Under conditions of stress, e.g., nitrogen deficiency, Eremosphaera viridis De Bary (Chlorophyceae, Chlorococcales) synthesized secondary carotenoids and large amounts of triacylglycerols forming orange-red, cytosolic lipid bodies. Additionally, four polypeptides (28, 26, 25 and 23 kDa) as well as traces of chlorophyll a and b, of violaxanthin, neoxanthin and zeaxanthin, and of membrane lipids could be demonstrated in isolated lipid bodies. No membrane could be shown around the lipid bodies by the use of electron microscopy. The formation of lipid bodies in Eremosphaera is discussed as a bulging of the chloroplast envelope membrane

Eichenberger, Waldemar

Ruppel, Georg

Vechtel, Burkhard

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Plant Cell Physiol. 33(1): 41-48 (1992)JSPP © 1992Lipid Bodies in Eremosphaera viridis De Bary (Chlorophyceae)Burkhard Vechtel1, Waldemar Eichenberger2 and Hans Georg Ruppel11 Department of Biology, University of Bielefeld, D-4800 Bielefeld, F.R. Germany2 Department of Biochemistry, University of Bern, CH-3012 Bern, SwitzerlandUnder conditions of stress, e.g., nitrogen deficiency, Eremosphaera viridis De Bary (Chloro-phyceae, Chlorococcales) synthesized secondary carotenoids and large amounts oftriacylglycerols forming orange-red, cytosolic lipid bodies. Additionally, four polypeptides (28,26, 25 and 23 kDa) as well as traces of chlorophyll a and b, of violaxanthin, neoxanthin and zea-xanthin, and of membrane lipids could be demonstrated in isolated lipid bodies. No membranecould be shown around the lipid bodies by the use of electron microscopy. The formation oflipid bodies in Eremosphaera is discussed as a bulging of the chloroplast envelope membranes.Key words: Chloroplast — Eremosphaera viridis — Lipid body — Secondary carotenoids —Triacylglycerols.When stressed by nutrient limiting conditions, algaesynthesize large amounts of TAG and accumulate themmostly in discrete cytosolic droplets variously referred to asoil bodies, spherosomes, oleosomes or lipid bodies, theterm to be used here (Czygan 1968, Shifrin and Chisholm1981, Piorreck et al. 1984, Suen et al. 1987, Guckert andCooksey 1990). Our knowledge of algal lipids has turnedout to be surprisingly poor, and even less is known aboutthe chemical composition of lipid bodies in algae. Somemore information is available about oil bodies in higherplants, particulary in seeds.Higher plants store TAG in so called oil bodies,spherosomes or oleosomes (Gurr et al. 1974, Bergfeld et al.1978). They are associated with some characteristicproteins not extractable with detergents (Herman 1987,Murphy and Cummins 1989). It was suggested that oilbodies are surrounded by a half-unit-membrane (Yatsu andJacks 1972, Moreau et al. 1989), or denied (Bergfeld et al.1978). They are considered to form freely in the cytosol(Murphy et al. 1989), or to be derived from endoplasmaticreticulum or chloroplasts (Schwarzenbach 1971, Wanner etal. 1981). The oil bodies of seeds of higher plants serve asa source of energy during germination (Qu et al. 1986),Abbreviations: DGDG, digalactosyldiacylglycerol; DGTS,diacylglycerol-N,yV,yV-trimethylhomoserine; HPLC, high pressureliquid chromatography; MGDG, monogalactosyldiacylglycerol;PE, phosphatidylethanolamine; PG, phosphatidylglycerol; SC,secondary carotenoids; TAG, triacylglycerol; TLC, thin layerchromatography; d, day(s).whereas lipid bodies in algae are divided, not consumedduring regreening after nitrogen limitations (Shifrin andChisholm 1981).In the chlorococcalean green alga Eremosphaeraviridis De Bary (Chlorophyceae), under nitrogen deficiencynumerous lipid bodies occurred which contained SC(Vechtel et al. 1991a) suggested to be synthesized in chloro-plasts (Vechtel et al. 1991b). The purpose of this studywas to examine the chemical composition of the lipidbodies in order to elucidate their ontogeny and to find in-dications