The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138, USA

Spectral Response Curves for the Formation of Phycobiliproteins, Chlorophyll and o-Aminolevulinic Acid in Cyanidium caldarium

HANSJORG A. W. SCHNEIDER and LAWRENCE BOGORAD

With 4 figures

Received March 20, 1979 . Accepted April 30, 1979

Summary

Spectral response curves for the formation of phycobiliproteins and the chlorophyll both show maxima at 440, 575 and 645 nm in cells of the wild-type mutant III-D-2 of Cyani­dium caldarium. Chlorophyll formation occurs maximally in about 460 and 645 nm light in the phycobiliprotein-Iess mutint III-C. The chlorophyll-less mutant GGB forms phycobili­proteins maximally when illuminated with 440 nm light and a much smaller response peak occurs at 575 nm. These data indicate that there is one photoreceptor-biosynthetic system for chlorophyll formation and another for phycobiliprotein production. One is defective in GGB and another in III-C. In the wild-type-like mutant III-D-2, where spectral response curves for the two classes of tetrapyrrolic pigments are qualitatively indistinguishable, the two biosynthetic paths may be linked through common intermediates whose synthesis is regulated by light. The synthesis of b-aminolevulinic acid is maximally promoted by about the same wavelengths of light which stimulate chlorophyll or phycobiliprotein synthesis. -This spectral similarity may be caused by removal of feedback inhibitors or by a synchronous formation of various pigment synthesizing enzymes.

Glucose represses b-aminolevulinic acid and chlorophyll formation in dark-grown cells of strain III-D-2. These compounds form in darkness after heterotrophically grown cells are washed free of glucose. However, the cells produce no phycobiliproteins in darkness regardless of whether glucose is present or not. Together with results from other experi­ments, this observation inferes that phycobiliprotein formation requires control mechanisms besides the regulation of ALA synthesis.

Key words: Cyanidium caldarium, (j-aminolevulinic acid, chlorophyll, phycobiliproteins, spectral response curves.

Introduction

The regulation of chlorophyll biosynthesis by light in higher plants and some algae has been studied extensively (for reviews see BOGORAD, 1976; SCHNEIDER,

1979). In all organisms pigment synthesis depends on the production of sufficient

Abbreviations: ALA = b-aminolevulinic acid; APC = allophycocyanin; ChI = chlorophyll a; PBP = phycobiliproteins; PC = phycocyanin.

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450 HANSJORG A. W. SCHNEIDER and LAWRENCE BOGORAD

amounts of (j-aminolevulinic acid (ALA). Mostly, however, ALA is not formed in the dark. Illumination results in the reduction of protochlorophyllide in a complex with protochlorophyllide photoreductase and the relief of the block in ALA syn­thesis. The synthesis of pigment synthesizing enzymes and plastid proteins is a second light requiring process in the regulation of chlorophyll biosynthesis.

Some organisms accumulate chlorophyll a (ChI) plus the accessory photosynthetic phycobiliprotein (PBP) pigments phycocyanin, allophycocyanin and phycoerythrin, whose synthesis also depends on the availability of ALA (for a review see BOGORAD, 1975). Little, however, is known about processes controlling the coordinated distribution of this precursor if two biosynthetic pathways compete for it.

The single-celled alga Cyanidium caldarium produces both ChI and the PBPs phycocyanin and allophycocyanin when grown in the light but none of these pigments when grown heterotrophically on glucose in darkness. The spectral response curve for PBP production in the ChI-less mutant GGB of this alga has been reported (NICHOLS and BOGORAD, 1962). In the present work we have re-analyzed the spectral response curve for PBP production in this strain. We have also studied the effects of light of different wavelength on the production of ChI in the PBP-less mutant, III-C, and ChI and PBP in the wild-type like strain III-D-2. One objective of this research was to determine whether production of ChI and PBPs are regulated by a single photosystem. They are not. Another objective was to determine the spectral response curves for the production of ALA in the different mutants. These curves were hoped to give information about the relations between the synthesis of different types of tetrapyrrolic pigments and the control of ALA synthesis. Finally, we have found functional interconnections between the photoreceptor-biosynthetic system for ChI and PBP production.

