[24] Isolation of membranous chromoplasts from daffodil flowers

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[24] Isolation of M e m b r a n o u s C h r o m o p l a s t s f r o m Daffodil F l o w e r s B y BODO LIEDVOGEL

Introduction

Chromoplasts are the yellow- to red-colored plastids of many flower petals and fruits. Their fine structure represents the most heterogeneous category of plastids, l Most of the chromoplasts investigated so far by electron microscopic methods belong to the globulous, tubulous, membranous, or crystallinous type as determined by the carotenoid-bearing fine structural element prevailing in their final state of development. Of these types the membranous chromoplast is the rarest one. In its typical form, it has so far been found only in flowers--in particular in the cells of the bright yellow corona ("Nebenkrone") of the daffodil Narcissus pseudonarcissus L. 2-4 The corona represents the aroma-producing organ of the flower which probably attracts insects.S It exhibits the highest numbers of chromoplasts per cell whose plastids are all uniform in their final state of differentiation.6 Inside the double chromoplast envelope, the periphery of the plastid body is made up of numerous (up to 20), concentric, closely appressed double membranes, the thickness of a single membrane sheet being about 6 nm. The build-up of these extremely lipid-rich internal membranes probably originates from invaginations of the inner sheet of plastidal envelopes that are frequently observed by electron microscopy of ultrathin sections from early developmental stages. 4 Inside the membrane convolution a relatively dense stroma matrix material is embedded, sometimes crossed by a few flat or tubular membranes with numerous ribosome-like particles as well as some plastoglobules. A few less dense regions probably contain the chromoplasts DNA. 3 The multimembrane coat which surrounds the plastidal stroma proP. Sitte, H. Falk, and B. Liedvogel, in "Pigments in Plants" (F.-C. Czygan, ed.), p. 117. Fischer, Stuttgart, 1980. 2 H. H. Mollenhauer and C. Kogut, J. Microsc. (Paris) 7, 1045 (1968). 3 K. V. Kowallik and R. G. Herrmann, Protoplasma 74, 1 (1972). 4 B. Liedvogel, P. Sitte, and H. Falk, Cytobiologie 12, 155 (1976). 5 S. Vogel, Abh. Akad. Wiss. Lit. (Mainz), Math,-Nat. Kl. 10 (1962). 6 Besides of the daffodil, membranous chromoplasts have been found in some close relatives like Corbularia [J. F. Mesquita, Rev. Biol. (Lisbon) 10, 127 (1976)] and Sternbergia (H. Falk, personal communication).

METHODS IN ENZYMOLOGY, VOL. 148

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vides excellent mechanical as well as osmotic stability to these organelles and, additionally, outstanding preservation of the stroma enzymes during the isolation procedure.

Isolation Procedure for Daffodil Chromoplasts Daffodil flowers from Narcissus pseudanarcissus L. (var. "Golden Harvest") arc used. In the very early invcstigations wild-type plant material was also used which was collccted in the southern Vosges (France) near Gdrardmer. 4,7,s The cultivated forms of N . p s e u d o n a r c i s s u s grow taller and possess larger and more deeply colored fowers but the general chemical composition of the chromoplasts originating from both plant materials remains more or less unchanged. For chromoplast isolation the coronas ("Nebenkronen") were used exclusively. It has been shown by electron microscopy that the only plastid types present in all tissues of this part of the plant are chromoplasts. Because petals contain some chloroplasts in their basal veins, they have not been used in our investigations. Only freshly bloomed flowers or those just in the state of opening should be used because chromoplasts from fully expanded flowers are less active with respect to their lipid biosynthetic capabilities. 9 Furthermore, with older plant material.the chromoplasts sometimes exhibited a tendency to aggregate during the very early steps of isolation; therefore, they should be excluded from the beginning. The coronas were cut off at their bases and the stamens carefully removed. For smaller preparations (up to I00 coronas) the homogenization step was done with a knife homogenizer (rotating knife; B0hler, Tiibingen, FRG) in portions containing about I0 flowers. The coronas were first cut into small pieces with razor blades in a small amount of icecold isolation medium. For the routinely performed large-scale preparations (up to 1000 coronas/day) a rebuilt kitchen mixer equipped with two pairs of razor blades was used for the homogenization of the tissue, t° The isolation medium consists of 0.47 M sucrose, 5 mM MgC12, 0.2% (w/v) poly(vinylpyrrolidone) K 90, Mr ca. 360,000 (Roth KG, Karlsruhe, FRG), and 67 mM phosphate buffer (Soerensen type buffer; solution A: KH2PO4; solution B: Na2HPO4" 2H20; approximate mixture for pH 7.5:1 vol A: 5.7 vol B). For homogenization a tissue-to-medium ratio of 1 : 3 to 1:4 (w/v) was used. Homogenization was carried out by four short 7 B. Liedvogeland P. Sitte, Naturwissenschaften 61, 131 (1974). 8 H. Falk, B. Liedvogel, and P. Sitte, Z. Naturforsch., C: Biosci. 29C~541 (1974). 9 H. Kleinig and B. Licdvogel, Eur. J. Biochem, 83, 499 (1978). ~0C. G. Kannangara, S. P. Gough, B. Hansen, J. N. Rasmussen, and D. J. Simpson, Carlsberg Res. Commun. 42, 431 (1977).

