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Light control of amaranthin synthesis in isolated Amaranthus cotyledons
Phyrochemsrr?:
IY75. Vol
14, pp 479-481.
Pergamon Press. Prmted in England.
LIGHT CONTROL OF AMARANTHIN SYNTHESIS ISOLATED AA4ARANTHb’S COTYLEDONS”
IN
MARINA GIL~DICIIX NICOLA, VINCENZO AMIGO.
SIBASTIANO SCIWO and MARIO PIATTELLI Istituto
di Chimica
Organica
dell’UniversitA
(Kwircd
Key Word Index Arnura&u~~
tricolor:
A. cuudutus;
di Catania,
Catania,
ltalia
IO .JMlJ 1974) Amdranthaceae;
biosynthesis;
light:
phytochrome:
beta-
lains; amaranthin.
Abstract--The effect of light on the amaranthin synthesis stimulated by exogenous precursors has been studied in isolated cotyledons of A~~rauthus tricoh and A. cuudatus. The results indicate that light acts at the level of the formation of the dihydropyridine moiety of the pigment.
INTRODUCTION
of amaranthin
Betalains (I), the characteristic pigments of Centrospermae, may be divided into two groups, the red--violet betacyanins (e.g. 2a) and the yellow betaxanthins, in which the dihydropyridine moiety is bound to an amine or amino acid other than S-cycloDOPA (5,6-dihydroxy-2,3-dihydro-2S-indole-2-carboxylic acid). Tracer experiments [l-4] have shown that the dihydropyridine unit originates, possibly via betalamic acid, from DOPA (L-3,4_dihydroxyphenylalanine); this amino acid is also precursor of the dihydroxydihydroindole moiety of betacyanins [S]. Early studies by Garay and Towers [6] on the efficacy of various compounds on the production RO
-0,c
Y-Y----
Fx N
cogi
CO,H
A
il
(2)
(1)
(la) (lb) (1~)
(2a) R=H
;
R’= H R” = HO&-
CH2- CHz-CH
- COzH
;
R’= H R” = HOzC -
CHr
(2b)
R = (qlucuronic 9lucosyl
acid)-
CH - CO,H
R’= H ; R*= (oH)&H~-c&-CH~C’+CO@
* This work was supported Ricerche.
by the Consiglio
Nazionale
delle
(2b) in illuminated
seedlings
of
Amaranthus tricolor showed, however, that DOPA was less effective than tyrosine. To explain this unexpected result, it was supposed that any DOPA fed was largely oxidized by phenoloxidases to melanin-like black compounds. In the presence of ascorbic acid, blackening of DOPA-treated seedlings was inhibited, but amaranthin production was not enhanced. In fact, administration of ascorbic acid together with DOPA or tyrosine resulted in a decreased synthesis of amaranthin. Further experiments by French et al. [7] indicated that in seedlings of A. caudatus var. uiridis exogenously supplied DOPA elicits a moderate stimulation of dark synthesis of amaranthin, and tyrosine a still smaller promotion, while in illuminated seedlings the accumulation of pigment is considerably greater and of similar magnitude in the presence of either of these precursors. On the basis of this and related observations these authors suggested that light promotes at least two reactions in the biosynthetic pathway, one between tyrosine and DOPA and a second between DOPA and amaranthin. This communication describes a study on the amaranthin synthesis in the dark and in the light by isolated cotyledons of A. caudatus and A. tricolor., in the presence of exogenous precursors. The results show that formation of the dihydropyridine moiety from DOPA is light controlled, but do not confirm or contradict the assumption [7] that the reaction tyrosine-+ DOPA is stimulated by light.
M. Gtr.rwt
480
NICOLA.
