Planta (Berl.) 94, 78--90 (1970) 9 by Springer-Verlag 1970

The Forms of Native Chlorophyll in Chlamydobotrys stellata and their Changes during Adaptation from Photo-heterotrophic to Autotrophic Growth W. WIESS~EI~ and C. S. FRENCH* Department of Plant Biology, Carnegie Institution of Washington, Stanford, Calif. U.S.A. Received April 15/~ay 21, 1970

Summary. Absorption and fluorescence spectra were measured for Chlamydobotrys stellata cultured either photo-heterotrophically on acetate or autotrophically on CO2 as well as during adaptation from hetero- to autotrophic conditions. Curve analyses of the absorption spectra at liquid nitrogen temperature suggest the presence of chlorophyll-a forms with their main absorption peaks at 663, 670, 678, 685, 693 and 707 nm. The proportion of the longer wavelength forms, 685, 693 and 707 nm, decreases during adaptation to autotrophic growth. The chlorophyll-b content of the photo-heterotrophic culture was very low. Introduction

The phototrophic Volvoeale Chlamydobotrys stellata (Pringsheim and Wiessner, 1960, 1961; Wiessner, 1962) can grow either photo-heterotrophically in media containing acetate or photo-autotrophically in media supplied with carbon dioxide (Wiessner, 1969a, b). I t has been shown that the kind of carbon source - - acetate or carbon dioxide - - not only determines the metabolic activity of the organisms (Pringsheim and Wiessner, 1961 ; Wiessner, 1963, 1968, 1969a) but in addition influences the submicroscopic chloroplast structure (Wiessner and Amelunxen, 1969 a, b) and leads to differences in the absorption spectra of the algae (Wiessner, 1969a, b). Recently Merrett (1969) reported that photo-heterotrophieally cultivated Chlamydobotrys is not only incapable of photosynthetic CO 2 fixation, but is also unable to carry out a DCIP Hill reaction. Evidently the so called photosystem I I does not function in these algae even though the pigments of this system are present (Wiessner, 1965). We report here the absorption and fluorescence spectra of auto- and photo-heterotrophically grown cultures and of intermediate stages during * Dedicated to Prof. Dr. A. Pirson on the occasion of his 60th birthday.

Forms of Native Chlorophyll in Chtamydobotrys

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the adaptation from photo-heterophie to autotrophic growth. The fluorescence time courses of the chlorophyll in the two types of cultures are compared, and the changes in initial and variable fluorescence yield are given during the adaptation period. Curve analyses of the absorption spectra were made in order to attribute the changes in shape of the spectra to differential changes in the relative proportions of the major forms of chlorophyll. The changes in the fluorescence yield and in the emission spectra are explained by the theory that certain of the long wavelength forms of chlorophyll act as pseudo traps that degrade the migrating energy within chlorophyll units without emission of fluorescence. Materials and Methods

Chlamydobotrys steltata (Strain 10--1e from the Sammlung yon Algenkulturen des Pflanzenphysiologischen Instituts der Universiti~t G6ttingen) was cultivated either photo-autotrophieally (light -t- C02) or photo-heterotrophically (light -~ acerate) as described by Wiessner (1968). The light intensity was 6000 lux incandescent, and the temperature 30~ C. Measurements of absorption and of fluorescence emission were done with algae collected from the phase of exponential growth of the culture. The organisms were suspended in 0,025 M P04 buffer at pit 8.5, and were broken by extrusion through the needle valve of a French' Press. Large chloroplast particles were removed from the suspension by centrifugation at 5,000 g for 5 min. Absorption spectra were measured with the spectrophotometer of French and Lawrence (1968). Fluorescence emission spectra were recorded with the instrument described by French (1955), French and Koerper 0967). Wavelength of the excitation light was 435 nm. Results

Absorption Spectra and Curve Analysis. Fig. 1A presents room temperature absorption spectra of chloroplast fragments from photo-autotrophically and from photo-heterotrophically cultivated Chlamydobotrys stellata. Fig. 1 B shows the same spectra recorded a t - - 1 9 6 ~ C. Measurements at liquid nitrogen temperature produce much sharper spectra than measurements at room temperature. Evidently, chloroplast particles from autotrophieally cultivated C. stellata absorb less in the spectral region above X 680 nm (14,700 em -1) than do chloroplasts from photoheterotrophically grown organisms. However they do show a higher absorption around X 650 nm (15,400 em -1) due to more ehlorophyll-b. Thus the algae grown photo-heterotrophieatly with acetate have a chlorophyll spectrum like the one usually found for system 1 particles (Brown, 1969). A first approach to the comparison of these absorption spectra was carried out with the DSPEC-program (French et al., 1969a, 1969b). With the aid of this computer program spectra can be compared by snbstracting one curve from another, which leads to an approximate estimate of the

