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Variations in the production of carotenoids in Neurospora
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
70, 561-567 (1957)
Variations in the Production of Carotenoids in Neurosporal Marko Zalokar From the Department
of Microbiology,
Yale
Received
October
University,
New Haven,
Connecticut
22, 1956
INTRODUCTION
The production of carotenoids in microorganisms can be modified by a variety of conditions, such as changes in nutrition (l), availability of vitamins (2), aeration of the culture (3), or the addition of particular chemicals, e.g., diphenylamine (4). The present paper reports the use of similar methods to obtain significant variations in the production of different carotenoids in Neurospora. These variations may provide useful clues as to the pathway of carotenogenesis, its dependence on genes, and its variation in different cell types. MATERIAL
AND METHODS
The wild strain of Neurospora crassa, 5297a, was grown at 35°C. for 3 days on 25 ml. of Fries minimal medium containing one drop of polyoxyethylene sorbitan monooleate (Tween 80) to prevent the formation of conidia. The mycelium pads were collected on a Btichner funnel by filtration and exposed to air and light for the given period. Chemicals were added to the medium, or the mycelial pads were soaked in the appropriate solution and the excess liquid filtered off. The mycelia were treated three times with 10 ml. of absolute methanol to extract all carotenoids except spirilloxanthin, which was then extracted with benzene. The methanol extract was made alkaline with 2 ml. of 2Oyo KOH and shaken with 10 ml. of hexane. After separation of the resulting phases, alkaline alcohol contained the acidic pigments and hexane the remaining carotenoids. Phytoene was isolated by chromatographing the hexane fraction on a magnesium oxide (U.S.P., heavy powder) column. The column was developed with hexane, and the fractions containing phytoene were collected. A reasonable estimation of total colored carotenoids in the crude methanol extracts could be made by the light absorption at 464 mr, assuming the extinction coefficient B:,%. = 2600. The same extinction coefficient was used to estimate r-carotene, neurosporene, and lycopene (designated tarot. in the tables) found in the hexane phase after fractiona1 This investigation was supported by a research grant (PHS RG 4560 C) from the Division of Research Grants, U. S. Public Health Service. 561
562
ZALOKAR
tion. Spirilloxanthin was measured in benzene solution using the extinction coefficient of the heat-isomerized compound at 541 rnp, E:& = 1605 (5). Phytoene was determined by optical extinction at 297 rnp, [El’,%. = 1030 (6)1. The acidic pigments were estimated by the light absorption at 464 rnp, using the extinction coefficient of their major component, neurosporaxanthin (7) E1’,$. = 2680. RESULTS
AND
DISCUSSION
Lack of Oxygen and Enzyme Poisons The Neurospora mycelium grown in still cultures with a low oxygen supply contains only phytoene, phytofluene, and some l-carotene. After exposure to air and light, colored carotenoids are formed and the phytoene content is reduced (8). Neither exposure to air without photoactivation or photoactivation with subsequent lack of oxygen could initiate the synthesis of pigments or the disappearance of phytoene (Table I). Diphenylamine, added to the grown mycelium before or after photoTABLE
I
Effect of Air and Light on Phytoene Content of the Mycelium Three-day-old mycelium pads were exposed periments, six flasks each, are given. Exposed to:
Control Air, light Air, dark Nz , light
Time hr.
as indicated.
Dry weight/pad WT.
0 4 4 4
69.5 74.5 77.0 63.5 TABLE
Effect of Diphenylamine
folld pg.
of two ex-
Phytoene mg./lOO g.
8.4 5.9 10.1 9.8
12.1 8.0 13.1 15.5
II on Carotenogenesis
Three-day-old mycelia were exposed to air and light experiments, one flask each. Dry weight/pad
Control, no add. Diphenylamine (1:40,000) added 1 hr. before photoactivation Diphenylamine added 1 hr. after photoactivation Diphenylamine added 1 hr. before photoactivation, washed off after photoactivation
Averages
for 6 hr. Average
of three
Total pigments Extinction Amount mg./lOO g.