Materials and Methods The experiments were performed with mutants of Cyanidium caldarium. Mutant strain

III-D-2 produces phycocyanin (PC), allophycocyanin (APC), and chlorophyll i.e. the same, set of photosynthetic tetrapyrroles as wild-type cells, but grows more rapidly and is more intensely pigmented. Mutant III-C lacks PC and APC; mutant GGB produces phycobili­proteins but lacks chlorophyll (NICHOLS and BOGORAD, 1960, 1962). Cells were grown in 0.5 or 1.0 I cultures in 31 Fernbach flasks in darkness at 37 DC shaken at 100 rpm. Cells were harvested after 5-7 days growth, at the end of the logarithmic growth phase, by centrifugation for 5 min. at about 600 g. Pelle ted cells were resuspended in 0.1 M NaCI, recentrifugated and finally suspended in nutrient medium lacking glucose. Suspensions adjusted to 1.2 X 108 cells/ml, corresponding to 2.26 mg fresh weight/ml, were used in these experiments.

To measure response to monochromatic light, 40 ml aliquots of cell suspension were illuminated in water-jacketed cylindrical glass vessels covered with a glass lid. Temperature was maintained at 37 DC by passing water through the jacket. Light was supplied by 500 watt quartz iodide lamps and narrow band interference filters. The quantum flux was adjusted to 43 pE/cm2 X sec by adjustment of the lamp current.

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Formation of phycobiliproteins and chlorophyll 451

An ISCO model SR spectroradiometer was used to determine incident light intensities. These were measured at the position occupied by the center of the water-jacked vessels during the experiment. At the most effective wavelengths the intensity chosen gave 40-50 0/0

of the response obtained with saturating, non-bleaching intensities of white light. The ISCO radiometer was calibrated by an ISCO model SRC standard lamp unit.

To provide aeration and to prevent settling, cells were stirred during illumination. Since the stirring velocity and the shape of the vessels influenced pigmentation, vessels and stirring positions were periodically interchanged.

All experimental results are expressed relative to response by aliquots of cells held simultaneously in darkness on the one hand and under illumination with 427 nm light on the other.

Pigments were measured by their absorption of light in situ using an Aminco DW 2 spectrophotometer. Suspensions of unilluminated (control) cells were used as blanks for measuring absorption.

Absolute amounts of pigments were computed using the following extinction coefficients (mllmg· cm):

PC APC ChI

626nm

5.9 2.7

12.6

653 nm

1.2 5.7

10.4

and the respective formulae:

678 nm

1.2 75

APC Cug/ml) = - 23.2 OD678 + 200.5 OD653 40.8 OD626 PC Cug/ml) = 13.8 OD678 - 3.8 OD653 + 0.8 OD626 ChI Cug/ml) = - 17.8 OD678 - 84.0 OD653 + 186.6 OD6~6

These formulae allow only an approximation of the relative amounts of the pigments present because in vivo extinction coefficients of Cyanidium pigments are not available at present. Maximum extinction coefficients were taken from the literature (BENNETT and BOGORAD, 1973; MAC KINNEY, 1940) and transferred to the absorbance maxima of Cyanidium or from the spectra run with mutants only producing phycobiliproteins or chlorophyll.

Four ml of the 40 ml aliquot were used to determine pigment content. The remaining 36 ml were used to determine the ALA-forming capacity of the cells. The 4 ml of suspension removed for pigment determination was replaced with 4 ml of 2 X 10-1 M levulinic acid adjusted to pH 4, and incubated with shaking in the dark or light for 5 hrs at 37°C. The ALA content of the culture was then determined by the method of MAUZERALL and GRANICK (1956). Cells were precipitated by centrifugation, suspended in 2.5 ml of 0.5 M sodium acetate (pH 4.6), 0.05 ml of acetyl acetone was added and the mixture was incubated at 100°C for 10 min. After cooling and centrifugation, an equal volume of modified Ehrlich's reagent was added to an aliquot and the absorbance was read 10 min later at 555 nm (E = 62 . 103 11M . cm). The supernatants mostly contained negligible amounts of ALA.

Because little ALA accumulated in GGB cultures, levulinic acid (final concentration 5 mM) was added to the sample before the 40 hrs illumination with monochromatic light. ALA from the whole sample (cells plus supernatant) was converted to ALA pyrrole in the usual way. The pyrrole was measured as described before.

Results

Dark-grown cells of the wild-type-like mutant III-D-2 of Cyanidium caldarium contain only traces of chlorophyll. If such cells are washed and transferred to a

z. Pjlanzenphysiol. Ed. 94. S. 449-459. 1979.