on the means of transport of SC from chloro-plasts into these cytosolic lipid bodies.Materials and MethodsAlgal culture—Eremosphaera viridis De Bary (AlgalColl. Goettingen F.R.G., SAG LB-221) was grown as de-scribed by Weidinger and Ruppel (1985). The N-deficientconditions of algal culture were employed according toVechtel et al. (1991a).Lipid bodies were isolated from algae grown under N-deficiency for 40 d according to Vechtel et al. (1991a) and,if necessary, incubated for 30 min at 25°C with 0.25% SDSor 0.61% Triton X-100 according to Murphy and Cum-mins (1989). Isolated lipid bodies were extracted twicewith acetone and centrifuged at 4,300 x g for 5 min. Thepellet was solubilized and used for SDS-PAGE to separateproteins according to Laemmli (1970). The proteins werestained by a combined commassie blue-silver stain pro-cedure (De Moreno et al. 1985). The supernatant was41B. Vechtel, W. Eichenberger and H. G. Ruppeldried under N2, and the residue redissolved in chloroformfor lipid analysis.Lipid analysis—Lipids were determined on silica gelTLC plates (Merck, F.R.G.). For separation, the follow-ing eluents and sprays, resp., were used: for TAG: petrolether (60-80°KP)/diethylether/acetic acid (90: 10 : 1 v/v),and bromothymol blue ( 0 . 4 m g m r ' 0.1 N NaOH), resp.;for phospholipids: chloroform/methanol/H2O (65 : 25 : 4v/v), and molybdenum blue, resp.; for glycolipids: chloro-form/methanol/acetic acid/H2O (85 : 15 : 10 : 3.5 v/v),and phosphomolybdic acid, resp. All spray reagents wereobtained from Sigma, F.R.G.Fatty acid methyl esters from lipids isolated by TLCwere obtained and identified according to Giroud et al.(1988). Chlorophyll content was determined according toSchmid (1971). Secondary carotenoids were separated byTLC and HPLC according to Vechtel et al. (1991a). IR-spektra from isolated lipids were made with a MattsonPolaris FT IR-spectronic.Electron microscopy—For electron microscopy thinsections were prepared according to Weidinger and Ruppel(1985). Additionally, samples were fixed with 5% KMnO4for 30-60 min without staining with uranyl acetate or leadcitrate. Thin sections were observed and recorded on aHitachi H 500 electron microscope at 75 kV.ResultsEremosphaera viridis De Bary contained about 200chloroplasts within its giant cell of 100-200//m diameter.Thin sections of Eremosphaera cells grown in completemedium showed chloroplasts similar to those of higherplants (Fig. 1). Most of the thylakoids appeared as pairsthroughout the chloroplasts. Over a wide range granastacks of about six thylakoids could be seen, whereasstroma-thylakoids were observed rarely. Starch grainsoccurred exclusively within the chloroplasts. Undernitrogen-free conditions thylakoids disintegrated and onlya few could be found after 15 d of nitrogen deficiency,mostly in pairs as stroma-thylakoids (Fig. 2A). Starchgrains often filled chloroplasts almost completely and lipidbodies appeared in the cytosol next to plastids. After 40 dof N-deficiency thylakoids were reduced to a minimum andlipid bodies reached or even surpassed the size of chloro-plasts. After fixing with glutaraldehyde/OsO4, no mem-brane or boundary could be detected around the lipidbodies from Eremosphaera (Fig. 2B). However, in cells ofEremosphaera fixed with KMnO4, each lipid body was sur-rounded by a electron-dense coat, known as a depositionmembrane of pyrolusite, and with marked differences froma normal bilayer membrane (Fig. 3A and B).Fig. 1 Thin sections of cells of Eremosphaera grown in complete medium, fixed with glutaraldehyde/OsO4 (bar=1 fim).chloroplast, MT=mitochondrion, S=starch grain.CHL=Lipid bodies of Eremosphaera viridis 43In isolated lipid bodies of Eremosphaera, TAG repre- xanthophylls (neoxanthin, violaxanthin and zeaxanthin)sented the main part (Table 1) with oleic acid (18:1) as the and small amounts of MGDG, DGDG and PG could be de-main fatty acid (Table 2). Most of the SC, synthesized tected. PE and the betaine lipid DGTS both of which areunder N-deficiency, ^-carotene and lutein were found in iso- present in whole cells of this alga, could not be detected inlated lipid bodies. Traces of chlorophyll (a/b ratio of 1.5), lipid bodies. Four polypeptides of 28, 26, 25 and 23 kDaFig. 