III
V. AMIGO. S. Scrr TO and M. PIAITI LLI
In addition to t\rosine and DOPA. the effect of cqcloDOPA has been oxamincd. When in preliminary expcriment~ intact seedlings of .4. fr.i~~o/or.cv. Illumination and J. CLU/&/~~/.S WCK used. the precursors being administcrcd in aqueous solution via the roots. formation of melanin-like pigment was observed both inside the seedlings and in the incubation medium. partic~llarl~ with DOPA and cycIoDOPA. Hlackcning was also obscrvcd when prccursor5 \vcrc supplied to de-rooted seedlings through the cul end. Howcwr. melanin formation was not visually dctcctablc using cotyledons isolatcd from cytiolated Z-day-old seedlings, which WCI~C thorcforc utili& throughout this work. Since it was obscrvcd that crude extracts cjbtaincd from cotyledons fed with precursors contained large amounts of DOPAchrotnc. it proved necessary to purify hy chromatography the amaranthin formed in cac11 individual experiment bcforc its quantitation 131 spcctroyhotometr4. In cxpcriments with light, the illumination period was no longer than 6 hr. to prc~nt complications due to the onset of photosynthetic activity. The results. summari& in Table 1. clearly show that in both ,A. [ricdor. which has an ahsolute light rcquiremcnt for pigment s) nthesis. and in A. CLUItltrtu.~ which is capal~l~ to s~nthcsira in darkness,
______
Treatment compound (concn in tng,:ml)
______
Dark C‘ontrol Tjrosinc (0.5) DOPA (0.5) C‘~cloDOPA (O-5) I5 Illill Nhik light T> rosinc (0.51 DOPA (0.5) (‘~cloDOPA (O-5) 6 hr whtti‘ lb&t Tyrwinc (0.05) Tjros~nc (0.1) Tyt-wine IO.51 DOPA (O.U5) DOPA (0.1) DOPA (0-j) C‘~cloDoP:~ (0.5) __
_
~~~___~__~.. ___
_______
(10
Amaranthin ‘I mol swdling)
1. cxrur/o/i/\
-.____
__--_
1.75 4.42 7.34 2.70 4.06 5.YS
10~37 4, IO
IO.XS I I ,.;h 14.3
11.r~ico/oJ~
0~00 0.‘) 5 2.43 0.00 I.21 2.36 4.9 I I-24 x.7.5 I 0.‘)0 I J-34
I (YS?
iFi.37
13.46 3ON 37.10 I (!.75
12-24
.._~.
.---
17.85 23.7 I x.70
_._____~~___
DOPA has a stimtlloting clfcct grcatcr than that of tyrosinc under all the ckpcrimcntal conditions employed. From the litct that thcsc amino acids are apt to stimulate the dark synlhcsis it can lx deduced that cotyledons. all thcx from the spctics with compulsory light mluircment. lv~css the complete biochcmicnl apparatus necessary I’OI their utilimtion in the amaranthin h>nthcsis. The activit!, of this apparatus is strongly enhanced hy a 6 hr irradiation Mith u.hitc light. ;I> sho\vn 17~the fact that in illuminated cot!lcdons fed with t)rosine or DOPA the amaranthin accumulation is much greater than in dark controls supplied with the same precursors. Tllis anJ ohscrvution that cycloDOPA failed to enha~~ce amaranthin production in the dark indicate that light acts on the rcaction(s) Lading from DOPA to the dihqdrop!ridinc moicfy. C’onsidcring that ;t I5 Inin illumination period. which is in gcncral suliicicnt to tlic photoactivation of phytochromc. has a much smaller stimulatory effect, it appears rcasonablc to assume. in agretmcnt with previous findings LX]. thn t phytochromc is not the onI> photoreceptor mediating this action of light. In the course of amaranthin isolation it was observed that small amounts of a yellow pigment were prcscnt in cot$cdons illuminated and,‘or fed with DOPA. This pigment was shown to he a mixture of a number of betaxanthins. among them vulgaxanthin II (la), and miraxanthin II (lb) [9, IO]; DOPAxanthin (1~) [i I] was cntircly absent. even when cotyledons wcrc supplied with DOPA. and this dcmonstratcs that iu riro ;I spontaneous condensation of DOPA with hctalamic acid or ;I chemical equivalent dots not t:lke place. In conclusion. the results prcscntcd hcrc suggest that the photocontrol of amaranthin sbnthcsis occurs at the level of the formation of the dihydropyridine portion ofthc molcculc. and that the light requirerncnt is not rclatcd to the low-encrgb phytochromc system alone. There is no e~idencc that light influcnccs other points on the bias! nthetic pathway. e.g. the reaction tyrosinc + DOPA as claimed by French (21rrl. 171. neither do the data reported in the prcscnt paper denq this possibilit>.
Light control
of amaranthin
dishes on 2 layers of tilter paper moistened with tap water and used when 2-days-old. Chrrl7icals. Chemicals were obtained commercially. except for S-cycloDOPA HCI which was synthesized according to the method of Wyler and Chiovini [12]. Fecdirry. Cotyledons were isolated from seedlings selected on the basis of uniform size and allowed to absorb the requisite compound (L-tyrosinc. L-DOPA or S-cycloDOPA) for 6 hr. At the end of the feeding period cotyledons were, where necessary, illuminated with fluorescent white light (5000 lx) for the desired length of time and returned to darkness. Pigment extraction was performed 24 hr after the onset of the experiment. Mrthorls r?/ rrnrr/,r:si.s.At the end of each treatment an equivalent number of cotyledons were homogenized in acetate buffer pH 4.5 and the homogenate. clarified by centrifugation (IOOOO~~). was passed through a column of Dowex 5OW-X2 (H’). After washing with O.l”,, HCI, pigment was eluted with H,O and amaranthin estimated by I’V spectrophotometry. Chromdtographic analysis of the eluate showed it to contain. in addition to amaranthin. small amounts of betaxanthins; the maior yellow pigments were identified as vulgaxanthin II and miraxanthin II by comparison (co-TLC, co-electrophoresis and degradation experiments) with authentic samples. DOPAxanthin was not present in the pigment mixture.
synthesis
481
REFERENCES I. Minalc.