80

W. Wiessner and C. S. French:

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Fig. 1 A--C. Absorption spectra of autotrophically and photo-heterotrophically grown Chlamydobotrys stellata. A Measured at room temperature. B At liquid N2 temperatures. C Difference spectra calculated from B. The heterotrophic cells have more long wavelength absorption

wavelengths peak, the halfwidth, and the shape of components that differ in their quantities in the two spectra. Fig. 1 C shows a family of difference spectra in which the absorption spectrum of chloroplast particles (Fig. 1 B) from autotrophieally grown algae, has been substracted from the one obtained with particles from photo-heterotrophically cultivated algae. Before substraeting one curve from another ten different scale factors were applied because the correct factors are unknown and m a y be different for each component. The interpretation of these difference spectra is not entirely clear but they do show that the two curves differ in their relative contents of chlorophyll-b and in several forms of chlorophyll-a as well.

Forms of Native Chlorophyll in Chlamydobotrys

81

Curve analyses of the absorption spectra were carried out to determine the extent to which the different native chlorophylls contribute to the overall absorption spectra. We now consider t h a t the major forms of ehlorophyll-a previously called "Ca 670" and "Ca 680" are in fact both mixtures of two narrower components. The present series of curve analyses assumes the existance of at least three major forms of chlorophyll-a : Ca 670, Ca 678 and Ca 685. General discussions of the forms of chlorophyll as deduced by curve analysis are given in French (1958, 1966, 1968); French et al., (1969b); French and Prager (1969). Using the Resolv program (French et al., 1969a) we tried to match the absorption spectra by summing simple hypothetical Gaussian curves. Each of the major bands are thought to represent the main absorption peak of one of the distinctive native forms of chlorophyll. The results of curve analysis for t h e - - 1 9 6 ~ C absorption speetra of chloroplast particles from photo- autotrophically and photo-heterotrophieally grown C. stellata are shown in Fig. 2. The band positions, widths and heights of these Gaussian curves are compared in Table 1. The six bands found at approximately 707, 693, 685, 678, 670, and 663 nm are main bands of different ehlorophyll-a forms. The 654, 630, and 594 nm bands we assume to be collective sums of a secondary and a tertiary band for each of the chlorophyll forms present. The data of Table 1 show very clearly t h a t the positions and the widths of the Gaussian curves matching the overall absorption spectra are almost identical in both spectra. Significant differences result only from differences in the relative heights of the Gaussian curves. Photo-heterotrophieally cultivated C. stellata has higher concentrations of the native ehlorophylls-a represented by the curves 707,693 and 685 than do the autotrophically grown Chlamydobotrys. The areas of the six bands identifying specific forms of chlorphyll-a as a percent of their total are shown in Table 1. If we assume the extinction coefficients of all the comparable native chlorophyll-a forms to be more or less identical than the height or better the area of each Gaussian curve is a measure of the total amount of the native chlorophyll. Contrary to the behavior of the longer wavelength forms, chlorophy]ls-a 670 and 678 are apparently much less affected by nutritional differences. Judged from the results of curve analysis their concentrations in chloroplasts from autotrophicMly and photo-hetcrotrophically grown organisms differ by less than 10 per cent. The uncertainty of curve analysis in the spectral region below 680 nm, where main and side bands overlap, however, calls for a very cautious interpretation of the curve heights. Adaptation /rom Photo-hetero- to Autotrophic Growth. Absorption spectra of chloroplast particles from previously photo-heterotrophica]ly grown C. stellata adapting to autotrophic nutrition and the curve analy-

82

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c o n s t a n t l y . I n photo-heterotrophic algae the variable fluorescence cont r i b u t e s a b o u t 30 per cent to the t o t a l fluorescence, i n completely a d a p t e d organisms a b o u t 40 per cent.