73.; 64.5
0.290 0.051
36.6 7.7
79.8
0.092
11.1
68.4
0.247
34.8
CAROTENOIDS
IN
563
NEUROSPORA
TABLE III Egect of Sodium A.&de on Carotenogenesis Mycelia were exposed to air and light for 6 hr. Chemical added just before photoactivation. Average of two experiments, three flasks each. Additions
No add. NaNa 0.001% NaNa 0.002% NaNa 0. O04yo NaNS 0.008%
Dry weight/pad m.
65 55 51 53 55
Acidic mg.j100
Carot. g.
24.6 19.9 5.1 4.7 3.2
mg./lOO
5.8 5.7 6.4 5.9 5.2
Spiril. g.
mg./100
g.
3.2 3.0 2.3 1.5 1.3
activation in amounts known to inhibit the formation of colored polyenes (9), had a similar effect to that of oxygen lack. When this chemical was present during photoactivation and then washed out, pigmentation subsequently proceeded in the dark at a rate only slightly lower than in untreated controls (Table II). This shows that the chemical does not interfere with photoactivation but with the processes following it, probably by acting as a respiratory enzyme poison. Most respiratory enzyme poisons were toxic to the mycelium without specifically altering pigmentation. Sodium azide differed in that, at appropriate concentrations, it inhibited growth but allowed pigmentation to proceed. Pigment analyses revealed that sodium aside inhibited the formation of neurosporaxanthin, and, to a lesser degree, of spirilloxanthin, but had no effect on the production of other colored polyenes (Table III). As sodium azide is an inhibitor of cytochrome oxidase, this would indicate that a fully active cytochrome system was not required for the production of carotenoids, with the possible exception of acidic pigments. This last possibility can be ruled out by the observation that cytochrome oxidase-deficient strains of Neurospora [poky and C-117 (lo)] contain a normal array of carotenoids not showing any decrease in acidic pigments. Vitamins
and Nutrients
Biotin is the only vitamin required by wild-type Neurospora. Cultures deficient in biotin (containing 0.2 pg. biotin/l. of medium) showed a marked increase in carotenes and spirilloxanthin, and a decrease in acidic pigments (Table IV). Whereas in the controls the spirilloxanthin content rose to a peak in 2 hr. after photoactivation and then decreased, in biotin-deficient cultures it continued to rise for the succeeding 2 hr. (Fig. 1). This suggests that one function of biotin is to form a system
564
ZALOKAR
TABLE IV Effect of Nutrition on Carotenogenesis Mycelia were grown on given media for 3 days, then exposed to air and light for 6 hr. Average of two experiments, three flasks each. Medium
Dry weight/pad m.
Acidic g./lOO g.
Carat. mg./lOO g.
Spiril. ?ng./mo 8.
70 27.3 6.1 1.05 Minimal (5 pg./l. biotin) 50 17.3 16.5 13.2 -biotin (0.2 pg./l. biotin) 34 15.7 13.4 32.8 -biotin + glycine 0.01 N 45 32.0 12.3 20.2 -biotin + biotin (100 pg./ l.)O 54 16.1 7.8 2.2 Minimalb 45 22.0 14.0 3.2 Min. + glycine 0.01 Mb 56 5.5 3.2 2.0 Min. + peptone 0.50Job 58 10.3 8.6 5.5 Min. + peptone 0.5% + glycine 0.01 Mb 0 Added to grown mycelium before exposure to air and light. b Experiments performed at another time, whence different dry weight and pigment values.
I
2
3
4
5
6
hours
Fro. 1. Spirilloxanthin (s) and carotenes (c) (including neurosporaxanthin) found in normal (+ open circles) and biotin-deficient (- black circles) mycelia at different times after exposure to light and air.