452 HANS]ORG A. W. SCHNEIDER and LAWRENCE BOGORAD

medium lacking glucose, about a 5-fold increase in chlorophyll occurs within 36-48 hrs. The increase in chlorophyll is preceded by an increase in the capacity to form ALA. Production of ALA reaches a maximum about 12 hrs after transfer to glu­cose-free medium. Maximal chlorophyll synthesis lags just behind the great increase in ALA formation and plateaus at about 36-48 hrs (Fig. 1). Although ALA is also the precursor of phycobilins (TROXLER and BOGORAD, 1966; TROXLER and BROWN, 1970), these pigments are not detectable in dark-grown cultures (Fig. 1 insert) either before or after removal of exogenous glucose.

0.6 Fig. I

0.5 ~,

0.3 I , I ,

I ,

E I , :::l I ,

~ 0.4 I , :s::

I l> ..c I , ALA u I Q2 r C) 0.3 I ,,~ l>

I '-~

I , , ~ I , 0.2

I , :r I , , I ,

I , 0.1 , , "" '20 .'" 700 ~

0.1 , ,

~---------- ----12 24 36 1.8 60 h

Fig. 1: ALA synthesis and chlorophyll formation in 1 ml of dark growing culture of Cyani­dium, mutant III-D-2 (1.2· lOB cells) after the transfer of the cells from the culture medium containing glucose to a medium free of glucose. (The points representing the accumulated amounts of ALA have been displaced from the point of sampling for 5 hours because of the subsequent 5 hours of incubation.) Insert: Absorption spectrum of the culture after 60 hours.

Fig. 2: The relative effectiveness of different light colours on the absorption of a Cyanidium III-D-2 culture (1.2· lOB cells/ml) at the maxima of phycocyanin and chlorophyll absorption and on ALA accumulation. Values have been normalized with respect to the response at 427 nm. Vertical lines represent the standard error of the mean. Each point represents values from 4 to 16 experiments. Equal amounts of quanta were applied corresponding to 43 pEl cm2 • sec. Illumination time was 22 hours.

a) Typical Light-dark difference spectrum after 22 hours of illumination with 427 nm. -

b) The absorbance of the culture at 626 nm in relation to the wavelengths applied. 100 per cent absorbance correspond to an absorbance of 0.043 for 1 cm light path. - c) The absorbance of the culture at 678 nm in relation to the wavelengths applied. 100 per cent absorbance correspond to an absorbance of 0.037 for 1 cm light path. - d) The amounts of accumulated ALA in relation to the wavelengths applied. 100 per cent of ALA correspond to about 0.162 nM of ALA produced/h and of ml culture. The amounts of ALA formed in the dark have been subtracted.

z. Pjlanzenphysiol. Bd. 94. S. 449-459. 1979.

Formation of phycobiliproteins and dJ.lorophyll 453

ALA synthesis by C. caldarium is enhanced by preillumination. The enhancement is not dependent upon continuous illumination as in the case of higher plants. Cells illuminated with red light for 22 hrs in glucose-free medium accumulated about equal

S50 600 650

Fig.2b

Z. Pjlanzenphysiol. Bd. 94. S. 449-459. 1979.

454 HANSJORG A. W. SCHNEIDER and LAWRENCE BOGORAD

amounts of ALA regardless of whether they were illuminated or kept in darkness during the ensuing 5 hrs. In two experiments with 3 replicates each, ALA accu­mulation in darkness after preillumination was 83-98 % of the ALA accumulated under illumination.

Illumination not only enhances ALA and chlorophyll formation but also starts and promotes phycobiliprotein synthesis. Figure 2 a shows the difference spectra between two cultures of III-D-2; one illuminated at 427 nm for 22 hrs minus the one maintained in darkness. The prominent peak at about 625 nm indicates the presence of PC. The shape of the spectrum of figure 2 a does not change dramatically if the cultures are illuminated with other light qualities. The ratio of the amounts of ChI and PBPs, however, vary slightly but significantly with the color of light under which the III-C-2 cells are grown. Both ChI and PBP production is maximal at about 440, 575 and 645 nm, but the ratios absorption at 625 nm/absorption at 678 nm (compare figures 2 band 2 c) or PBP/Chl (compare figures 3 d and 3 a) decrease from the blue to the red region. Minimal responses occurred at about 500 nm and the wavelengths beyond 700 nm. The graphs of the figure 3 a and 3 d have been computed (see methods) using the absorbance data of figures 2 band 2 c and similar ones for the absorbances at 653 nm (APC). Figures 3 a and 3 d show a strong and sharp peak of PBP accumulation at 440 nm and a broad peak including the 460 nm region for ChI production. ALA synthesis in III-D-2 is also maximal at about 575 and 645 nm but the major response to blue region is at about 460 nm (figure 2 d). All the responses shown in figure 2 are relative to that resulting from exposure to 427 nm light.

