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Anthocyanin biosynthesis in Celosia seedlings
ARCHIVES
OF
BIOCHEMISTRY
AND
Anthocyanin
BIOPHYSICS
56-66 (1966)
Biosynthesis
I. Locus of Anthocyanin
in Celosia
Formation
33. MALAVIYA Department
114,
AND
Seedlings
and Effect of Seedling
Age
M. M. LALORAYA
of Botany, Allahabad University, Allahabad,
India
Received July 1, 1965 Anthoeyanin formation in the seedlings of Celosia plumosa was studied in order to locate the site and the mode of its production. It is shown that seedlings grown in dark fail to produce any anthocyanin. Exposure of the dark-grown seedling to light induces snthocyanin formation. The capacity to produce anthocyanin, however, gradually declines and exhibits a linear relationship to the period of growth in dark; 5-day-old seedlings were unable to synthesize any anthocyanin. Floating the seedlings on l-2’% sucrose failed to restore the loss in capacity to induce anthocyanin formation. The light effects, and the decline in the formation of anthocyanin with the growth of the seedling in dark, therefore, are independent of photosynthetic carbo-
hydrates. Removal of cotyledons from the seedlings resulted in complete loss of capacity of anthocyanin formation in the hypocotyl, but the excision of the roots had no effect. Isolated hypocotyl sections, when floated on 14% sucrose, also failed to form anthocyanin. It is concluded that the cotyledons are the site of anthocyanin formation in Celosia seedlings. Further, the anthoeyailin formation is dependent on some factor other than a carbohydrate in its requirement of light, and this factor is rapidly lost during continued growth of seedling in the dark.
Formation of anthocyanin has been studied in different plant systems, and existing literature (5-8, 10, 12, 18-21, 24; also see 2 and 4) reveals that these plant systems differ in several important aspects from one another. Differences in the action spectra of anthocyanin production in different systems have been brought to Light (10, 11, 15, 17, 25). This would be expected when one considers that formation of anthocyanins in plants is genetically controlled (15, 2, 3, 9) and is possibly linked to RNA synthesis (22, 23). Studies on biosynthesis of anthocyanin in seedlings are as numerous as in other systems, and isolated hypocotyls of Impatiens and Buckwheat have been shown to possess the capacit,y to synthesize anthocyanins when exposed to light. Studies described in this paper, however, show that while light-grown Celosia seedlings show a
high concentration of anthocyanin in the hypocotyl-the entire hypocotyl length turning dark reddish purpl~isolated hypocotyls from seedlings previously grown in the dark are incapable of synthesizing any anthocyanin. MATERIAL
AND METHODS
Seeds of Celoaia ~l~rnosa were sprinkled on moist filter paper placed in lo-cm Petri dishes and were allowed to germinate at 27’ f 2°C in dark or light as was required. Pre~ration of rna~~rial. Intact seedlings in different stages of growth were either directly transferred to light by transferring the Petri dishes or, in those cases where anthocyanin formation in separated plant parts was to be studied, were transferred to light after completing the operations under a green safe-lamp. In an attempt to study the site of anthocyanin production in the seedlings, either the cotyledons or root alone or both were removed before exposure to white light. 56
ANTROCYANIN Anthocyanin formation in entire seedlings was measured in all cases, and in the case of excised h~oeotyls t,he entire length of hypocotyl at any given seedling age was used for experiment and analysis. Duplicate samples comprising 50 seedlings each were taken for each analysis. Light treatnlent. Seedlings and seedling parts were exposed to light of 500 Lux, made available by six ~~Iorescent tube lights (40 W, 6500 K), and the samples were taken at desired periods of incubation in light. For determming the minimum inductive light period required for anthocyanin formation, seedlings grown for 40 hours in the dark were exposed t,o light periods varying from 1 to 10,000 seconds, and the total anthocyanin formed after 3 days of incubation in the dark was determined. The incubation was carried out at 27” f 2°C. Sucrose feeding. One per cent sucrose solution was supplied to intact seedlings, just after bringing them into the light, by transferring 20 ml of the solution to Petri dishes aft,er decanting any excess water. This sucrose was available chiefly through roots. In other experiments (Table I), the intact seedlings or isolated sections were floated on sucrose solution. Determination of anthocyanin. Fifty seedlings or hypocotyls with cotyledons were dropped into 5 ml of 0.3 iv HCl, and the extraction was carried
20.
