Cornelius Lütz

Biochemical and Cytological Observations on Chloroplast Development VI. Chlorophylls and Saponins in Prolamellar Bodies and Prothylakoids Separated from Etioplasts of Etiolated Avena sativa L. Leaves

Summary Prothylakoids (PTs) were separated from prolamellar bodies (PLBs) of etioplasts obtained from leaves of Avena seedlings grown in complete darkness of after 3–5 min. illumination. In part of the leaves protochlorophyll (ide) content was increased by treatment with δ-aminolevulinic acid (ALA) prior to the isolation of the etioplasts. Protochlorophyl(ide), chlorophyll (ide) and saponin content (the latter as an indication for presence of tubules from the PLBs) of the fractions was determined. In the isolated PLB and PT fractions about 75 % of the saponin content of the «broken etioplast» (BE) fraction could be recovered. The PT fraction contained only 4.6 % of the saponins, indicating a high degree of purity. The PLB fraction was enriched in PLBs, but contained an unknown amount of PTs. About 80 % of the chlorophyllous pigments in the BE fraction were recovered in the PT and PLB fractions independent of light and/or ALA treatment of the plants. Thus, both endogenous PChl(ide) as well as the ALA-induced PChl(ide) are bound to membranes which can be sedimented at relatively low g-values. About 1/3 of the endogenous PChl(ide) and Chl(ide) and of the ALA-induced PChl(ide) was found consistently in the PT fraction, independent of light treatment. Since the ratio chlorophlls/saponin in the PTs was about 6 times higher in the PT fraction than in the PLB fraction it is evident that in Avena etioplasts the pigment content of the PT membrane is much higher than that of the PLB tubules. The possibility is discussed that most of the chlorophyll may be located in the prothylakoids only.

Biochemische und cytologische untersuchungen zur chloroplastenentwicklung I. Die chemische Charakterisierung der Prolamellarkörper aus Etioplasten von Avena sativa L.

Summary Etioplasts were prepared from primary leaves of etiolated Avena seedlings. The prolamellar bodies could be separated after osmotic shock. After extraction of the isolated prolamellar bodies with water and chelating agents the remaining ≪structural proteins≫ (SP) could be dissolved in a strong SDS 1 ) buffer. The dissolved SP were then separated by gel electrophoresis into 7 polypeptides (SP-1 to SP-7). The determined molecular weights are between 22000 (SP-1) and 68000 (SP-7). SP-1 represents 40–50% of the total SP. By staining for carbohydrate SP-1 could be designated as the own glycoprotein among the SP. The lipid composition of isolated prolamellar bodies could be identified by thin-layer chromatography: Mono- and Digalactosyldiglyceride, Acylsterylglycoside, Cerebroside, Phosphatidylcholine, Phosphatidylethanolamine, Phosphatidylinosite and Phosphatidic acid. To compare the SP of prolamellar bodies of etioplasts with the SP of thylakoids of chloroplasts we isolated the thylakoid structural proteins. Their gel electrophoretic separation showed the same SP found in the prolamellar bodies and an additional polypeptide (MW 24000).

The Localization of Saponins in Prolamellar Bodies Mainly Depends on the Isolation of Etioplasts

Summary For some years it was assumed, that the prolamellar bodies (PLBs) of well developed etioplasts are mainly built up by two saponins, called avenacosides A and B. Recently published experiments indicate however, that these compounds are very quickly formed during homogenisation of plants from preexisting, not etioplast bound precursors. Experiments are described for isolation of etioplasts from glutaraldehyde prefixed leaves, and from untreated ones. The plastids are intact and very low in their content of the above mentioned saponins. High yields in these etioplasts are obtained using Percoll gradients run for short times in a high speed vertical rotor. The separated fractions are desribed by means of electron microscopy and of lipid analysis. The high saponin content found especially in oat etioplasts, can be explained by a fast and specific adsorption of these lipids to the PLB-tubules. It is shown, that this only occurs with damaged plastids or free PLBs. Thus, the results support the opinion, that PLBs are not built up by saponins.