Chlorophyll production by C. caldarium III-C, a strain which does not form PBPs, shows one maximum at about 460 nm and another maximal response at about 645 nm (Fig. 3 b). These maxima coincide with the maxima for ALA production in the same mutant.

NICHOLS and BOGORAD (1962) reported that PBP synthesis by C. caldarium GGB was maximal at about 427 nm; a minor response was seen at about 575 nm. In the present experiments we have found the maximum response by GGB to be at about 440 nm (Fig. 3 c). Illumination of GGB for 22 hrs and 46 hrs gave almost identical results expressed in responses normalized to illumination at 427 nm. ALA assays at different wavelengths reconfirmed the finding that high ALA production occurs in each strain at about the same wavelength which promote maximum production of tetrapyrrolic pigments. Tetrapyrrole synthesis in mutant strain III-C and especially in GGB is considerably lower than in III-D-2 (see legends of Figures 2 d and 3). The usual 5 hr incubation period used for assessing ALA synthesis in other C. caldarium strains was insufficient to detect ALA production in suspensions of GGB. For this mutant results are given for 40 hrs incubation with levulinic acid and simulanous monochromatic illumination. In addition to the reduced ALA production in GGB, the effect of light on the synthesis of ALA is small. In light (427 nm) -.dark comparison, ALA accumulation increased only about 23 per cent as a result of

Z. Pflanzenphysiol. Rd. 94. S. 449-459. 1979.

./.

./.

./.

./.

Formation of phycobiliproteins and mlorophyll 455

120

80 Fig. 30

40

OL-T----I·~--~--------r-----~~~~

160 400

120

80

40

120

80

40

400

160

120

80

40

, , , ,

500 600

500

W-C Chi 0 ALA I

GGB PC+APC 0 ALA I

m-O-2 PC -­APe ----

600

600

700 nm

Fig.3b

700 nm

Fig.3c

700 nm

Fig. 3d

OL-~---------r--------~~------~~--~ 400 500 600 700 nm

Fig. 3: The relative effectiveness of different wavelengths on pigment and ALA synthesis in the mutants III-D-2, III-C and GGB of Cyanidium. a) Light-mediated chlorophyll synthesis in III-D-2. 100 0/0 ChI = 0.43 pg or 0.48 nM/ml . 22 hours. - b) Light-mediated chlorophyll and ALA synthesis in III-C. 100 010 ChI = 0.09 flg or 0.1 nM/ml· 22 hours. 100 Ufo ALA = 0.08 nM/ml· h. - c) Light-mediated phycobiliprotein and ALA synthesis in GGB. 100 Ufo PC + APC = 1.0 pg (or 0.08 nm)/ml· 22 hours, respectively 3.6 flg (or 0.29 nM)"/ml . ml . 46 hours. 100 Ufo ALA = 0.009 mI· h. - d) Light­mediated phycocyanin and allophycocyanin synthesis in III-D-2. 100 Ufo PC = 4.75 flg (or 0.38 nM)*/ml . 22 hours. 100 Ufo APC = 3.36 ,ug (or 0.27 nm':'/ml . 22 hours.

':. An average of 12,500 daltons of protein per chromophore has been assumed.

456 HANSrORG A. W. SCHNEIDER and LAWRENCE BOGORAD

illumination while cells of III-D-2 or III-C produced about 3 times more ALA in light than in darkness.

Discussion

The spectral response curves obtained with phycobiliprotein-Iess (III-C) and chlo­rophyll-less (GGB) mutants of C. caldarium indicate that both have photoreceptive systems for control of tetrapyrrole biosynthesis. The spectral response curve for phycobiliprotein synthesis in strain GGB shows a strong maximum at 440 nm and a much smaller response at 575 nm. NICHOLS and BOGORAD (1962) reported the blue maximum for this strain to be a few nm to the blue, at 427 nm. they suggested that the photoreceptor might be a hemoprotein. The spectral response curve for chloro­phyll production by strain III-C shows a maximum in the blue at about 440-460 nm and another fairly strong maximum response at 645 nm. This action spectrum resembles the absorption spectrum of protochlorphyllide holochrome, i.e. the enzyme substrate complexe between protochlorophyllide and its photoreductase, if a single pigment is indeed responsible.

