Planta 9 by Springer-Verlag 1978

Planta 141, 253 -258 (1978)

Incorporation of 14C'-'t.)2 in Prenylquinones of Chlorella pyrenoidosa K.H. Grumbach and H.K. Lichtenthaler Botanisches Institut (Pflanzenphysiologie)der Universitfit Karlsruhe, KaiserstraBe 12, D-7500 Karlsruhe, Federai Republic of Germany

Abstract. Incorporation and release of 14C-label in

prenylquinones of C h l o r e l l a was investigated under steady state conditions. After one hour of 14CO 2photosynthesis all plastid quinones investigated were labeled. The highest label was found in phylloquinone (18%) while ~-tocopherol exhibits the lowest label (0.38%). Among the plastoquinones, plastohydroquinone-9 shows a higher labeling degree (5.1%) and a faster labeling kinetic than plastoquinone-9 (1.6%). After replacement of 14CO2against Z2CO2 the total radioactivity in plastohydroquinone-9, c~-tocopherol and phylloquinone decreases but in ~-tocoquinone and plastoquinone-9 proceeds further. From this labeling kinetic we conclude, that newly synthesized [14C]c~-tocopherol molecules are converted to [14C]c~tocoquinone and [l~C]plastohydroquinone-9 molecules to [t4C]plastoquinone-9. From their z~C-incorporation kinetic half-lives could be calculated for all prenylquinones in the same range as previously found for the chlorophylls and carotenoids (Grumbach et al., 1978). Half-lives are shorter in plastohydroquinone-9 (30 rain) and plastoquinone-9 (40 rain) than in phylloquinone (55min), 7-tocoquinone (50 rain) and ~-tocopherol (220 min). This means that all prenyl-lipids such as chlorophyll a, c~- and fi-carotene, plastohydroquinone-9 and plastoquinone-9 which are more directly involved in the process of photosynthesis are subject to a continuous and higher turnover than the xanthophylls and c~-tocopherol. From the fast labeling kinetic and short half-lives of c~-tocoquinone and especially phylloquinone with a labeling degree of 12% after one hour of 14CO2 photosynthesis we suppose that perhaps these two prenylquinones are also involved in the photosynthetic activity of chloroplasts. Key words: C h l o r e l l a - Photosynthesis (prenylquinone synthesis) - Prenylquinones.

Introduction

Prenylquinones play an important role as potential electron carriers during energy conservation in the chloroplast and mitochondria membrane (Trebst and Hauska, 1974). All prenylquinones can be stored inside the chloroplast in the osmiophilic plastoglobuli as well as in the thylakoid membrane (Lichtenthaler, 1969; Grumbach and Lichtenthaler, 1975). Except plastoquinone-9 and ubiquinone-9 or UQ-10 whose function is now clearly established (Amesz, 1977; Trebst, 1974; Redfearn, 1966; Siggel, 1976), it has been shown by use of restoration experiments with heptan extracted chloroplasts, as well as with a S c e n e d e s m u s mutant lacking ~-tocopherol, that a-tocopherol is involved in the photosynthetic activity and photophosphorylation of chloroplasts (Baszynski and Tukendorf, 1975 ; Baszynski, 1974; Bishop, 1974; Barr and Crane, 1977). The biological function of e-tocoquinone and phylloquinone, also supposed to be involved in the photosynthetic activity of chloroplasts (Bishop, 1958b; Pfister, 1976), is until now still unknown. There are also no observations about the metabolism and biological half-life of the different prenylquinones within the chloroplast. Although the intracellular distribution (Bucke et al., 1966; Dilley and Crane, 1963) and the biosynthesis of the plastoquinones, tocopherols, tocoquinones and phylloquinone have been elucidated (Goodwin, 1977; Threlfall et al., 1967, 1968; Whistance and Threlfall, 1966b, 1967, 1968, 1971), there is no knowledge of the mechanism and rate of labeling of single prenylquinones with 14CO2 via the Calvin cycle and the isoprenoid pathway. Furthermore it is not known how fast prenylquinones are metabolized in a fully green chloroplast during photosynthesis and how the continuous flow of precursor molecules to the different prenyl-lipid classes,

0032-0935/78/0141/0253/$01.20

254

K.H. Grumbach and H.K. Lichtenthaler: Incorporation of 14COz in Prenylquinones

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Fig. l a anti b. Tracing of a two dimensional separated thin layer chromatogramm of a Chlorella prenyl-lipid fraction after one hour of ~4CO2 photosynthesis a Radioautogramm of two dimensional separated Chlorella prenylquinones after one hour of 14CO2 photosynthesis b Car. = carotenes, PQ-9 = plastoquinone-9, PQ-9 Hz = plastohydroqninone-9, a-T = c~-tocopherol, 7-TQ = ~-tocoquinone, K t = phylloquinone, Chl. =chlorophylls, S a-s = not yet identified unknown terpenoids, X1.11 = not yet identified unknown lipids

chlorophylls, carotenoids and prenylquinones is regulated. In a previous paper we have shown in algae that the chlorophylls and all carotenoids are labeled within one hour from 14CO2via the Calvin cycle and subject to a continuous turnover in the thylakoid membrane with shorter half-life times in chlorophyll a and the carotenes than in chlorophyll b and the xanthophylls (Grumbach et al.). In this paper we have investigated, how besides the chloroplast pigments the prenylquinones are labeled and metabolized during 14C02photosynthesis in order to acquire further information about their function and biosynthetic relations.

water=20:1 as the solvent system for the second direction. As shown in Figure I a and 1 b all Chlorella prenylquinones could be separated and purified except c~-tocoquinone which needed further purification procedures. Besides the prenylquinones plastoquinone-9, plastohydroquinone-9, c~-tocopherol, c~-tocoquinone and phylloquinone many so far unidentified lipids with a very high 14C incorporation could be located.

Results Prenyl-lipid Composition of Chlorella Chloroplasts The prenyl-lipid comosition of Chlorella chloroplasts is very similar to that of Scenedesmus obliquus and Table 1. Prenyllipid composition of autotrophically grown Chlo-

Materials and Methods Autotrophically grown ChIorella pyrenoidosa cells were exposed to 1r for two hours in a steady state apparatus. Thereafter 1'~CO2 was blown out and replaced by 12COa (2.2 vol%) and the algae kept under these conditions for further four hours. At different times (1, 2, 21/2, 3, 4, 6 h) after the beginning of the experiment 2.2• 101~ (80 ml) Chlorella cells were removed from the steady state apparatus and the prenylquinones extracted. Cultivation of algae, extraction of ChloreIIa prenyl-lipids and further experimental details are described in a previous paper (Grumbach et al., 1978).

Chromatography of Chlorella Prenylquinones Prenylquinones of Chlorella pyrenoidosa were separated and purified by use of adsorption and partition chromatography (Lichtenthaler, 1968, 1977; Grumbach, 1976). Two dimensional chromatography on silica gel plates was used with light petrol: diethylether = 5:1 for the first direction. After impregnation with 5 % liquid paraffin in light petrol the plates were developed in acetone:

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Chlorophyll a Chlorophyll b Chlorophylls Carotenes Zeaxanthin Antheraxanthin Violaxanthin Neoxanthin Lutein Carotenoids Plastoquinone-9 Plastohydroquinone-9 c~-Tocopherol c~-Tocoquinone Phylloquinone Prenylquinones

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12.6 7.6 7.2 6.3 1.6 61.1 96.4 49.7 3.8 11.3 6.2 0.8 71.8

K.H. Grumbach and H.K. Lichtenthaler: Incorporation of ~CO: in Prenylquinones 12C0 2

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