2.
3. 4. 5. 6.
L.. Piattelli. M. and Nicolaus. R. A. (1965) P/IJTOchumistrj~ 4, 593. Miller. H. E.. Rosier. H., Wohlpart, A.. Wyler-, H.. Wilcox, M. E.. Frohofer-. H.. Mabrv. T. J. and Drcidine. A. S. (1968) Heft. Chirn. Actu 51, 1470: Fischer. N. and Dreiding. A. S. (1968) Heir. Chir77. Acta 55, 649. Impellizzeri. G. and Piattelli, M. (1972) Ph~tocl7r1r7i,st1~~ II, 2499. Sciuto. S.. Orientc. G.. Piattelli. M.. Impellizzeri, G. and Amico. V. (I 974) Ph~foc/7~~,,~i,sr~~~ 13, 947. _ Garay, A. S. and Towers. G. H. N. (1966) Carl. J. Botm) 44,231.
7. French. C. J.. Pccket. R. C. and Smith. H. (1973) Phyrocl7r~77isfry 12, 2887. 8. Giudici de Nicola, M. Piattelli. M. and Amico, V. (1972) Pl7!~toch~r~ri.stry 11, 1005. 9. Piattclli. M.. Minale. L. and Prota. G. (1965) Ph!~toche~nisrry4, 121. 10. Piattelli. M.. Minale. L. and Nicolaus. R. A. (1965) Pl7ytochc,77isfry 4, 8 17. I I. lmpellizzeri. G.. Piattelli, M. and Sciuto, S. (1973) Phyrocl7ru7isrr!~ 12, 2293. 12. Wylcr. H. and Chiovini, J. (1968) He/r. Chirn. Actu 51, 1476.
IY75. Vol
14, pp 479-481.
Pergamon Press. Prmted in England.
LIGHT CONTROL OF AMARANTHIN SYNTHESIS ISOLATED AA4ARANTHb’S COTYLEDONS”
IN
MARINA GIL~DICIIX NICOLA, VINCENZO AMIGO.
SIBASTIANO SCIWO and MARIO PIATTELLI Istituto
di Chimica
Organica
dell’UniversitA
(Kwircd
Key Word Index Arnura&u~~
tricolor:
A. cuudutus;
di Catania,
Catania,
ltalia
IO .JMlJ 1974) Amdranthaceae;
biosynthesis;
light:
phytochrome:
beta-
lains; amaranthin.
Abstract--The effect of light on the amaranthin synthesis stimulated by exogenous precursors has been studied in isolated cotyledons of A~~rauthus tricoh and A. cuudatus. The results indicate that light acts at the level of the formation of the dihydropyridine moiety of the pigment.
INTRODUCTION
of amaranthin
Betalains (I), the characteristic pigments of Centrospermae, may be divided into two groups, the red--violet betacyanins (e.g. 2a) and the yellow betaxanthins, in which the dihydropyridine moiety is bound to an amine or amino acid other than S-cycloDOPA (5,6-dihydroxy-2,3-dihydro-2S-indole-2-carboxylic acid). Tracer experiments [l-4] have shown that the dihydropyridine unit originates, possibly via betalamic acid, from DOPA (L-3,4_dihydroxyphenylalanine); this amino acid is also precursor of the dihydroxydihydroindole moiety of betacyanins [S]. Early studies by Garay and Towers [6] on the efficacy of various compounds on the production RO
-0,c
Y-Y----
Fx N
cogi
CO,H
A
il
(2)
(1)
(la) (lb) (1~)
(2a) R=H
;
R’= H R” = HO&-
CH2- CHz-CH
- COzH
;
R’= H R” = HOzC -
CHr
(2b)
R = (qlucuronic 9lucosyl
acid)-
CH - CO,H
R’= H ; R*= (oH)&H~-c&-CH~C’+CO@
* This work was supported Ricerche.