In contrast to the distinctively different results obtained with strain III-C and GGB, spectral response curves for production of chlorophyll a and phycobiliproteins in the wild-type-like III-D-2 are virtually indistinguishable with regard to position of maxima. Responses are strongest at about 440, 575 and 645 nm. However, there are quantitative differences: the responses are about equally strong at all three wavelengths for chlorophyll formation but for PC and APC synthesis the blue response is 2 or 3 times greater to green or red light.

The three maxima in the spectral response curves for PBP and ChI biosynthesis in III-D-2 appear to be the sum of the maxima for chlorophyll synthesis in III-C at 440-460 and 645 nm plus the maxima for PC and APC synthesis at 440 and 575 nm in GGB. The most obvious conclusion is that PBP and ChI production are regulated by different photoreceptor-biosynthetic systems - one operating in III-C, another in GGB, both in III-D-2. There is no basis for judging whether the lesion in GGB or III-C is in the photoreceptor or in an enzyme. One possible explanation taking into account the interdependence between tetrapyrrolic photoreceptors and tet­rapyrrole synthesizing enzymes is given in figure 4.

The great similarity - and perhaps identity - of spectral response patterns for the synthesis of ChI and PBPs in III-D-2 compared with results with GGB and III-C is a striking outcome of the present experiments. The capacity of either photo­receptive system to drive the other biosynthetic chain could be by production either of a common intermediate that could leak across or of a cofactor required in both chains. If this is the case, the failure of III -C to form PBPs and of GGB to produce chlorophyll would have to result from blocks in the synthesis of enzymes late in the chain. Small intermediates would be expected to accumulate. None have been detected. They are either labile or are not formed because of secondary effects of genetic lesions.

z. Pf/anzenphysiol. Bd. 94. S. 449-459. 1979.

Formation of phycobiliproteins and dtlorophyll 457

III-C

PROTEII- --SYNTH.--

ENZYHE SYNTH.

I----+--t ALAE-SYNTH.- ALA , ENZYHE -SYNTH.

PROTEIN-SYNTH. I I I I I I L _____ • _______________________________ 1

r--------------------------------------I I 1 I I

I PROTEIN -: -SYNTH. 1

1 1 1 1

r-----i :--PRI 1------1 L _____ J .... _

ENZYME -: -SYNTH. I t

ALAE-SYNTH.- ALA , -ENZYHE -- -SYNTH.-- . ---------.,

"-.l-k:Hl;PROT.' -PROTEII-- -~YNTH.- " I,; ____ -' ___ :!

GGB Fig. 4: A tentative model on the regulation of tetrapyrrole synthesis in the mutants III-C and GGB of Cyanidium caldarium. The model takes into account the interdependence be­tween tetrapyrrolic photoreceptors, the synthesis of tetrapyrrole synthesizing enzymes and properly constructed plastids. As demonstrated by the experimental results, photoreceptor I (PR I: possibly a protochlorophyllide reductase complex) is mainly connected with the synthesis of ChI, photoreceptor II (PR II: probably a hemoprotein) with the synthesis of PBPs. In cells without lesions the twO photoreceptor systems may be linked by energy transfer or by their action on common reaction chains or common intermediates. In GGB an action of PR I is not detectable; in consequence there is no synthesis of Chl. PR II allows only the synthesis of very moderate amounts of ALA and phycobiliproteins. In III-C PR II may be present (see Fig. 3 b) but its action may be diminished by structural or bio­chemical lesions in the plastids. The formation of the photoreceptors may have features of selfcatalytic cycles. The heavy lines with the heading III-C and GGB indicate possible sites of lesions in the respective mutants. ALAE: ALA synthesizing enzymes.