Discussion As shown b y the curve analyses a decrease in c o n c e n t r a t i o n of n a t i v e long wavelengths forms of chlorophyll takes place if photoheterotrophic cells a d a p t to a u t o t r o p h y . This change is n o t paralleled

88

W. Wiessner and C. S. French:

by a decrease in total chlorophyll-a concentration which makes it reasonable to believe that Ca 685, Ca 693 and Ca 707 have changed into short wavelength chlorophylls, probable those predominating in photosystem II. This phenomenon could be similar to the transition of chlorophyll-a 695 into chlorophyll-a670 which appears to take place in Euglena gracili8 (Brown and Michel-Wolwertz, 1968). According to the results of curve analysis, this transition does not lead to twice the concentration of chlorophylls of photosystem II. A change that large would be required to account for the doubling of fluorescence intensity in the cells adapted to autotrophy. Even though a chlorophyll-a transformation might contribute to the higher photosystem I I fluorescence of autotrophic cells it cannot be the only reason. As a rough approximation we may adopt the current ideas that F 685 comes primarily from system II pigments including energy transfer from Cb 650 while F 730 is more attributable to system I (Boardman et al., 1966; Govindjec and u 1966; Murata et al., 1966). Adaptation to autotrophy increases the amount of chlorophyll-b and also the proportion of chlorophyll-a forms in system II thus accounting in part for the increased F 685 of autotrophically adapting cells. However the doubling of F 685 is too large to be attributed entirely to the increase of chlorophyll-a in system I I pigments. A possible interpretation of this effect is that long wavelength chlorphyll-a forms may act as traps themselves having low fluorescence yields that can capture and degrade some of the energy absorbed by the system II pigments thus reducing system I I fluorescence. The necessity for a mechanism quenching system II fluorescence results from the requirement for a very efficient photosystem I mediated cyclic electron transport in organisms which depend on cyclic photophosphorylation. Photo-heterotrophically grown Chlamydobotrys stellata is such an organism. I t has been shown with chloroplast preparations that cyclic photophosphorylation can proceed with highest efficiency only if the electron transport from photosystem II to photosystem I is reduced, in other words if photosystcm II activity is low (Heber, 1969; Tagawa et al., 1963; Trebst et al., 1963). Therefore such a photosystem II quenching mechanism as outlined above appears to be a very suitable way to facilitate highest rates of photosystem I depcdent cyclic photophosphorylation in photo-hcterotrophically grown C. stellata. This research was supported by the Deutsche Forschungsgemeinschaft. We wish to thank also Dr. D. D. Tunnicliff of the Shell Development Laboratory, Emeryville, California, for the curve analysis program and the National Science Foundation for Grant No. GB-8,630 which paid the costs of the computer use.

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References Brown, J. : Absorption and fluorescence of chlorophyll a in particle fractions from different plants. Biophys. J. 9, 1542--1552 (1969). --Michel-Wolwertz, M. 1~.: Chlorophyll fluorescence near 720 m~ in Euglena extracts. Biochim. biophys. Acta (Amst.) 153, 288--290 (1968). Boardman, N. K., Thorne, S. W., Anderson, Jan M. : Fluorescence properties of particles obtained by digitonin fragmentation of spinach chloroplasts. Proc. nat. Acad. Sci. (Wash.) 56, 586--593 (1966). Duysens, L. N. M., Sweers, H. E.: Mechanism of two photochemical reactions in algae as studied by means of fluorescence. In: Studies on microalgae and photosynthetic bacteria, p. 353--372. Special issue of Plant and cell physiology. Tokoyo: Tokyo University Press 1963. French, C. S.: Fluorescence spectrophotometry of photosynthetic pigments. In: The luminescence of biological systems (Frank H. Johnson, ed.), Washington D. C.: AAAS, 1955. - - Variability of chlorophyll in plants. In: Photobiology, p. 52--64. Nineteenth Annual Biology Colloquium Corvallis Oregon 1958. - - Chloroplast pigments. In: Biochemistry of chloroplasts (T. W. Goodwin, ed.), vol. 1, p. 377--386. London and New York: Acad. Press 1966. - - Absorption spectra of chlorophyll a in algae. Carnegie Inst. Wash. Year Book 6 6 , 177--186 (1968). - - Brown, J. S., Prager, L., Lawrence, M. : Analysis of spectra of natural chlorophyll complexes. Carnegie Inst. Wash. Year Book 67, 536--546 (1969a). - - Koerper, M. : Fluorescence spectra of photosynthetic pigments. Carnegie Inst. Wash. Year Book 65, 492--498 (1967). - - Lawrence, M. : A spectrophotometer primarily for light scattering samples at low temperature. Carnegie Inst. Wash. Year Book 6 6 , 175--177 (1968). - - Michel-Wolwertz, M. R., Michel, J.M., Brown, J. S., Prager, L. K. : Naturally occuring chlorophyll types and their function in photosynthesis. In: The Biochemical Society Symposium, No. 28, Porphyrins and related compounds (T. W. Goodwin, ed.), p. 147--162. London and New York: Acad. Press 1969b. - - Prager, L. : Absorption spectra for different forms of chlorophyll. In: Progress in photosynthesis research (H. Metzner, ed.), vol. 2, p. 555--564. Tiibingen: H. Haupt jr. 1969. Govindjee, Yang, L. : Structure of the red fluorescence band in chloroplasts. J. gen. Physiol. 49, 763--780 (1966). Heber, U. : Control of photosystem I mediated cyclic electron transfer by photosystem II and electron acceptors. In: Progess in photosynthesis research (H. Metzner, ed.), vol. 2, p. 1082--1090. Tiibingen: H. Haupt jr. 1969. Hind, G., Olson, J. M. : Electron transport pathways in photosynthesis. Ann. I~ev. Plant Physiol. 19, 249--276 (1968). Loach, P. A., Sekura, D. L., ttadsell, R. M., Sterner, S. : Quantitative dissolution of the membrane and preparation of subunits from Rhodopseudomonas spheroides. Biochemistry 9, 724--733 (1970). Merrett, M.: Observation on the fine structure of Chlamydobotrys stellata with particular reference to its unusual chloroplast structure. Arch. Mikrobiol. 65, 1--11 (1969). Murata, N., Nishimura, M., Takamiya, A. : Fluorescence of chlorophyll in photosynthetic systems. III. Emission and action spectra of fluorescence. Three emission bands of chlorophyll a and the energy transfer between two pigment systems. Biochim. biophys. Acta (Amst.) 126, 234--243 (1966).