Distribution of Carotenoids and Lipids in Separated Prolamellar Bodies and Prothylakoids of Etioplasts from Avena sativa L.

Summary Isolated etioplasts of primary leaves from Avena sativa have been used to prepare a «broken etioplast» (BE) fraction after discarding envelopes and stroma. This fraction, containing prolamellar body-prothylakoid-complexes was sonicated and run on density gradients. Two bands could be isolated, the «prolamellar body» (PLB) fraction and the «prothylakoid» (PT) fraction. The three fractions prepared (BE, PLB, PT) have been subjected to lipid and carotenoid analysis. The aim of this work is to elucidate the localization of carotenoids, of the glycolipids MGDG, DGDG and SQDG and of the phospholipids PC and PG in either PT-membranes or in the tubular system of PLBs. The results obtained give a clear localisation for the glycolipids and for the carotenoids: they are bound exclusively to the PT-membranes. However, PC and PG display a different bond: c. 8% of PG and c. 19% of PC determined in the fractions are considered to be fixed in the PLB-complexes. Thus the PLB cannot be seen as a storage for lipids and carotenoids which has to be used for thylakoid formation during greening. The phospholipids found in part in PLBs could indicate an affinity in assembly between PC, PG and the saponins constructing mainly the tubules of PLBs.

Biochemical and Cytological Observations on Chloroplast Development V. Reaggregations of Prolamellar Body Tubules without Protein Participation

Summary Prolamellar bodies (PLBs) of Avena sativa L. were completely dissolved by heating in buffers containing sodium dodecyl sulfate (SDS). The dissolved building units were separated into 2 fractions: one containing only proteins, the other containing lipids and pigments with one protein only. The fractions were then tested for their ability to reaggregate into PLB-like structures. Only the lipid-fraction with pigments and one protein was able to form tubules resembling the structure of native PLBs. Disintegration of the protein by a proteolytic enzyme did not influence the ability to reaggregate; protcins do not participate in reaggregation of dissolved PLBs. Artifical tubules are built up only by lipids, pigments or a mixture of these substances. These experiments lead to the assumption that the building units of this artifical system also playa main role in the construction of natural PLBs.

Amino acid composition of the enzyme NADPH: Protochlorophyllide oxidoreductase

The enzyme protochlorophyllide oxidoreductase (PCR) could be purified from Avena sativa plants by means of column chromatography and preparative gel electrophoresis. The amino acid composition of the protein was determined. The enzyme consists of 346 amino acids and has a molecular weight of approximately 37 800. Tryptophan is absent, and only one cysteine participates in the active site. The high content of basic amino acids explains the basic pI of approximately 8.5, observed by gel electrophoresis. The amino acid composition favours a theory explaining the observed association of the enzyme with the lipid environment of prolamellar bodies. Furthermore, the protein contains hydrophobic/hydrophilic amino acids in amounts similar as is known for intrinsic pigment binding proteins of the mature thylakoid membrane.

Biochemische und cytologische Untersuchungen zur Chloroplastenentwicklung: II. Isolierung und Charakterisierung eines Glycoproteins aus den Prolamellarkörpern der Etioplasten von Avena sativa L.