CAROTENOIDS
IN
565
NEUROSPORA
transforming this pigment. The addition of biotin after photoactivation cannot restore this system, since it increases rather than supresses the accumulation of spirilloxanthin. A different function of biotin is manifest in the decrease of the acidic pigments in deficient mycelia; this can be restored to normal when the vitamin is added afterphotoactivation. Of complex nutrients and ammo acids tried, yeast extract, hydrolyzed casein, and most individual amino acids had no effect on pigmentation in Neurospora. Peptone decreased, while glycine increased all carotenoids. Glycine restored pigmentation in peptone cultures, increasing particularly spirilloxanthin. The spirilloxanthin-promoting action of glycine was also found in biotin-deficient cultures. Valine and leucine, which stimulate carotenogenesis in Phycomyces (11)) had no effect in Neurospora, or depressed pigmentation when used as the sole nitrogen source. TABLE
V
Disappearance of Phytoene and Production Pigments
of Carotenoid
after Photoactivation
Polyenes were extracted from mycelia combined from six flasks at 0 hr. and at 4 hr. after exposure to air and light.
-
DIY reight/ pad
Growth medium or sddn. after photoactivation
hr.
Minimal,
no addn.
Minimal, add. NaN 0.002% -biotin
(0.2 pg./l.)
0
ms.
Phytoene
mg./lOO
70 72 76 74 62 60 -___
16.8 16.2 7.8 8.3 13.7 13.7
46 46 47 45
22.2 22.4 15.4 13.4
g.
Acidic
ng./lOO g. -
17.8
19.0 7.6 7.2
T
Carot.
Spiril.
L&/100 $
lg./loo g
-
-
6.8 6.6 8.4 8.4
5.3 4.6 3.4 3.6
-
0.5%
60 65 62
Total pigments
‘hytoem? used
ng./lOO g.
100 g.
mg./
8.5 19.3
2.8
31.5
7.8
_-
-
-
-
7.2 10.2
17.1 17.8
12.3 18.3 _-
fpeptone
I
Average
7.7 4.5 4.7
11.4 14.2
-
-
3.5 4.0
1.2 1.6 -
566
ZALOKAR
Loss of Phytoene and Pigmentation Previous studies (8) posed the question of whether the phytoene which disappeared during the first 4 hr. after photoactivation was converted into a colored carotenoid. If this was the case, we may expect that in pigment variants the amount of phytoene used would remain proportional to the amount of pigment formed. The carotenoid content of mycelia subjected to different conditions was measured before and 4 hr. after exposure to air and light (Table V). The same amount of phytoene disappeared in the normal and in the biotin-deficient cultures, although the former produced twice as much neurosporaxanthin and less than half as much carotenes and spirilloxanthin as the latter. The loss of phytoene was much smaller in cultures poisoned with sodium azide and in cultures grown on peptone, but the former produced more carotenes and less acidic pigment, while the latter produced as much or more of the acidic pigment as the biotin-deficient mycelia. The disappearance of phytoene therefore cannot be related to the formation of any particular carotenoid. The fact that the amount of pigments formed exceeded in all cases that of phytoene used shows that the existing phytoene can be, if at all, only a partial source of colored polyenes. New carotenoids must be synthesized from precursors which were present in the mycelium before photoactivation or produced as a result of photoactivation. Conclusions The various means of inducing changes of mycelium pigmentation show that carotenogenesis is widely subject to environmental control. This raises the question of whether in different pigment mutants the genes act directly on the biosynthetic chain of these pigments or indirectly by providing a metabolic environment enhancing or inhibiting the formation of a particular polyene. The existence of the albino mutant, producing phytoene and no other pigments (12), does not necessarily require a direct block in the conversion of phytoene to colored polyenes; the same effect could be obtained by a deficiency in the oxidation mechanism, preventing the conversion of unknown precursors into colored polyenes. The lack of oxygen or the action of diphenylamine provides good phenocopies for such a gene action. Sodium azide produces a phenocopy of the “yellow” mutant (12) which lacks acidic pigments. Similarly, the pigment changes occuring in differentiated cells could be a result of a particular metabolic environment, without