BIOSYNTHESIS.
57
I
ii 28
48 HR IN LIGHT
120 HR IN LIGHT
AFTER 24 HRIN LIGHT
DAYS OF DARK GROWTH FIG. 2. Relative amounts of anthocyanin synthesized after different periods of illuxnination in the seedlings germinated and grown in darkness. 24, After 24 hours; 48, after 48 hours of illumination; and 120, after 120 hours of illumination. (Figs. 1 and 2 are from two differeut experiments.)
out in dark at 5°C for I8 hours. No maceration was needed for extraction because of the delicate nature of the seedlings, which were very permeable to the acid. The color was measured in a Klett calorimeter equipped with a 540 rnp filter, and the number of anthoeyanin units was calculated according to the formula of Thimann and Edmondson (19) : Anthocyanin
units
per seedling
Klett 2s.
reading Number
;I01
part
X ml of extract of seedlings
RESULTS
15 t i B 8% E 4
or, seedling
3-D IN DARK 24 43 7; Gij HOwiS IN LIGHT
126
FIG. 1. Relative amounts of anthooyanin synthesized in the seedlings grown in darkness for different periods of time and then brought to light. C, Throughout in light; 48, germinated and grown in darkness for 48 hours; 72, for 72 hours; 96, for 96 hours; and, 120, for 120 hours.
~4nthoc~~~~n ~5rrn~~~n and the age of the seedling. Anthocyanin formation in Celosia seedlings is significantly altered by the duration of the seedling growth in dark prior to light exposure. Figures 1 and 2 show the results of two separate experinlents. Intact seedlings of diierent age groups growing in dark were exposed to light of different periods, and the anthocyanin content was determined after 24, 48, 72, or 120 hours. It is clear from the figures that the capacity of seedlings to synthesize anthoeyanin declines sharply as the duration of growth in
55
MALAVIYA
AND
I
24
4% 72
HOURS IN
96 120
LIGHT
FIG. 3. Relative amount,s of anthocyanin synthesized after 48 hours of illumination in the seedlings previously grown in darkness for different periods of time and fioated on water or 1% sucrose solution.
dark increases. The anthocyanin formation in continuous light shows an optimum at 3 days of growth, after which it exhibits a slight decrease. A similar pattern is shown by the seedlings previously grown for 2 and 3 days in dark, although the anthocyanin formed is only 60 and 45%, respectively, of the continuous light set. Seedlings grown for 4-s days in dark show very poor synthesis of anthocyanin, which even declines during subsequent exposure to light. Although maximum anthocyanin formation takes place in the seedlings continuously exposed to light,, the anthocyanin formed during the first, 48 hours of exposure to light in seedlings grown in dark for 48 hours is about the same as that formed in continuous light. Figure 2 shows that seedlings grown for 48 hours in dark produce more anthocyanin than those grown for 24 hours in dark on subsequent exposure to 48 hours of light, but at 120 hours the latter group of seedlings ha.s more antho~ya~n than the former. A linear decline in anthocyanin formation wit,h the age of the seedling is evident at 120 hours of light. E.fect oj suwose on anthocyanin jormation in seedlings previously grown in dark. It was of interest to study whether this loss
LALORAYA
of capacity of anthocyanin formation in the dark grown seedlings was due to loss of carbohydrates and could be restored by floating the seedlings on sucrose solution. Figure 3 shows t,he results of a typical experiment. Addition of 1% sucrose to the seedlings failed to restore this lost capacity to produce anthocyanin, indicating that the factor controlling antho~yanin formation was other than the availability of sugars. Loci of anthocyanin formation. In the seedlings grown in light and those transferred to light after 48 hours of dark growth, it was observed that the anthocyanin formation started in the cotyledons first. Only subsequently did the hypocotyl show its presence; the hypocotyl portion just below the cotyledon was t’he first to develop anthocyanin, which proceeded from the tip do~vnwards, appearing as though anthocyanin produced in