dark in A. caudatus (Bigot 1968). The same inhibitory effect was observed both for GA 3 and GAg with A. tricolor seedlings cultivated in the dark or in red light (Colomas et al. 1973). For Stobart et al. (1970), Kinsman et al. (1975), and Stobart and Kinsman (1977), GA 3 overcomes the light induced biosynthesis but does not affect that induced by kinetin. The above-mentioned authors, although often using species synthetizing several betalains, studied the quantative variations induced under the influence of different treatments only at the amaranthin level. We thought it would be interesting, using A. caudatus seedlings which are able to synthetize both betacyanins and betaxanthins (French et al. 1974; Giudici de Nicola et al. 1975; Colomas 1977), to examine the influence of DOPA, light, kinetin, and GA3 at the different pigment levels. This work was made possible by the separation method previously described (Colomas 1977).
J, Bianco-Colomas: Betalain Biosynthesis in
Amaranthus
177
Material and Methods
Results
Seeds (10,000) of A. eaudatus L. var. pendula were allowed to germinate for 24 h on moistened filter paper in Petri dishes in the dark at 26 ~ C. The seedlings were then kept in water or transferred into kinetin, DOPA, or GAs solutions. For the next 96 h they were exposed to light, fluorescent light giving 12.5 W m -2 at seedlings level, or maintained in the dark, in both cases at 23 ~ C. The concentrations chosen were 10-5M for kinetin (Bigot 1968), 5.10 6M for GA3 (Colomas et al. 1973), and 2.5.10 aM for DOPA (Colomas 1977). Seedlings, five days old, were homogenized at 5~ C in a mortar in presence of BHT. The homogenate was centrifuged at 18,000 g for 10 min and the supernatant adjusted to pH 3 with 1 M HC1. After a second centrifugation, the supernatant was adjusted to pH Z3 with 1 M citric acid. The crude extracts were submitted to Sephadex G-15 column chromatography as previously described (Colomas 1977). The column was eluted with an acetate buffer (M, pH4.5) at a flow rate of 6 ml per hour. The collected fractions (1 mi) were studied using a Varian spectrophotometer (605 M). For each fraction the absorption maximum was determined and the absorbance measured. Each experiment was repeated at least five times. In some cases incubation experiments were performed using crude extracts obtained from seedlings maintained for five days in the dark on distilled water. One ml crude extract without BHT added and one ml aqueous DOPA solution at 5.10- 3 M were introduced in glass vials of 24 ml capacity stoppered with cotton plugs. The vials were shaken (100 rev min 1) and exposed to light at 23 ~ C. The experiments lasted either 30 or 120 min.
1. Qualitative Changes ( T a b l e ) Seedlings Grown without DOPA. T h e t a b l e s h o w s t h a t s e e d l i n g s g r o w n o n d i s t i l l e d w a t e r in t h e d a r k o r in the light synthetize only one betacyanin (fractions 14 t o 22). T h i s p i g m e n t w a s f o r m e r l y i d e n t i f i e d as a m a r a n t h i n ( C o l o m a s 1977). T h e a d d i t i o n o f G A 3 caused the disappearance of this pigment in seedlings cultivated in the dark. A d d i n g k i n e t i n t o s e e d l i n g s e x p o s e d t o l i g h t ind u c e d t h e s y n t h e s i s o f a n o t h e r b e t a c y a n i n f o u n d in t h e f r a c t i o n s f r o m 2 4 t o 28. T h i s p i g m e n t h a s f o r m e r ly b e e n i d e n t i f i e d as b e t a n i n ( C o l o m a s 1977). W h e n both kinetin and GA 3 were applied, the qualitative results were very similar to those obtained with kinetin only. Seedlings Grown with DOPA. W h a t e v e r t h e t r e a t m e n t a p p l i e d (light, k i n e t i n , G A 3 ) , a m a r a n t h i n a n d b e t a n i n w e r e a l w a y s p r e s e n t i n t h e f r a c t i o n s f r o m 14 t o 22 a n d 24 t o 28, r e s p e c t i v e l y . In addition, the DOPA treatment induced the form a t i o n o f b e t a x a n t h i n s , f r a c t i o n s 32 t o 39, w h i c h h a v e p r e v i o u s l y b e e n i d e n t i f i e d as v u l g a x a n t h i n a n d
Table 1. Separation by Sephadex gel G-15 chromatography of the pigments synthesized in the different experimental conditions Fractions 14 22 2m,x 540 n m amaranthin
17 22 2max475 n m dopachrome
24 28 )~max 540 nm betanin
32-39 2m,x 475 nm betaxanthins
Water
Darkness Light
+ +
nd" nd
nd nd
nd nd
Kinetin
Darkness Light
+ +
nd nd
nd +
nd nd
GA3
Darkness Light
nd +
nd nd
nd nd
nd nd
Kinetin + GA 3
Darkness Light
+ +
nd nd
nd +
nd nd
DOPA
Darkness Light
+ +
+ +
+ +
+ +
DOPA + Kinetin
Darkness Light
+ +
+ +
+ +
+ +
DOPA +GA3
Darkness Light
+ +
+ +
+ +
nd +
DOPA +Kinetin + GA 3
Darkness
+
+
+
nd
Light
+
+
+
+
and = n o t detectable
J. Bianco-Colomas: Betalain Biosynthesis in Amaranthus
178
miraxanthin (Colomas 1977). They did n,ot appear when GA3 was applied to seedlings grown in darkness, with or without kinetin. Whatever treatment the seedlings received, the presence of another substance showing an absorption maximum at 475 nm was detected in the fractions from 17 to 22. A compound exhibiting the same characteristics (2 max and elution volume) was detected in the crude extract after incubation for 30 rain with DOPA at 23 ~ C. When the incubation lasted a longer time, 120 rain, the coloration became progressively darker and finally black. Since dopachrome, one of the intermediaries of melanogenesis, is orange colored and shows an absorption maximum at 475 rim, we propose to assimilate to it the compound found in the fractions from 17 to 22.
2. Quantitative Changes Seedlings Grown without DOPA. In the dark, on distilled water, the amaranthin synthesis was very low. Under the influence of light it increased markedly (Fig. 1A). Whereas GA3 completely inhibited the amaranthin synthesis in the dark, it induced a significant decrease in the light (Fig. 1 A). In seedlings maintained in darkness as well in those exposed to light, kinetin clearly increased the amaranthin production. Betanin appeared in small quantities under light conditions only (Fig. 1 A). GA3 inhibited significantly the kinetin induced synthesis. On the other hand, the production of betanin in very low quantities was only very slightly affected by GA 3 (Fig. 1 B).
Seedlings Grown with DOPA. In darkness, without growth regulators, the amaranthin synthesis was greatly increased under the influence of DOPA (Fig. 2A). Betanin and betaxanthins were found in small quantities, respectively, in the fractions from 24 to 28 and 32 to 39. The quantity of dopachrome found in fractions 17 to 22 was somewhat less than that of amaranthin. Under light conditions, the synthesis of all these pigments was very markedly stimulated (Fig. 2B).
% 1,o
A 9 dw, D
Z~
E E
~)"%
0 ~o 0(-
~
i
l
i
t
0 dw, L
~A
A K,L X GA3, L 9 K,D
k
0.5 i_ 0 o~
< o 14
o
16
18
20
Fraction
22
24
26
28
number
A
E 1.0
B
/~z~
L~
* K,D
I1~
A
[3 K G A 3 , L
t-' m 0.5 n .D