CAROTENOIDS IN NEUROSPORA
567
direct involvement of genes.The action of glycine and of biotin deficiency can serve as a model for the metabolic state of conidia, where spirilloxanthin is the predominant pigment (8). SUMMARY
The formation of pigments after photoactivation was inhibited by lack of oxygen or addition of diphenylamine; sodium azide inhibited only the formation of acidic pigments. Deficiency of biotin or addition of glycine increased spirilloxanthin content; the addition of peptone decreased all carotenoids. The disappearance of phytoene could not be related to the formation of any particular carotenoid in these pigment variants. REFERENCES 1. GOODWIN, T. W., “The Comparative
Biochemistry of the Carotenoids.” Chapman & Hall Ltd., London, 1952. 2. STARR, M. P., AND SAPERSTEIN, S., Arch. Biochem. Biophys. 43, 157 (1953). 3. VAN NIEL, C. B., Antonie van Leeuwenhoek J. Microbial. Serol. 12, 156 (1947). 4. TIJRIAN, G., Helv. Chim. Acta 33, 1988 (1950). 5. POLQ~R, A., VAN NIEL, C. B., AND ZECHMEISTER, L., Arch. Biochem. 6, 243 (1944). 6. RABOURN, W. J., QUACBENBUSH, F. W., AND PORTER, J. W., Arch. Biochem. Biophys. 43, 267 (1954). 7. ZALOKAR, M., Arch. Biochem. Biophys. 70, 568 (1957). 8. ZALOKAR, M., Arch. Biochem. Biophys. 60, 71 (1954). 9. TURIAN, G., AND HAXO, F., J. Bacterial. 63, 690 (1952). 10. TISSIERES, A., AND MITCHELL, H. K., J. Biol. Chem. 206, 241 (1954). 11. GOODWIN, T. W., AND LIJINSKY, W., Biochem. J. 60, 268 (1951). 12. HAXO, F., Fortschr. Chem. org. Naturstoffe 12, 169 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
70, 561-567 (1957)
Variations in the Production of Carotenoids in Neurosporal Marko Zalokar From the Department
of Microbiology,
Yale
Received
October
University,
New Haven,
Connecticut
22, 1956
INTRODUCTION
The production of carotenoids in microorganisms can be modified by a variety of conditions, such as changes in nutrition (l), availability of vitamins (2), aeration of the culture (3), or the addition of particular chemicals, e.g., diphenylamine (4). The present paper reports the use of similar methods to obtain significant variations in the production of different carotenoids in Neurospora. These variations may provide useful clues as to the pathway of carotenogenesis, its dependence on genes, and its variation in different cell types. MATERIAL
AND METHODS
The wild strain of Neurospora crassa, 5297a, was grown at 35°C. for 3 days on 25 ml. of Fries minimal medium containing one drop of polyoxyethylene sorbitan monooleate (Tween 80) to prevent the formation of conidia. The mycelium pads were collected on a Btichner funnel by filtration and exposed to air and light for the given period. Chemicals were added to the medium, or the mycelial pads were soaked in the appropriate solution and the excess liquid filtered off. The mycelia were treated three times with 10 ml. of absolute methanol to extract all carotenoids except spirilloxanthin, which was then extracted with benzene. The methanol extract was made alkaline with 2 ml. of 2Oyo KOH and shaken with 10 ml. of hexane. After separation of the resulting phases, alkaline alcohol contained the acidic pigments and hexane the remaining carotenoids. Phytoene was isolated by chromatographing the hexane fraction on a magnesium oxide (U.S.P., heavy powder) column. The column was developed with hexane, and the fractions containing phytoene were collected. A reasonable estimation of total colored carotenoids in the crude methanol extracts could be made by the light absorption at 464 mr, assuming the extinction coefficient B:,%. = 2600. The same extinction coefficient was used to estimate r-carotene, neurosporene, and lycopene (designated tarot. in the tables) found in the hexane phase after fractiona1 This investigation was supported by a research grant (PHS RG 4560 C) from the Division of Research Grants, U. S. Public Health Service. 561
562
ZALOKAR