the cotyledons was gradually translocated down to t,he hypocotyl. To find out whether the hypocotyl could synthesize anthocyanin, independently of cotyledons, the cotyledons together with the shoot-apex of the seedlings grown for 48 hours in dark were decapitated under a green safe-lamp, and the hypocotyl, along with the root and the excised hypocotyl free of root, were floated on distilled water or on sucrose solution in light. The anthocyanin formed in 72 hours after t,ransferring to light in the different sets was measured. Table I shows the results. It will be observed that, whereas removal of roots from the seedlings did not significantly alter anthocyanin formation, removal of cotyledons from hypocotyls resulted in complete
TABLE RELATIVE
I
AMOUNTS OF ANTHOCYANIN PRODUCED~
Mdhl
Water 1% sucrose
18.6 26.4
0 The seedlings hours; they were light and brought anthoayanin units
15.9 27.3
0.0 0.0
0.0 0.0
were grown in the dark for 48 excised in very dim green safe to light. Data are expressed as per seedling.
ANTHOCYANIN
IO 100 loo0 loo00 LIGHT INDUCTION PERIOD IN SEC,
FIG. 4. Relative amounts of anthocyanin produced after different induction periods of light, and subsequent transfer to 72 hours of darkness.
absence of anthocyanin formation in the hypocotyls up to 48 hours or even longer periods of exposure to light. Thus the hypocotyl of CeEosiczseems to be lacking in the precursors of anthocya~n format,ion. That the factor involved was other than some photosynthetic product is evident from the fact that floating the hypocotyl sections in I or 2% sucrose solution failed to induce anthocyanin format,ion in this organ.
Inductive light period. Brief exposures to light ranging from a second to 2000 seconds showed a log-linear relationship between duration of light exposure and anthocyanin formed (Fig. 4). A longer exposure of 10,000 seconds shows a deviation from linearity, the curve following a marked upward turn. However, it will be observed that during the linear part of the curve, the anthocyanin formation is only slight and the upward curvature is characteristic of a trigger release in the biosynthesis of anthocyanin format.ion during longer exposure periods. To check whether anthoeyanin itself is translocated to the hypocotyl after its synthesis in the cotyledon, or some precursor formed in light is released which subsequently produces anthocyanin in t,he hypocot,yl, the seedlings were exposed to an inductive light period sufficient to cause
BIOSYNTHESIS.
I
59
antho~y~in formation during subsequent 72 hours of incubation in dark but without any anthocyanin formation in the hypocotyl during the inductive light period. In experiments where cotyledons were excised after 5 hours (18,~~ seconds) of inductive light period and the seedlings and seedling parts subsequently exposed to 72 hours of light or 72 hours of dark, the seedlings with cotyledons formed anthocyanin both in cotyledons and in the hypocotyl, in both light and dark; excising the cotyledon from the hypocotyl resulted in failure of any anthocyanin formation in the hypocoty1 in light as we11 as in dark. The excised cotyledons which were separateIy floated in Petri dishes on water, however, showed anthocyanin formation both in light and dark, and, during 72 hours of incubation about 10 mm or more of hypocot~yl was regenerate(~ from t,he base of the cotyledons, of which the portion adjacent to t,he cotyledons contained anthocyanin. In view of the above results it appears likely that the locus of anthocyanin formation is in the cotyledons, from where it is transloeated to the hypocotyl, and that the hypocotyl itself lacks the metabolic machinery needed for anthoeyanin formation. A comparison of anthocyanin formation in Celosia seedlings and seedling parts with other seedling systems reflected some important differences. While the isolated sections of Balsam and Buck~~l~eat hypocotyls are able to synthesize anthocyanin, the isolated hypocotyls of Celosia, even in association with roots but without cotyledons, are unable to form any anthocya~n when exposed to light and in presence of sucrose in the medium. However, if the cotyledons were left together with the hypocotyl, and the root alone was removed, no effect on the ant,hocyanin formation could be observed. Troyer, working on Buckwheat (24), also reported enhancement of anthocyanin formation by added sucrose. Sucrose would appear to stimulate anthocyanin formation in t,hose tissues which possess the capacit.y to synthesize it. The Celosiu root does not appear t,o play any