Summary In the prolamellar bodies of the etioplasts of Avena sativa seedlings we found a glycoprotein (MW 22,000) among the structural proteins (SP). After disintegration of the prolamellar bodies with detergent this polypeptide (SP-1) could be separated from the mixture of other SP working with exclusion chromatography on Sephadex. After digesting SP-1 with trypsin over 34 peptides were separated by «fingerprinting». The determination of the amino acid content of SP-1 showed a high yield of hydrophobic amino acids (54,9 mol-%). The ratio from basic to acidic amino acids is 1.15. The remaining residue after trypsin treatment has a higher content of hydrophobic amino acids (64,3 mol-%), compared with total SP-1; in the same way the ratio of basic to acidic amino acids has a higher value (2.03). In this trypsin-resistant residue we find the carbohydrate moiety of the glycoprotein and 8–10% of the total protein content. Amino acid and carbohydrate analysis gave a minimal molecular weight of 19,500 for SP-1, this agrees with the value of 22,000 calculated after gel electrophoresis. In the thylakoid preparation of Avena we found a glycoprotein corresponding to SP-1. After purification and tryptic digestion we compared the «fingerprint» analyses of this glycoprotein and SP-1. The opposition proved that the two glycoproteins are identical.

Biochemische und cytologische Untersuchungen zur Chloroplastenentwicklung III. Superoxiddismutase in Etioplasten von Avena sativa L.

Summary Soluble extracts from etioplasts,chloroplasts of Avena sativa L. contain a single, electrophoretically distinct, superoxide dismutase (SOD). Besides this soluble form another SOD activity is associated with membranes of prolamellar bodies,thylakoids. The membrane-bound SOD was solubilized by digitonin treatment of the membranes. Both the soluble,the bound enzyme have the same Rf-value (0.68) in gelelectrophoresis,are both sensitive to cyanide indicating a Cu/Zn containing SOD. Soluble extracts of mitochondria from Avena sativa L. contain also a single, electrophoretically distinct, SOD. In mitochondria no membrane-bound SOD-activity could be detected. The soluble SOD of mitochondria is sensitive to cyanide indicating a Cu/Zn containing enzyme.

Biochemical and Cytological Observations on Chloroplast Development

Summary Prolamellar bodies (PLBs) of etioplasts of Avena sativa are made up of tubules, which compose a highly organized crystal-like system. After isolation of pure PLBs we solubilized the PLB-membranes completely with Na-dodecylsulfate (SDS). Using the -negative stain- technique we again observed in the electron microscope the presence of tubules a short time after the dissolution of the lipoprotein complexes. The first examinations of this phenomenon led to the following results: 1. The PLBs are completely dissolved by heating in a solution with a high ratio of detergent to protein. 2. The dissolved components can reaggregate into tubules during undisturbed incubation of the solution even in the presence of detergent. The speed of reaggregation is de- pendent upon the protein/SDS - ratio and the temperature of the incubation. The aggregates display the same structures and inner diameters as the native tubules. 3. Depending on incubation time and temperature of the solution, changes in the most predominantly visible structures are as follows: a) Temperatures between 12 °C and 20 °C: In the first days the growth of mostly straight and of only a few branched tubules was visible. Often these tubules bind laterally. b) Temperatures between 25 °C and 40 °C: The straight unbranched tubules are less often visible. Soon (3 rd-5 th day), branched aggregates appear which can form large tubular complexes. In the course of a longer incubation time (about 30 days) aggregates of fusing netlike structures and long tubules are formed. Experiments of other authors have shown, that PLBs were reconstructed into elongated tubules when exposed to light during cold treat- ment, which only very slowly fuse into thylakoids. In addition, our system shows the presence of mainly elongated tubules at low temperatures. The strong tendency of the

Reaggregation of etioplast lipids and the formation of prolamellar bodies and thylakoids: An ultrastructural study.

Etioplasts of dark-grownAvena sativa plants were used to prepare either saponin-free or saponin-containing prolamellar bodies. Lipid extracts from both fractions were studied in reaggregation experiments: extracts containing saponins showed liposomes as well as tubules, while saponin-free samples formed only liposomes. Purified PLB lipids in reaggregation experiments were either studied in the presence or in the absence of saponins. Best tubule formation was found with samples containing MGDG+saponin. However, the reconstruction of PLB-like structures was not possible. The long tubules, protruding from isolated PLBs, are seen as a result of the reaction of saponins (originally located in vacuoles) with MGDG.