tion. Spirilloxanthin was measured in benzene solution using the extinction coefficient of the heat-isomerized compound at 541 rnp, E:& = 1605 (5). Phytoene was determined by optical extinction at 297 rnp, [El’,%. = 1030 (6)1. The acidic pigments were estimated by the light absorption at 464 rnp, using the extinction coefficient of their major component, neurosporaxanthin (7) E1’,$. = 2680. RESULTS
AND
DISCUSSION
Lack of Oxygen and Enzyme Poisons The Neurospora mycelium grown in still cultures with a low oxygen supply contains only phytoene, phytofluene, and some l-carotene. After exposure to air and light, colored carotenoids are formed and the phytoene content is reduced (8). Neither exposure to air without photoactivation or photoactivation with subsequent lack of oxygen could initiate the synthesis of pigments or the disappearance of phytoene (Table I). Diphenylamine, added to the grown mycelium before or after photoTABLE
I
Effect of Air and Light on Phytoene Content of the Mycelium Three-day-old mycelium pads were exposed periments, six flasks each, are given. Exposed to:
Control Air, light Air, dark Nz , light
Time hr.
as indicated.
Dry weight/pad WT.
0 4 4 4
69.5 74.5 77.0 63.5 TABLE
Effect of Diphenylamine
folld pg.
of two ex-
Phytoene mg./lOO g.
8.4 5.9 10.1 9.8
12.1 8.0 13.1 15.5
II on Carotenogenesis
Three-day-old mycelia were exposed to air and light experiments, one flask each. Dry weight/pad
Control, no add. Diphenylamine (1:40,000) added 1 hr. before photoactivation Diphenylamine added 1 hr. after photoactivation Diphenylamine added 1 hr. before photoactivation, washed off after photoactivation
Averages
for 6 hr. Average
of three
Total pigments Extinction Amount mg./lOO g.
73.; 64.5
0.290 0.051
36.6 7.7
79.8
0.092
11.1
68.4
0.247
34.8
CAROTENOIDS
IN
563
NEUROSPORA
TABLE III Egect of Sodium A.&de on Carotenogenesis Mycelia were exposed to air and light for 6 hr. Chemical added just before photoactivation. Average of two experiments, three flasks each. Additions
No add. NaNa 0.001% NaNa 0.002% NaNa 0. O04yo NaNS 0.008%
Dry weight/pad m.
65 55 51 53 55
Acidic mg.j100
Carot. g.
24.6 19.9 5.1 4.7 3.2
mg./lOO
5.8 5.7 6.4 5.9 5.2
Spiril. g.
mg./100
g.
3.2 3.0 2.3 1.5 1.3
activation in amounts known to inhibit the formation of colored polyenes (9), had a similar effect to that of oxygen lack. When this chemical was present during photoactivation and then washed out, pigmentation subsequently proceeded in the dark at a rate only slightly lower than in untreated controls (Table II). This shows that the chemical does not interfere with photoactivation but with the processes following it, probably by acting as a respiratory enzyme poison. Most respiratory enzyme poisons were toxic to the mycelium without specifically altering pigmentation. Sodium azide differed in that, at appropriate concentrations, it inhibited growth but allowed pigmentation to proceed. Pigment analyses revealed that sodium aside inhibited the formation of neurosporaxanthin, and, to a lesser degree, of spirilloxanthin, but had no effect on the production of other colored polyenes (Table III). As sodium azide is an inhibitor of cytochrome oxidase, this would indicate that a fully active cytochrome system was not required for the production of carotenoids, with the possible exception of acidic pigments. This last possibility can be ruled out by the observation that cytochrome oxidase-deficient strains of Neurospora [poky and C-117 (lo)] contain a normal array of carotenoids not showing any decrease in acidic pigments. Vitamins
and Nutrients
Biotin is the only vitamin required by wild-type Neurospora. Cultures deficient in biotin (containing 0.2 pg. biotin/l. of medium) showed a marked increase in carotenes and spirilloxanthin, and a decrease in acidic pigments (Table IV). Whereas in the controls the spirilloxanthin content rose to a peak in 2 hr. after photoactivation and then decreased, in biotin-deficient cultures it continued to rise for the succeeding 2 hr. (Fig. 1). This suggests that one function of biotin is to form a system
564
ZALOKAR
TABLE IV Effect of Nutrition on Carotenogenesis Mycelia were grown on given media for 3 days, then exposed to air and light for 6 hr. Average of two experiments, three flasks each. Medium
Dry weight/pad m.