60
MALAVIYA
AND LALORAYA
Dart in the initial al~thocvanin formation in seedlings. Bachelard and Stowe (1) have demonstrsted a possible link between root initiation and anthocyanin formation in Acer rubrum, but not with the growth of the roots. The locus of anthocyanin production in CeEosia seedlings is in the cotyledons, and the experiments described in this paper tend to suggest that anthocyanin synthesized in the cotyledons is subsequently t,ransported to the hypocotyl, so that the presence of anthocyanin in the hypocotyl of intact seedlings does not necessarily indicate its capacity to synthesize anthocyanin. That the ability to form anthocya~n in the hypocotyls of seedlings is gradually lost with the age of the seedling has been demonstrated in Buckwheat (24). Maximum anthocyanin formation has been observed in seedlings grown for 6-7 days in dark before being given brief light exposures, and with longer periods the capacity to form anthocyanin declines. Our results with Celoaicz indicate that during the first 48 hours of growth in dark and subsequent exposure to a light period of 48 hours, the anthocyanin formed per hypocotyl is about as much as in the seedlings germinated and grown directly for 48 hours in light. At 72 and 120 hours of exposure to light a linear decline in anthocyanin formation with the age of the seedlings is observed (Figs. 1 and 2). The data expressed are per seedling; calculat,ed as per unit weight the dechne would be even much more marked. It is pertinent to infer that the factor responsible for anthocyanin formation in the cotyledons of Celosia seedlings is rapidly lost during the seedling growth in dark and that this is not a carbohydrate. The nature of this factor is not known. However, it has been shown by Thimann and Radner that the synthesis of anthocyanin is in some way dependent on RNA synthesis, since the metabolic inhibi~rs which block RNA synthesis also inhibit anthocyanin formation (22). It is possible that the RNA and protein (enzymes) responsible for anthocyanin formation have been degraded during growth in the dark, thus dis~pting the over-all machinery for anthocyanin format’ion.
ACKNOWLEDGMENT The authors wish to thank the Head of the Botany Department for providing the laboratory facilities. One of us (B. M.) is a Fool Scientist to the Council of Scientific and Industrial Research, New Delhi, the financial assistance of which is
gratefully acknowledged.
REFERENCES 1. BACHELARD, E. P., AND STOWE, B. B., ~at~~e
194, 209 (1962). 2. BLANK, F., in “Encyclopaedia of Plant Physiology” X. W. Ruhland, ed.), p. 300. Springer Verlag, Berlin (1958). 3. BOGDAN, A. V., Meredith 18, 364 (1963). 4. BOGORAD, L., Ann. Rev. Plant Physiol. 9, 417 (1958). 5. DOWNS, R. J., AND SIEGELMAN, H. W., Plant Pkysiol. 88, 25 (1963). 6. EBERHARDT, E., Planta 48, 253 (1954). 7. EDDY, B. P., AND MAPSON, L. pi., &o&em. J. 49, 694 (1951). 8. FREY-WYSSLING, A., AND BLANK, F., Ber. Schweiz. Botan. Ges. 53 A, 550 (1943). 9. HOEN, WT.,fVa~~rw~ssensha~e~ 60, 527 (1963). 10. KLEIN, A. O., AND HAQEN, C. W., JR., PEant Physiol. 86, 1 (1961). 11. KLEIN, W. H., WITHRO~, R. B., ELSTAD, V., AND PRICE, L., Am. J. Bot. 44, 15 (1957). 12. MOHR, H., Planta 49, 389 (1957). 13. NEYLAND, M., NG, Y. L., AND THIMANN, K. V., Plant Physiol. 89, 447 (1963). 14. NG, Y. L., AND TRIMANN, K. V., Arch Biochem. Biophys. 107, 550, (1964). 15. ONSLOW, M. W., “The Anthocyanin Pigments of Plants,” University Press, Cambridge (1925). 16. PIRINGER, A. A., AND HEINZE, P. II., Plant Physiol. 29, 467 (1954). 17. RADNER, B. S., AND THTMANN, K. V., Arch. B&hem.