by the Consiglio
Nazionale
delle
(2b) in illuminated
seedlings
of
Amaranthus tricolor showed, however, that DOPA was less effective than tyrosine. To explain this unexpected result, it was supposed that any DOPA fed was largely oxidized by phenoloxidases to melanin-like black compounds. In the presence of ascorbic acid, blackening of DOPA-treated seedlings was inhibited, but amaranthin production was not enhanced. In fact, administration of ascorbic acid together with DOPA or tyrosine resulted in a decreased synthesis of amaranthin. Further experiments by French et al. [7] indicated that in seedlings of A. caudatus var. uiridis exogenously supplied DOPA elicits a moderate stimulation of dark synthesis of amaranthin, and tyrosine a still smaller promotion, while in illuminated seedlings the accumulation of pigment is considerably greater and of similar magnitude in the presence of either of these precursors. On the basis of this and related observations these authors suggested that light promotes at least two reactions in the biosynthetic pathway, one between tyrosine and DOPA and a second between DOPA and amaranthin. This communication describes a study on the amaranthin synthesis in the dark and in the light by isolated cotyledons of A. caudatus and A. tricolor., in the presence of exogenous precursors. The results show that formation of the dihydropyridine moiety from DOPA is light controlled, but do not confirm or contradict the assumption [7] that the reaction tyrosine-+ DOPA is stimulated by light.
M. Gtr.rwt
480
NICOLA.
III
V. AMIGO. S. Scrr TO and M. PIAITI LLI
In addition to t\rosine and DOPA. the effect of cqcloDOPA has been oxamincd. When in preliminary expcriment~ intact seedlings of .4. fr.i~~o/or.cv. Illumination and J. CLU/&/~~/.S WCK used. the precursors being administcrcd in aqueous solution via the roots. formation of melanin-like pigment was observed both inside the seedlings and in the incubation medium. partic~llarl~ with DOPA and cycIoDOPA. Hlackcning was also obscrvcd when prccursor5 \vcrc supplied to de-rooted seedlings through the cul end. Howcwr. melanin formation was not visually dctcctablc using cotyledons isolatcd from cytiolated Z-day-old seedlings, which WCI~C thorcforc utili& throughout this work. Since it was obscrvcd that crude extracts cjbtaincd from cotyledons fed with precursors contained large amounts of DOPAchrotnc. it proved necessary to purify hy chromatography the amaranthin formed in cac11 individual experiment bcforc its quantitation 131 spcctroyhotometr4. In cxpcriments with light, the illumination period was no longer than 6 hr. to prc~nt complications due to the onset of photosynthetic activity. The results. summari& in Table 1. clearly show that in both ,A. [ricdor. which has an ahsolute light rcquiremcnt for pigment s) nthesis. and in A. CLUItltrtu.~ which is capal~l~ to s~nthcsira in darkness,
______
Treatment compound (concn in tng,:ml)
______
Dark C‘ontrol Tjrosinc (0.5) DOPA (0.5) C‘~cloDOPA (O-5) I5 Illill Nhik light T> rosinc (0.51 DOPA (0.5) (‘~cloDOPA (O-5) 6 hr whtti‘ lb&t Tyrwinc (0.05) Tjros~nc (0.1) Tyt-wine IO.51 DOPA (O.U5) DOPA (0.1) DOPA (0-j) C‘~cloDoP:~ (0.5) __
_
~~~___~__~.. ___
_______
(10
Amaranthin ‘I mol swdling)
1. cxrur/o/i/\
-.____
__--_
1.75 4.42 7.34 2.70 4.06 5.YS
10~37 4, IO
IO.XS I I ,.;h 14.3
11.r~ico/oJ~
0~00 0.‘) 5 2.43 0.00 I.21 2.36 4.9 I I-24 x.7.5 I 0.‘)0 I J-34
I (YS?
iFi.37
13.46 3ON 37.10 I (!.75
12-24
.._~.