Acidic g./lOO g.
Carat. mg./lOO g.
Spiril. ?ng./mo 8.
70 27.3 6.1 1.05 Minimal (5 pg./l. biotin) 50 17.3 16.5 13.2 -biotin (0.2 pg./l. biotin) 34 15.7 13.4 32.8 -biotin + glycine 0.01 N 45 32.0 12.3 20.2 -biotin + biotin (100 pg./ l.)O 54 16.1 7.8 2.2 Minimalb 45 22.0 14.0 3.2 Min. + glycine 0.01 Mb 56 5.5 3.2 2.0 Min. + peptone 0.50Job 58 10.3 8.6 5.5 Min. + peptone 0.5% + glycine 0.01 Mb 0 Added to grown mycelium before exposure to air and light. b Experiments performed at another time, whence different dry weight and pigment values.
I
2
3
4
5
6
hours
Fro. 1. Spirilloxanthin (s) and carotenes (c) (including neurosporaxanthin) found in normal (+ open circles) and biotin-deficient (- black circles) mycelia at different times after exposure to light and air.
CAROTENOIDS
IN
565
NEUROSPORA
transforming this pigment. The addition of biotin after photoactivation cannot restore this system, since it increases rather than supresses the accumulation of spirilloxanthin. A different function of biotin is manifest in the decrease of the acidic pigments in deficient mycelia; this can be restored to normal when the vitamin is added afterphotoactivation. Of complex nutrients and ammo acids tried, yeast extract, hydrolyzed casein, and most individual amino acids had no effect on pigmentation in Neurospora. Peptone decreased, while glycine increased all carotenoids. Glycine restored pigmentation in peptone cultures, increasing particularly spirilloxanthin. The spirilloxanthin-promoting action of glycine was also found in biotin-deficient cultures. Valine and leucine, which stimulate carotenogenesis in Phycomyces (11)) had no effect in Neurospora, or depressed pigmentation when used as the sole nitrogen source. TABLE
V
Disappearance of Phytoene and Production Pigments
of Carotenoid
after Photoactivation
Polyenes were extracted from mycelia combined from six flasks at 0 hr. and at 4 hr. after exposure to air and light.
-
DIY reight/ pad
Growth medium or sddn. after photoactivation
hr.
Minimal,
no addn.
Minimal, add. NaN 0.002% -biotin
(0.2 pg./l.)
0
ms.
Phytoene
mg./lOO
70 72 76 74 62 60 -___
16.8 16.2 7.8 8.3 13.7 13.7
46 46 47 45
22.2 22.4 15.4 13.4
g.
Acidic
ng./lOO g. -
17.8
19.0 7.6 7.2
T
Carot.
Spiril.
L&/100 $
lg./loo g
-
-
6.8 6.6 8.4 8.4
5.3 4.6 3.4 3.6
-
0.5%
60 65 62
Total pigments
‘hytoem? used
ng./lOO g.