Biophys.
102, 93 (1963).
18. SIEGELMAN, H. W., AND HENDRICBS, PEant Physiol. 88, 185 (1953). 19. THIMANN, K. V., AND EDMONDSON, Y. H., Arch. Biochem. 22, 33 (1949). 20. THIMANN, K. V., AND RADNER, B. S., Arch. Bioehem. Biophys. 68, 484 (1955). 21. THIMANN, K. V., AND RADNER, B. S., Arch. Bioehem. Biophys. 69, 511 (1955). 22. THIMANN, II. V., AND RADNER, B. S., Arch. Biochem. Biophys. 96, 270 (1962). 23. THIMANN, K. V., AND RADNER, B. S., Arch. Biochem.
3~ophys.
102, 92 (1963).
24. TBOYER, J. R., Paant PhysioE. 89, 913 (1964). 25. WITHROW, R. B., KLEIN, W. H., PRICE, L., AND ELSTAD, V., Plant Physiol. 99, 1 (1953).
OF
BIOCHEMISTRY
AND
Anthocyanin
BIOPHYSICS
56-66 (1966)
Biosynthesis
I. Locus of Anthocyanin
in Celosia
Formation
33. MALAVIYA Department
114,
AND
Seedlings
and Effect of Seedling
Age
M. M. LALORAYA
of Botany, Allahabad University, Allahabad,
India
Received July 1, 1965 Anthoeyanin formation in the seedlings of Celosia plumosa was studied in order to locate the site and the mode of its production. It is shown that seedlings grown in dark fail to produce any anthocyanin. Exposure of the dark-grown seedling to light induces snthocyanin formation. The capacity to produce anthocyanin, however, gradually declines and exhibits a linear relationship to the period of growth in dark; 5-day-old seedlings were unable to synthesize any anthocyanin. Floating the seedlings on l-2’% sucrose failed to restore the loss in capacity to induce anthocyanin formation. The light effects, and the decline in the formation of anthocyanin with the growth of the seedling in dark, therefore, are independent of photosynthetic carbo-
hydrates. Removal of cotyledons from the seedlings resulted in complete loss of capacity of anthocyanin formation in the hypocotyl, but the excision of the roots had no effect. Isolated hypocotyl sections, when floated on 14% sucrose, also failed to form anthocyanin. It is concluded that the cotyledons are the site of anthocyanin formation in Celosia seedlings. Further, the anthoeyailin formation is dependent on some factor other than a carbohydrate in its requirement of light, and this factor is rapidly lost during continued growth of seedling in the dark.