2 As Fig. 1, grown in nitrogen-free medium for 9 d (A) and 40 d (B). (bar=l//m). LB=lipid body.44 B. Vechtel, W. Eichenberger and H. G. RuppelFig. 3 Thin sections of cells of Eremosphaera grown in nitrogen-free medium for 40 d, fixed with KMnO4. A: lipid bodies (1//m —bar), B: detail of border zone between lipid body and chloroplast (0.04^m=bar). CHL=chloroplast, CYT=cytosol, IE=innerenvelope, LB=lipid body, M=lipid body membrane, OE=outer envelope, S=starch grain.Lipid bodies of Eremosphaera viridis 45Table 1 Chemical composition of lipid bodies isolatedfrom cells of Eremosphaera grown under N-deficiency for40dTriacylglycerolsTrialkylethersSecondary carotenoidsChlorophyllsPhophatidylglycerolMonogalactosyldiacylglycerolDigalactosyldiacylglycerolPolypeptides: 28, 26, 25, 23 kDa80%10%7%1 0 /Percentage of w/w, estimated.molecular weight could be shown to be associated with theisolated lipid bodies (Fig. 4). Additionally, a fraction wasisolated from lipid bodies by TLC containing ether bondsdetected by IR-spectra (Fig. 5). We assume that thesetrialkylethers are derivatives from the phytols liberated bychlorophyll degradation during N-deficiency (Vechtel et al.1991a).Incubation with detergents, SDS or Triton X-100, didnot result in fusion of the isolated lipid bodies, but led tothe eluation of the proteins chlorophylls, xanthophylls andphospho- and glycolipids into the rinsing buffer. In con-trast, TAG, trialkylethers and secondary carotenoids re-mained within the lipid bodies where they were demon-strated by TLC.To investigate the process of lipid body formation,cells of Eremosphaera were grown under N-deficiency forseveral days and periodically observed by electron micros-copy. From day 3 on, cytosolic lipid bodies appeared,13B C D EFig. 4 SDS-PAGE of proteins attached to isolated lipidbodies. lane A: of isolated lipid bodies, lane B: after twoand lane C after four times of washing of isolated lipid bodies,lane D: after extraction from rinsing buffer containingdetergents. E: of isolated lipid bodies after incubation withdetergents.Table 2 Fatty acid composition in per cent (w/w) oftriacylglycerols of lipid bodies isolated from cells ofEremosphaera grown under N-deficiency for 40 dFatty acid composition of triacylglycerols[% mol of total fatty acids]C-14:0C-16:0C-16:lC-16:3C-18:0C-18:lC-18:2C-18:3C-18:4C-20:4C-20:50.9%8.0%0.7%1.5%67.0%16.6%1.1%0.9%0.6%always having a distinct minimum of size, and smaller onescould not be detected. On the other hand, after two daysof growing in nitrogen-free medium, cells showed a "bulg-ing" of chloroplast envelope membranes, especially of theouter one (Fig. 6A and B). Vesicles germinating from thy-lakoids seemed to migrate towards the "bulging site"(Fig. 6B), possibly one of the first stages of developing lipidbodies. Often the inner envelope was clearly to be seen,whereas the outer one began to disintegrate (Fig. 6B).DiscussionIn Eremosphaera the accumulation of TAG under N-deficiency leads to the formation of lipid bodies, commonto other algae as well (Czygan 1968, Shifrin and Chisholm1981, Ben-Amotz et al. 1982, Cooksey et al. 1987, Suen etal. 1987, Lers et al. 1990). Like in the other algae studied,the TAG of Eremosphaera mainly consists of C-18:l fattyB. Vechtel, W. Eichenberger and H. G. RuppelFig. 