100 g.
mg./
8.5 19.3
2.8
31.5
7.8
_-
-
-
-
7.2 10.2
17.1 17.8
12.3 18.3 _-
fpeptone
I
Average
7.7 4.5 4.7
11.4 14.2
-
-
3.5 4.0
1.2 1.6 -
566
ZALOKAR
Loss of Phytoene and Pigmentation Previous studies (8) posed the question of whether the phytoene which disappeared during the first 4 hr. after photoactivation was converted into a colored carotenoid. If this was the case, we may expect that in pigment variants the amount of phytoene used would remain proportional to the amount of pigment formed. The carotenoid content of mycelia subjected to different conditions was measured before and 4 hr. after exposure to air and light (Table V). The same amount of phytoene disappeared in the normal and in the biotin-deficient cultures, although the former produced twice as much neurosporaxanthin and less than half as much carotenes and spirilloxanthin as the latter. The loss of phytoene was much smaller in cultures poisoned with sodium azide and in cultures grown on peptone, but the former produced more carotenes and less acidic pigment, while the latter produced as much or more of the acidic pigment as the biotin-deficient mycelia. The disappearance of phytoene therefore cannot be related to the formation of any particular carotenoid. The fact that the amount of pigments formed exceeded in all cases that of phytoene used shows that the existing phytoene can be, if at all, only a partial source of colored polyenes. New carotenoids must be synthesized from precursors which were present in the mycelium before photoactivation or produced as a result of photoactivation. Conclusions The various means of inducing changes of mycelium pigmentation show that carotenogenesis is widely subject to environmental control. This raises the question of whether in different pigment mutants the genes act directly on the biosynthetic chain of these pigments or indirectly by providing a metabolic environment enhancing or inhibiting the formation of a particular polyene. The existence of the albino mutant, producing phytoene and no other pigments (12), does not necessarily require a direct block in the conversion of phytoene to colored polyenes; the same effect could be obtained by a deficiency in the oxidation mechanism, preventing the conversion of unknown precursors into colored polyenes. The lack of oxygen or the action of diphenylamine provides good phenocopies for such a gene action. Sodium azide produces a phenocopy of the “yellow” mutant (12) which lacks acidic pigments. Similarly, the pigment changes occuring in differentiated cells could be a result of a particular metabolic environment, without
CAROTENOIDS IN NEUROSPORA
567
direct involvement of genes.The action of glycine and of biotin deficiency can serve as a model for the metabolic state of conidia, where spirilloxanthin is the predominant pigment (8). SUMMARY
The formation of pigments after photoactivation was inhibited by lack of oxygen or addition of diphenylamine; sodium azide inhibited only the formation of acidic pigments. Deficiency of biotin or addition of glycine increased spirilloxanthin content; the addition of peptone decreased all carotenoids. The disappearance of phytoene could not be related to the formation of any particular carotenoid in these pigment variants. REFERENCES 1. GOODWIN, T. W., “The Comparative
Biochemistry of the Carotenoids.” Chapman & Hall Ltd., London, 1952. 2. STARR, M. P., AND SAPERSTEIN, S., Arch. Biochem. Biophys. 43, 157 (1953). 3. VAN NIEL, C. B., Antonie van Leeuwenhoek J. Microbial. Serol. 12, 156 (1947). 4. TIJRIAN, G., Helv. Chim. Acta 33, 1988 (1950). 5. POLQ~R, A., VAN NIEL, C. B., AND ZECHMEISTER, L., Arch. Biochem. 6, 243 (1944). 6. RABOURN, W. J., QUACBENBUSH, F. W., AND PORTER, J. W., Arch. Biochem. Biophys. 43, 267 (1954). 7. ZALOKAR, M., Arch. Biochem. Biophys. 70, 568 (1957). 8. ZALOKAR, M., Arch. Biochem. Biophys. 60, 71 (1954). 9. TURIAN, G., AND HAXO, F., J. Bacterial. 63, 690 (1952). 10. TISSIERES, A., AND MITCHELL, H. K., J. Biol. Chem. 206, 241 (1954). 11. GOODWIN, T. W., AND LIJINSKY, W., Biochem. J. 60, 268 (1951). 12. HAXO, F., Fortschr. Chem. org. Naturstoffe 12, 169 (1955).