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strokes (5 sec each; in the case of the Btihler homogenizer at half-maximum speed, ca. 35,000 rpm). The tissue brei was filtered through four layers of nylon cloth (40-tLm mesh), and cellular debris, nuclei, and cell wall material were removed by a low-speed centrifugation step (15 min, 1000 g, 4°). Chromoplasts were sedimented from the supernatant by a subsequent centrifugation step (20 min, 16,500 g). This crude chromoplast pellet also contained mitochondria, which have to be removed from the plastids during a sucrose gradient centrifugation step. The pellets were gently resuspended with the aid of a rather loosefitting Dounce glass homogenizer in 67 mM Soerensen buffer, pH 7.5, containing 50% (w/v) sucrose and 5 mM MgC12. The resuspended organelles were placed in centrifugation tubes to form a layer at the bottom (ca. 8 ml, if 34-ml swing-out tubes were used, or ca. 14 ml in the case of 60-ml tubes). The organelle suspension was carefully overlaid with equal volumes of 40, 30, and 15% (w/v) sucrose in the same buffer to form a discontinuous gradient. It was centrifuged for 1 hr in a 3 × 34 ml and 3 × 60 ml swinging bucket rotor (Weinkauf, Brandau, FRG), respectively, at 50,000 g. The chromoplasts, concentrated by flotation between 40 and 30%, and between 30 and 15% sucrose, respectively, were carefully removed with a Pasteur pipet. It should be mentioned that by conventional centrifugation, i.e., migration of the organelles from top to bottom of the gradient, such a high degree of purity of the fraction could not be achieved; the necessary elimination of mitochondrial contaminants was not very successful by this method. Therefore, flotation-like centrifugation of the gradient was an indispensable requirement in order to obtain pure chromoplast fractions. The combined fractions from the two interphases were slowly diluted with 67 mM phosphate buffer, pH 7.5, 5 mM MgCI2 to a final concentration of 15% sucrose, as measured from the refractive index by an Abbe refractometer. A pure chromoplast pellet was obtained by a final centrifugation step (20 min, 16,500 g). All steps of the isolation procedure were carried out in the cold (4°). The chromoplast fraction was used immediately after isolation when investigating acyl- and galactosyltransferases or the fatty acid-synthesizing machinery of plastids. For studies on carotenogenic enzymes, 11 the chromoplasts were diluted in the appropriate buffer, treated in a French pressure cell, and then stored at - 7 0 ° until use. (There was no substantial loss with respect to the enzymatic activity for several months.) In this context it should be remarked that it is also possible to isolate-in addition to the chromoplasts--the mitochondria of the corona cells. These organelles are found in the 40%/50% sucrose interphase of the tl p. Beyer, this v o l u m e [36].

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FIG. 1. Ultrathin section of isolated chromoplastsof Narcissus pseudonarcissus. Survey of the fraction (×6000; bar, 5 /zm). The electron micrograph was kindly provided by Dr. H. Falk. gradient, but still contaminated with chromoplasts. The method for mitochondria purification is given elsewhere, n Basing on the procedure presented above, a modification has been described which allows the simultaneous isolation of chromoplasts, as well as mitochondria, from daffodil coronas.~3 Criteria for the Purity of the Chromoplast Fraction The isolation procedure described above resulted in a clean fraction, rich in unbroken chromoplasts (Fig. 1). The morphometric evaluation of a number of electron micrographs of several different preparations is summarized in Table I. More than 92% and probably as much as 97% of the 12 F. Ltitke-Brinkhaus, B. Liedvogel,and H. Kleinig, Eur. J. Biochem. 141, 537 (1984). ~3p. Hansmann and P. Sitte, Z. Naturforsch., C: Biosci. 39C, 758 (1984).