5 As Fig. 3, grown in nitrogen-free medium for 2 d.thylakoid vesicles.A and B. possible stages of lipid body formation (bar=0.15 fim). TV=Fig. 6 IR-spectra of trialkylethers isolated by TLC from lipidbodies of cells of Eremosphaera grown under N-deficiency for 40d. A: 2,960, 2,930 and 2,860 cm"1 (CH2 and CH3);B: 1,120cm"1 (C-O-C ether).acid which under conditions of stress appears in even largerproportions than under normal conditions (Piorreck et al.1984). It was suggested that inhibition of cell division andinterruption of the cell cycle at a stage of increased lipidsynthesis leads to the formation of lipid bodies (Coleman etal. 1988).Although lipid bodies of Dunaliella bardawil (Ben-Amotz et al. 1982) and of Eremosphaera seem not to be sur-rounded by a membrane, some indications for remnants ofmembrane compounds exist. Membrane lipids, togetherwith proteins and chlorophylls are found in lipid bodiesof Dunaliella (Ben-Amotz et al. 1982) and in those ofEremosphaera as well. This could be explained as a con-tamination caused by their attachment to the surface of thelipid bodies during isolation (Steinmuller and Tevini1985). On the other hand, the proteins, together with thechlorophylls and the xanthophylls might be derived fromlight-harvesting complexes from the chloroplasts. Addi-tionally, MGDG and DGDG which are exclusively locatedLipid bodies of Eremosphaera viridisin chloroplast membranes (Joyard and Douce 1987) arepresent in isolated lipid bodies and the trialkylethers, ifthey are derived from phytols of degraded chlorophylls,also have plastidic origin. Since, on the other hand, PEand DGTS both of which are constituents characteristicof the ER (Eichenberger and Gerber 1987), are absentfrom lipid bodies, these results indicate an involvement ofplastids in the formation of lipid bodies in Eremosphaera.It is accepted today that plastids play a distinctive rolein lipid biosynthesis. Chloroplast envelopes are postulatedas a major site for biosynthesis of membrane lipids like gly-co- and phospholipids or of TAG (for review see: Douceand Joyard 1990). In addition, there is the possibility thatplastids convert diacylglycerols into TAG as describedfrom plastids of spinach leaves under stress conditions(Sakaki et al. 1990).In 1981, Wanner et al. published their model of lipidbody formation by plastids in oil plants suggesting buddingoff from the outer envelope. In agreement with this hy-pothesis, we now assume that in Eremosphaera the in-creased synthesis, together with a conversion of diacylglyce-rols to TAG, leads to the accumulation of neutral lipidsbetween inner and outer envelope resulting in bulgingespecially of the outer membrane. Then the envelope mem-branes disintegrate.—Additionally, the TAG content ofchloroplasts isolated from cells grown under N-deficiencyfor 3 d was higher than of chloroplasts from normal growncells (data not shown).—Thereafter, lipid bodies of a par-ticular size could be demonstrated in close proximity tochloroplasts by electron microscopy. Unfortunately, thetransient stage of lipid body formation between bulgingsites and individual droplets has not yet been observed.Our hypothesis is in accordance with observations byLichtenthaler and Peveling (1966), describing lipid globulesleaving chloroplasts of Hoya carnosa, and Miyake et al.(1989), reporting plastoglobuli containing TAG releasedfrom plastids into the vacuole.Our assumption explains the transport of SC out ofchloroplasts into cytosolic lipid bodies as a simple diffusionof carotenodis into the hydrophobic bulging sites of theplastids, lateron forming individual lipid bodies.We wish to thank Dr. Bodo Muller, Department of Biology,University of Bielefeld, for critical reading of manuscript.ReferencesBen-Amotz, A., Katz, A. and Avron, M. (1982) Accumulationof ^-carotene in halotolerant algae: purification and characteri-zation of /?-carotene-rich globules from Dunaliella bardawil.J. Phycol. 18: 529-537.Bergfeld, R., Hong, Y. N., Kiihnl, T. and Schopfer, P. 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Lipid Bodies in Eremosphaera viridis De Bary (Chlorophyceae)

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