Formation of anthocyanin has been studied in different plant systems, and existing literature (5-8, 10, 12, 18-21, 24; also see 2 and 4) reveals that these plant systems differ in several important aspects from one another. Differences in the action spectra of anthocyanin production in different systems have been brought to Light (10, 11, 15, 17, 25). This would be expected when one considers that formation of anthocyanins in plants is genetically controlled (15, 2, 3, 9) and is possibly linked to RNA synthesis (22, 23). Studies on biosynthesis of anthocyanin in seedlings are as numerous as in other systems, and isolated hypocotyls of Impatiens and Buckwheat have been shown to possess the capacit,y to synthesize anthocyanins when exposed to light. Studies described in this paper, however, show that while light-grown Celosia seedlings show a
high concentration of anthocyanin in the hypocotyl-the entire hypocotyl length turning dark reddish purpl~isolated hypocotyls from seedlings previously grown in the dark are incapable of synthesizing any anthocyanin. MATERIAL
AND METHODS
Seeds of Celoaia ~l~rnosa were sprinkled on moist filter paper placed in lo-cm Petri dishes and were allowed to germinate at 27’ f 2°C in dark or light as was required. Pre~ration of rna~~rial. Intact seedlings in different stages of growth were either directly transferred to light by transferring the Petri dishes or, in those cases where anthocyanin formation in separated plant parts was to be studied, were transferred to light after completing the operations under a green safe-lamp. In an attempt to study the site of anthocyanin production in the seedlings, either the cotyledons or root alone or both were removed before exposure to white light. 56
ANTROCYANIN Anthocyanin formation in entire seedlings was measured in all cases, and in the case of excised h~oeotyls t,he entire length of hypocotyl at any given seedling age was used for experiment and analysis. Duplicate samples comprising 50 seedlings each were taken for each analysis. Light treatnlent. Seedlings and seedling parts were exposed to light of 500 Lux, made available by six ~~Iorescent tube lights (40 W, 6500 K), and the samples were taken at desired periods of incubation in light. For determming the minimum inductive light period required for anthocyanin formation, seedlings grown for 40 hours in the dark were exposed t,o light periods varying from 1 to 10,000 seconds, and the total anthocyanin formed after 3 days of incubation in the dark was determined. The incubation was carried out at 27” f 2°C. Sucrose feeding. One per cent sucrose solution was supplied to intact seedlings, just after bringing them into the light, by transferring 20 ml of the solution to Petri dishes aft,er decanting any excess water. This sucrose was available chiefly through roots. In other experiments (Table I), the intact seedlings or isolated sections were floated on sucrose solution. Determination of anthocyanin. Fifty seedlings or hypocotyls with cotyledons were dropped into 5 ml of 0.3 iv HCl, and the extraction was carried
20.
BIOSYNTHESIS.
57
I
ii 28
48 HR IN LIGHT
120 HR IN LIGHT
AFTER 24 HRIN LIGHT
DAYS OF DARK GROWTH FIG. 2. Relative amounts of anthocyanin synthesized after different periods of illuxnination in the seedlings germinated and grown in darkness. 24, After 24 hours; 48, after 48 hours of illumination; and 120, after 120 hours of illumination. (Figs. 1 and 2 are from two differeut experiments.)
out in dark at 5°C for I8 hours. No maceration was needed for extraction because of the delicate nature of the seedlings, which were very permeable to the acid. The color was measured in a Klett calorimeter equipped with a 540 rnp filter, and the number of anthoeyanin units was calculated according to the formula of Thimann and Edmondson (19) : Anthocyanin
units
per seedling
Klett 2s.
reading Number
;I01
part
X ml of extract of seedlings
RESULTS
15 t i B 8% E 4
or, seedling
3-D IN DARK 24 43 7; Gij HOwiS IN LIGHT
126
FIG. 1. Relative amounts of anthooyanin synthesized in the seedlings grown in darkness for different periods of time and then brought to light. C, Throughout in light; 48, germinated and grown in darkness for 48 hours; 72, for 72 hours; 96, for 96 hours; and, 120, for 120 hours.
~4nthoc~~~~n ~5rrn~~~n and the age of the seedling. Anthocyanin formation in Celosia seedlings is significantly altered by the duration of the seedling growth in dark prior to light exposure. Figures 1 and 2 show the results of two separate experinlents. Intact seedlings of diierent age groups growing in dark were exposed to light of different periods, and the anthocyanin content was determined after 24, 48, 72, or 120 hours. It is clear from the figures that the capacity of seedlings to synthesize anthoeyanin declines sharply as the duration of growth in
55
MALAVIYA
AND
I
24
4% 72
HOURS IN
96 120
LIGHT
FIG. 3. Relative amount,s of anthocyanin synthesized after 48 hours of illumination in the seedlings previously grown in darkness for different periods of time and fioated on water or 1% sucrose solution.