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TABLE I CHARACTERIZATIONOF Narcissus pseudonarcissus CORONA CHROMOPLASTFRACTION BY PARTICLE COUNTING ON ULTRATHIN SECTIONS

Particle type Chromoplasts Mitochondria Microbodies Nuclei, and nuclear envelope fragments Small plasma droplets with inclusions Bacterial cells Other particles, not identifiablea

Total counted particles (%) 92.3 0.6 0.5 0.8 0.6 5.2

a None of these particles is mitochondria or bacteria, which are easily identified in sections. Part of this category is likely to be unusually formed or degraded chromoplasts.

TABLE II ENZYMATIC PROPERTIES OF ISOLATEDDAFFODIL CHROMOPLASTS A. Activities associated with chromoplast membranes 1. Reactions concerned with glycerolipid metabolism Glycerob3-phosphate acyltransferase TM Acylglycerol-3-phosphate acyltransferase 14 Phosphatidate phosphatase 14 Galactosyltransferases (galactosylating diacylglycerol and monogalactosyldiacylglycerol) 14-16 Acylation of phospholipids from acyl-CoA 14,~6 2. Further acyl and glycosyl transfer reactions Acylation of monogalactosyldiacylglyceroF~ Acylation of sterol glycoside 16 Glycosylation of sterols ~5,~6 3. Acyl-CoA synthetasC 4 and acyl-CoA hydrolase 9,~4A6 4. Prenyl lipid metabolism Carotenogenic enzymes (including phytoene synthase and prenyltransferases) H,~7-~9 Chlorophyll synthase 2° 5. Membrane translocators (phosphate and adenylate translocator) 21 B. Soluble chromoplast enzymes 1, Complete enzyme complement for long-chain fatty acid synthesis from acetate, including acetyl-CoA carboxylase 9,14,2223 2. Acetyl-CoA synthetase 24 3. Glycolytic enzymes (starting from dihydroxyacetone phosphate) 22 4. Pyruvate dehydrogenase complex 23,25

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particles counted on those micrographs were identified as chromoplasts. Less than 1% of the particles were mitochondria, microbodies, or bacterial cells. Contaminating droplets with cytoplasmic inclusions did not exceed 1%, and nuclei or nuclear envelope material were totally absent. Support for the latter statement came from investigations on the chromoplast DNAwlinear DNA strands exceeding a contour length of about 45 /zm, which is the size of a complete plastid DNA circle, were never found. 8 In accordance with the high purity of the chromoplast fraction determined by electron microscopy, marker enzymes typically associated with plant mitochondria and microbodies could not be detected. The membranous chromoplast of the daffodil has proved to be an outstanding example of a nonphotosynthesizing, energy-heterotrophic plastid exhibiting high activities for several metabolic pathways. In particular, lipid synthetic capabilities were investigated. Table II presents a survey of the rather extensively studied enzymatic properties of daffodil chromoplasts.14-25 14B. Liedvogel and H. Kleinig, Planta 144, 467 (1979). 15 B. Liedvogel and H. Kleinig, Planta 129, 19 (1976). 16 B. Liedvogel and H. Kleinig, Planta 133, 249 (1977). 17 p. Beyer, K. Kreuz, and H. Kleinig, Planta 150, 435 (1980). 18 K. Kreuz, P. Beyer, and H. Kleinig, Planta 154, 66 (1982). 19 p. Beyer, G. Weiss, and H. Kleinig, Eur. J. Biochem. 153, 341 (1985). 2o K. Kreuz and H. Kleinig, Plant Cell Rep. 1, 40 (1981). 21 B. Liedvogel and H. Kleinig, Planta 150, 170 (1980). 22 H. Kleinig and B. Liedvogel, Planta 150, 166 (1980). 23 B. Liedvogel and H. Kleinig, in "Biogenesis and Function of Plant Lipids" (P. Mazliak, P. Benveniste, C. Costes, and R. Douce, eds.), p. 107. Elsevier/North-Holland Biomedical Press, Amsterdam, 1980. 24 B. Liedvogel, Anal. Biochem. 148, 182 (1985). z~ B. Liedvogel, Z. Naturforsch., C: BioscL 40C, 182 (1985).

[25] S t r u c t u r e a n d F u n c t i o n o f t h e I n n e r M e m b r a n e S y s t e m s in E t i o p l a s t s B y SATORU MURAKAMI

In etiolated plants grown in total darkness the proplastids develop to etioplasts, which are devoid of chlorophyll (Chl) and contain substantial amounts of protochlorophyllide (Pchld) and carotenoids. Photosynthetic METHODSIN ENZYMOLOGY,VOL. 148

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