The spectral response curves for ALA synthesis largely coincide with those for PBP and ChI production in all Cyanidium strains tested. It seems highly unlikely that the sole role of each of the two photoreceptive systems in this alga is to independently and directly regulate a different enzyme for ALA synthesis because when ALA is administered to C. caldarium cells, including the mutants used here,

Z. Pjlanzenphysiol. Bd. 94. S. 449-459. 1979.

458 HANS]ORG A. W. SCHNEIDER and LAWRENCE BOGORAD

phycocyanobilin is produced but neither chlorophyll a nor PBPs are formed (TROX­LER and BOGORAD, 1966). It seems as likely, or perhaps more likely, that ALA syn­thesis is moderated via feedback inhibition by intermediates in the biosynthetic paths to ChI or phycocyanobilin. The photoreceptor regulating PBP synthesis (perhaps a hemoprotein or a free bile pigment) could directly regulate the production of a protein required in a middle or late step in PBP or phycocyanobilin synthesis; the chlorophyll synthesis regulating system (perhaps a protochlorophyllide-photoreduc­tase complex) could affect a late step in the chlorophyll biosynthetic path or the formation of an essential thylakoid protein. ALA synthesis would be affected indirectly.

Even if the regulation of ALA synthesis could be explained in this way, there would still be the following puzzles: If a step in ChI formation requires illumination at 645 nm to reduce an intermediate, how does illumination at 575 nm, which stimulates another photoreceptor, bring about the reduction of protochlorophyllide? Perhaps by generating a reductant which can serve to circumvent the photodriven step? The same photo-generated reductant would have to relieve the block in PBP biosynthesis. The converse question is: how does the photoregulated step in chlorophyll biosynthesis influence PBP production? If we assume that ChI biosynthesis is regu­lated through the reduction of protochlorophyllide (one possible interpretation of the spectral response curve), consumption of this compound could promote the syn­thesis of ALA which might then feed both biosynthesic chains. We know (TROXLER and BOGORAD, 1966) that administration of ALA to III-D-2 promotes phycocyanobilin and not PC or APC synthesis. Thus relief of the blockage in ALA synthesis cannot alone stimulate PBP synthesis.

Finally, it is noted that the maxima in the spectral response curve for ALA, chlorophyll, PC and APC synthesis in C. caldarium III-D-2 are at 440 (460), 575 and 645 nm. Maxima in the action spectra for PC synthesis in Fremyella diplosiphon are at 463. 575 and 641 nm (HAURY and BOGORAD, 1977). The similarities are striking. Under the conditions used by VOGELMANN and SCHEIBE (1978), however, PC synthesis in Fremyella has action maxima at about 645 and 370 nm. Another striking similarity should also be mentioned. The action maxima at 575 and 645 nm are exactly the same as the action maxima of the reversible absorbance changes of purified APC fractions (OHAD et aI., 1979, see also BJORN, 1978). However, the results of the present investigations give no indication that reversible absorbance changes of a phycobiliprotein are involved in the light-mediated synthesis of pig­ments of C. caldarium.

Acknowledgements

This research was aided in part by a travel grant from the Deutsche Forschungsgemein­schaft to Hj. A. W. S. It was supported in part by grant PCM-75-13456 from the National Science Foundation and by funds from the MARIA MOORS Cabot Foundation of Harvard

Z. Pflanzenphysiol. Bd. 94. S. 449-459. 1979.

Formation of phycobiliproteins and chlorophyll 459

University to L. B. We are indebted to SUZANNE GROET for her skilled technical assistance in the conduct of this work.

References

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McKINNEY, G.: Criteria for purity of chlorophyll preparations. J. Bio!. Chern. 132, 91-109 (1940).

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SCHNEIDER, HJ. A. W.: Chlorophyll biosynthesis. Enzymes and regulation of enzyme activities. In: CZYGAN, F.-C.: Pigments in plants. In the press Stuttgart 1979.

TROXLER, R. F. and L. BOGORAD: Studies on the formation of phycocyanin, porphyrins, and a blue phycobilin by wild-type and mutant strains of Cyanidium caldarium. Plant Physio!. 41, 491-499 (1966).

TROXLER, R. F. and A. BROWN: Biosynthesis of phycocyanin in vivo. Biochim. Biophy. Acta 215,503-511 (1970).

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Prof. Dr. LAWRENCE BOGORAD, Harvard University, The Biological Laboratories, 16 Divinity Avenue, Cambridge, Mass. 02138, USA.

Prof. Dr. HANSJORG SCHNEIDER, Botanisches Institut der Universidit zu Kaln, 5 Cologne 41, W.-Germany.

z. Pjlanzenphysiol. Bd. 94. s. 449-459. 1979.