dark increases. The anthocyanin formation in continuous light shows an optimum at 3 days of growth, after which it exhibits a slight decrease. A similar pattern is shown by the seedlings previously grown for 2 and 3 days in dark, although the anthocyanin formed is only 60 and 45%, respectively, of the continuous light set. Seedlings grown for 4-s days in dark show very poor synthesis of anthocyanin, which even declines during subsequent exposure to light. Although maximum anthocyanin formation takes place in the seedlings continuously exposed to light,, the anthocyanin formed during the first, 48 hours of exposure to light in seedlings grown in dark for 48 hours is about the same as that formed in continuous light. Figure 2 shows that seedlings grown for 48 hours in dark produce more anthocyanin than those grown for 24 hours in dark on subsequent exposure to 48 hours of light, but at 120 hours the latter group of seedlings ha.s more antho~ya~n than the former. A linear decline in anthocyanin formation wit,h the age of the seedling is evident at 120 hours of light. E.fect oj suwose on anthocyanin jormation in seedlings previously grown in dark. It was of interest to study whether this loss
LALORAYA
of capacity of anthocyanin formation in the dark grown seedlings was due to loss of carbohydrates and could be restored by floating the seedlings on sucrose solution. Figure 3 shows t,he results of a typical experiment. Addition of 1% sucrose to the seedlings failed to restore this lost capacity to produce anthocyanin, indicating that the factor controlling antho~yanin formation was other than the availability of sugars. Loci of anthocyanin formation. In the seedlings grown in light and those transferred to light after 48 hours of dark growth, it was observed that the anthocyanin formation started in the cotyledons first. Only subsequently did the hypocotyl show its presence; the hypocotyl portion just below the cotyledon was t’he first to develop anthocyanin, which proceeded from the tip do~vnwards, appearing as though anthocyanin produced in the cotyledons was gradually translocated down to t,he hypocotyl. To find out whether the hypocotyl could synthesize anthocyanin, independently of cotyledons, the cotyledons together with the shoot-apex of the seedlings grown for 48 hours in dark were decapitated under a green safe-lamp, and the hypocotyl, along with the root and the excised hypocotyl free of root, were floated on distilled water or on sucrose solution in light. The anthocyanin formed in 72 hours after t,ransferring to light in the different sets was measured. Table I shows the results. It will be observed that, whereas removal of roots from the seedlings did not significantly alter anthocyanin formation, removal of cotyledons from hypocotyls resulted in complete
TABLE RELATIVE
I
AMOUNTS OF ANTHOCYANIN PRODUCED~
Mdhl
Water 1% sucrose
18.6 26.4
0 The seedlings hours; they were light and brought anthoayanin units
15.9 27.3
0.0 0.0
0.0 0.0
were grown in the dark for 48 excised in very dim green safe to light. Data are expressed as per seedling.
ANTHOCYANIN
IO 100 loo0 loo00 LIGHT INDUCTION PERIOD IN SEC,
FIG. 4. Relative amounts of anthocyanin produced after different induction periods of light, and subsequent transfer to 72 hours of darkness.
absence of anthocyanin formation in the hypocotyls up to 48 hours or even longer periods of exposure to light. Thus the hypocotyl of CeEosiczseems to be lacking in the precursors of anthocya~n format,ion. That the factor involved was other than some photosynthetic product is evident from the fact that floating the hypocotyl sections in I or 2% sucrose solution failed to induce anthocyanin format,ion in this organ.
Inductive light period. Brief exposures to light ranging from a second to 2000 seconds showed a log-linear relationship between duration of light exposure and anthocyanin formed (Fig. 4). A longer exposure of 10,000 seconds shows a deviation from linearity, the curve following a marked upward turn. However, it will be observed that during the linear part of the curve, the anthocyanin formation is only slight and the upward curvature is characteristic of a trigger release in the biosynthesis of anthocyanin format.ion during longer exposure periods. To check whether anthoeyanin itself is translocated to the hypocotyl after its synthesis in the cotyledon, or some precursor formed in light is released which subsequently produces anthocyanin in t,he hypocot,yl, the seedlings were exposed to an inductive light period sufficient to cause
BIOSYNTHESIS.