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Pringsheim, E. G., Wiessner, W. : Photo-assimilation of acetate by green organisms. Nature (Lond.) 188, 919--921 (1960). - - Wiessner, W. : Ern~hrung und Stoffwechsel yon Chlamydobotrys stellata (Volvocales). Arch. Mikrobiol. 40, 231--246 (1961). Tagawa, K., Tsujimoto, H. Y., Arnon, D. I. : Role of chloroplast ferredoxin in the energy conversion process of photosynthesis. Proe. nat. Aead. Sci. (Wash.) 49, 567--572 (1963). Trebst, A., Eck, It., Wagner, S.: Effects of quinones and oxygen in the electron transport system of chloroplasts. In: Photosynthetic mechanisms of green plants. Publ. 1145, p. 174--194. Natl. Acad. Sci.-Nat. Res. Council, Washington D. C. 1963. Wiessner, W. : Kohlenstoffassimilation yon Chlamydobotrys (Volvocales). Arch. Mikrobiol. 48, 4 0 2 4 1 1 (1962). - - Stoffwechselleistung und Enzymaktivit/~t bei Chlamydobotrys (Volvocales). Arch. Mikrobiol. 45, 33--45 (1963). Quantum requirement for acetate assimilation and its significance for quantum measurements in photophosphorylation. Nature (Lond.) 205, 56--57 (1965). Enzymaktivit~t und Kohlenstoffassimilation bei Griinalgen unterschiedlichen ern/~hrungsphysiologischen Typs. Planta (Berl.) 79, 92--98 (1968). - - Photo-assimilation and photosynthesis in green algae. Metabolic activities and correlated changes of the absorption spectrum of Chlamydobotrys stellata. Progress in photosynthesis research (H. Metzner, ed.), vol. 1, p. 4424--449. Tiibingen: H. Haupt jr. (1969 a). - - Effect of autotrophic or photo-heterotrophic growth conditions of in vivo absorption of visible light by green algae. Photosynthetica 3, 225--232 (1969b). - - Amelunxen, F. : Beziehungenzwischen submikroskopischer Chloroplastenstruktur und Art der Kohlenstoffquelle unter phototrophen Ern~ihrungsbedingungen bei Chlamydobotrys stellata. Arch. Mikrobiol. 66, 14--24 (1969a). - - - - Umwandlungen der submikroskopischen Chloroplastenstruktur parallel zur Ver~nderung der stoffwechselphysiologischen Leistung yon Chlamydobotrys stellata. Arch. Mikrobiol. 67, 357--369 (1969b). -

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Dr. W. Wiessner Pflanzenphysiologisches Institut D-3400 G5ttingen, Untere Karspfile 2