.---
17.85 23.7 I x.70
_._____~~___
DOPA has a stimtlloting clfcct grcatcr than that of tyrosinc under all the ckpcrimcntal conditions employed. From the litct that thcsc amino acids are apt to stimulate the dark synlhcsis it can lx deduced that cotyledons. all thcx from the spctics with compulsory light mluircment. lv~css the complete biochcmicnl apparatus necessary I’OI their utilimtion in the amaranthin h>nthcsis. The activit!, of this apparatus is strongly enhanced hy a 6 hr irradiation Mith u.hitc light. ;I> sho\vn 17~the fact that in illuminated cot!lcdons fed with t)rosine or DOPA the amaranthin accumulation is much greater than in dark controls supplied with the same precursors. Tllis anJ ohscrvution that cycloDOPA failed to enha~~ce amaranthin production in the dark indicate that light acts on the rcaction(s) Lading from DOPA to the dihqdrop!ridinc moicfy. C’onsidcring that ;t I5 Inin illumination period. which is in gcncral suliicicnt to tlic photoactivation of phytochromc. has a much smaller stimulatory effect, it appears rcasonablc to assume. in agretmcnt with previous findings LX]. thn t phytochromc is not the onI> photoreceptor mediating this action of light. In the course of amaranthin isolation it was observed that small amounts of a yellow pigment were prcscnt in cot$cdons illuminated and,‘or fed with DOPA. This pigment was shown to he a mixture of a number of betaxanthins. among them vulgaxanthin II (la), and miraxanthin II (lb) [9, IO]; DOPAxanthin (1~) [i I] was cntircly absent. even when cotyledons wcrc supplied with DOPA. and this dcmonstratcs that iu riro ;I spontaneous condensation of DOPA with hctalamic acid or ;I chemical equivalent dots not t:lke place. In conclusion. the results prcscntcd hcrc suggest that the photocontrol of amaranthin sbnthcsis occurs at the level of the formation of the dihydropyridine portion ofthc molcculc. and that the light requirerncnt is not rclatcd to the low-encrgb phytochromc system alone. There is no e~idencc that light influcnccs other points on the bias! nthetic pathway. e.g. the reaction tyrosinc + DOPA as claimed by French (21rrl. 171. neither do the data reported in the prcscnt paper denq this possibilit>.
Light control
of amaranthin
dishes on 2 layers of tilter paper moistened with tap water and used when 2-days-old. Chrrl7icals. Chemicals were obtained commercially. except for S-cycloDOPA HCI which was synthesized according to the method of Wyler and Chiovini [12]. Fecdirry. Cotyledons were isolated from seedlings selected on the basis of uniform size and allowed to absorb the requisite compound (L-tyrosinc. L-DOPA or S-cycloDOPA) for 6 hr. At the end of the feeding period cotyledons were, where necessary, illuminated with fluorescent white light (5000 lx) for the desired length of time and returned to darkness. Pigment extraction was performed 24 hr after the onset of the experiment. Mrthorls r?/ rrnrr/,r:si.s.At the end of each treatment an equivalent number of cotyledons were homogenized in acetate buffer pH 4.5 and the homogenate. clarified by centrifugation (IOOOO~~). was passed through a column of Dowex 5OW-X2 (H’). After washing with O.l”,, HCI, pigment was eluted with H,O and amaranthin estimated by I’V spectrophotometry. Chromdtographic analysis of the eluate showed it to contain. in addition to amaranthin. small amounts of betaxanthins; the maior yellow pigments were identified as vulgaxanthin II and miraxanthin II by comparison (co-TLC, co-electrophoresis and degradation experiments) with authentic samples. DOPAxanthin was not present in the pigment mixture.
synthesis
481
REFERENCES I. Minalc.
2.
3. 4. 5. 6.
L.. Piattelli. M. and Nicolaus. R. A. (1965) P/IJTOchumistrj~ 4, 593. Miller. H. E.. Rosier. H., Wohlpart, A.. Wyler-, H.. Wilcox, M. E.. Frohofer-. H.. Mabrv. T. J. and Drcidine. A. S. (1968) Heft. Chirn. Actu 51, 1470: Fischer. N. and Dreiding. A. S. (1968) Heir. Chir77. Acta 55, 649. Impellizzeri. G. and Piattelli, M. (1972) Ph~tocl7r1r7i,st1~~ II, 2499. Sciuto. S.. Orientc. G.. Piattelli. M.. Impellizzeri, G. and Amico. V. (I 974) Ph~foc/7~~,,~i,sr~~~ 13, 947. _ Garay, A. S. and Towers. G. H. N. (1966) Carl. J. Botm) 44,231.
7. French. C. J.. Pccket. R. C. and Smith. H. (1973) Phyrocl7r~77isfry 12, 2887. 8. Giudici de Nicola, M. Piattelli. M. and Amico, V. (1972) Pl7!~toch~r~ri.stry 11, 1005. 9. Piattclli. M.. Minale. L. and Prota. G. (1965) Ph!~toche~nisrry4, 121. 10. Piattelli. M.. Minale. L. and Nicolaus. R. A. (1965) Pl7ytochc,77isfry 4, 8 17. I I. lmpellizzeri. G.. Piattelli, M. and Sciuto, S. (1973) Phyrocl7ru7isrr!~ 12, 2293. 12. Wylcr. H. and Chiovini, J. (1968) He/r. Chirn. Actu 51, 1476.