I
59
antho~y~in formation during subsequent 72 hours of incubation in dark but without any anthocyanin formation in the hypocotyl during the inductive light period. In experiments where cotyledons were excised after 5 hours (18,~~ seconds) of inductive light period and the seedlings and seedling parts subsequently exposed to 72 hours of light or 72 hours of dark, the seedlings with cotyledons formed anthocyanin both in cotyledons and in the hypocotyl, in both light and dark; excising the cotyledon from the hypocotyl resulted in failure of any anthocyanin formation in the hypocoty1 in light as we11 as in dark. The excised cotyledons which were separateIy floated in Petri dishes on water, however, showed anthocyanin formation both in light and dark, and, during 72 hours of incubation about 10 mm or more of hypocot~yl was regenerate(~ from t,he base of the cotyledons, of which the portion adjacent to t,he cotyledons contained anthocyanin. In view of the above results it appears likely that the locus of anthocyanin formation is in the cotyledons, from where it is transloeated to the hypocotyl, and that the hypocotyl itself lacks the metabolic machinery needed for anthoeyanin formation. A comparison of anthocyanin formation in Celosia seedlings and seedling parts with other seedling systems reflected some important differences. While the isolated sections of Balsam and Buck~~l~eat hypocotyls are able to synthesize anthocyanin, the isolated hypocotyls of Celosia, even in association with roots but without cotyledons, are unable to form any anthocya~n when exposed to light and in presence of sucrose in the medium. However, if the cotyledons were left together with the hypocotyl, and the root alone was removed, no effect on the ant,hocyanin formation could be observed. Troyer, working on Buckwheat (24), also reported enhancement of anthocyanin formation by added sucrose. Sucrose would appear to stimulate anthocyanin formation in t,hose tissues which possess the capacit.y to synthesize it. The Celosiu root does not appear t,o play any
60
MALAVIYA
AND LALORAYA
Dart in the initial al~thocvanin formation in seedlings. Bachelard and Stowe (1) have demonstrsted a possible link between root initiation and anthocyanin formation in Acer rubrum, but not with the growth of the roots. The locus of anthocyanin production in CeEosia seedlings is in the cotyledons, and the experiments described in this paper tend to suggest that anthocyanin synthesized in the cotyledons is subsequently t,ransported to the hypocotyl, so that the presence of anthocyanin in the hypocotyl of intact seedlings does not necessarily indicate its capacity to synthesize anthocyanin. That the ability to form anthocya~n in the hypocotyls of seedlings is gradually lost with the age of the seedling has been demonstrated in Buckwheat (24). Maximum anthocyanin formation has been observed in seedlings grown for 6-7 days in dark before being given brief light exposures, and with longer periods the capacity to form anthocyanin declines. Our results with Celoaicz indicate that during the first 48 hours of growth in dark and subsequent exposure to a light period of 48 hours, the anthocyanin formed per hypocotyl is about as much as in the seedlings germinated and grown directly for 48 hours in light. At 72 and 120 hours of exposure to light a linear decline in anthocyanin formation with the age of the seedlings is observed (Figs. 1 and 2). The data expressed are per seedling; calculat,ed as per unit weight the dechne would be even much more marked. It is pertinent to infer that the factor responsible for anthocyanin formation in the cotyledons of Celosia seedlings is rapidly lost during the seedling growth in dark and that this is not a carbohydrate. The nature of this factor is not known. However, it has been shown by Thimann and Radner that the synthesis of anthocyanin is in some way dependent on RNA synthesis, since the metabolic inhibi~rs which block RNA synthesis also inhibit anthocyanin formation (22). It is possible that the RNA and protein (enzymes) responsible for anthocyanin formation have been degraded during growth in the dark, thus dis~pting the over-all machinery for anthocyanin format’ion.
ACKNOWLEDGMENT The authors wish to thank the Head of the Botany Department for providing the laboratory facilities. One of us (B. M.) is a Fool Scientist to the Council of Scientific and Industrial Research, New Delhi, the financial assistance of which is
gratefully acknowledged.
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