94

PLANT LIPIDS

Phospholipid transport in plant cells

The intracellular transport ofmembrane components and theircorrect targeting to the right des-

tinations are of primeval importance inliving cells. Protein transport and sort-ing have largely been studied in bacte-ria. fungi. plants. and animals but thetrafficking of lipids has been chieflystudied in animal cells (1-3). It isessentially during the last decade thatnew insights concerning the imracellu-lar transport of membrane lipids havebeen obtained from plant cells (3),

This short review will summarizethe arguments (membrane lipid com-positions. enzyme locations) underly-ing an intracellular lipid movement inplant cells and which mechanismscould be involved. The review willfocus on one of the most abundantlipid families: the phospholipids.

Phospholipid compositionof various plant membranesTable I presents the phospholipidcomposition of the principalendomembranes and the plasma mem-brane of some plant tissues.

It is clear from this table that mem-branes isolated from differentorganelles have distinct and specificphospholipid compositions.

For example. phosphat idyl-ethanolamine (PE) is an abundant

This article was prepared forINFORM by P. Moreau. J.J.Bessoule, and C. Cassagne of theLaboratoire de Btosenese M~m-branaire UMR 5544-CNRS. Univer-site Ylctor Segalen Bordeaux 2. 146rue Lio Saignot. 33076 BordeauxCMu. France.

desaturation and elongation reactionsof the fatty acids can occur in theendoplasmic reticulum (ER) andendomembranes (10, II).

Except for the specific phospho-lipids of the mitochondria [cardi-olipids (4)] and the synthetic capabili-ties of chloroplasts in PA and PG syn-thesis. the biosynthesis of the phos-pholipids of most membranes takesplace primarily in the ER (12). How-ever. partially purified Golgi mem-branes from pea seedlings (13) andmaize roots (14) are able to synthesizePC and PE to an extent that is notentirely attributable to ER contamina-tion. In contrast to this, Golgi mem-branes prepared from leek seedlings(5) or cotton seedlings (15) are notimportant sites for PC and PE biosyn-thesis. Therefore. some discrepanciesexist between various plant tissues. Inpea seedlings and maize roots, theGolgi apparatus might be partly inde-pendent of the phospholipid syntheticcapabilities of the ER. These charac-teristics, and the fact that ER-Golgiinteractions are not systematicallyobserved under the microscope, ques-lion the nature (if any) of the relation-ship between the ER and the Golgimembranes in some cell types (16).

The presence of PA in ER, plasmamembrane, and tonoplast (Table I)

phospholipid in the plasma mem-brane, the tonoplast. and the mito-chondria but is absent from thechloroplast envelope. Phosphatidylser-ine (PS) is abundant only in the plas-ma membrane (this is true for leekseedlings but not for maize roots),whereas phosphatidylglycerol (PG) isfound predominantly in chloroplastsand glyoxysomes. and phosphatidyl-choline (PC) is more ubiquitous. Car-diolipid (diphosphatidylglycerol) hasbeen omitted from the table, as it issynthesized and found exclusively inthe mitochondria (4). Finally. we haveobserved a rather high level of phos-phatidic acid (PA) in the plasma mem-brane which could not be attributed tolipid catabolism.

Location of enzymesof phospholipid metabolismThe de flOVO synthesis of fatty acidsoccurs in the plastids (10). Then, some

Table 1PhospholipId composition of different plant membranes (% of totai) from various piant tissues

Membnme I'A PC FE PC PI PSEndoplasmic reticulum" 0.4 53.4 27.5 6.' 8.8 3.4Plasma membrane" 7.0 26.6 42.7 3.2 8.9 11.6'Ionoptestc 1.4 30.3 46.7 2.3 15.3 2.5Glyoxysornesc 55.0 13.5 14.6 16.8Mitochondria ourer-mb" 61.0 27.0 3.' 8.0Chloroplast envelcpee 60.7 0.9 24.8 13.6 0

a Data obtained from leek seedlings by Moreau (5). Bertho (6). and unpublished results by B. Sturbois-Balcerzak and P. Moreaub Data obtained from sycamore cells by Tavernier et al. (7).C Data obtained from cotyledons of cotton seedlings by Chapman and Trelease (8).d Data combined from sycamore cells and mung bean hypocrxyls. from Douce (4).~ Data combined from spinach. pea. and cauliflower plastids. from Douce and Joyard (9).

PA: phosphatidic acid: PC: phosphatidylcholine; PE: phosph:nidylethanolamine: PO: phosphatidylglycerol: PI: phosphatidylinositol;PS: phosphatidylserine.

INFORM, Vol. 9, no. I (January 1998)

300r.r------------------,

200Plasma membrane

100

o 5 30 6015Chase time (min) following a 120 minlabeling period with 14C-acetate.

FIgure t. In vivo Irsn.'er 01 phospholipids from the ER to the plasma membnlne.a moni-tored by pUIM-ChaH experiment •• Leek seedlings were labeled wittl(14C) acetate 'or 120min and then chand with unlabeled acetate for 5, IS, 30, and 60 min. MembraneIractions were then Isolated, and thalr phospholipid labeling was determined. EA " Endo-pl.smlc reticulum

can be attributed 10 the lipid syntheticpathways in the ER and to the activityof a phospholipase D that has beendemonstrated 10 be present in thesemembranes (17),

Except for ER and in some casesthe Golgi apperurus. the other mem-branes are not able to synthesize theirentire population of phospholipids.Therefore. this fact and. to a lesserextent, the different phospholipidcompositions require that the lipidmolecules be more or less specificallytransported to different destinations inthe cell.

lipids are transferred from their mainsite of synthesis (the ER membranes)to the other membranes. Various pos-sibilities will now be scrutinized fortheir involvement in phospholipidtransport.

Phospho Iipi d-binding protei liS.

This puzzling family of proteins hasbeen recently reviewed (22). Theywere first thought to participate inmembrane biogenesis owing to theirproperties of lipid binding; therefore,their possible role in lipid transportwas a topic of speculation. However,the isolation of cDNA and sequenc-ing of genes have revealed that thecorresponding proteins are routed tothe secretory pathway. Indeed, someof them have been found in theextracellular space, thus theirinvolvement as vehicles in the trans-fer of phospholipids cannot be takenfor granted, and may even be unlike-ly. It has. however. been suggestedthai, when present inside the cells.

Mechanisms of phospholipidtransportLabeling and pulse-chase experimentshave strongly suggested that a phospho-lipid movement from the ER to othercellular membranes does occur in manyplant systems (3,5,6.8,18-21). Howev-er, it has not been easy 10 determineby which mechanisms the phospho-

they could be involved in the phos-phatidylinositol (PI) dynamics inplants (23). Further studies using acell-free system from soybeanseedlings led to the conclusion thatPI transfer from the ER to the cellsurface did not require any lipidtransfer proteins or intermediatevesicles (24). Thus PI transfer awaitsfurther examination.

vesicular transport. That intenne-diate vesicular structures could beinvolved in the transport of membranecomponents in plant cells was firstdeveloped by the endomembrane con-cept of Mcrre and Mollenhauer (25).

The relative importance of vesicu-lar ER-Golgi plasma membrane/tone-plast pathways has been extensivelydiscussed (16,26,27). Vesicular struc-tures potentially playing a role in post-Golgi events have been clearly idemi-ficd and isolated (26,27), but there areonly few reports on the presence ofintermediate vesicular structuresbetween the ER and the Golgi appara-tus of plant cells (16). The difficultyin visualizing these structures is prob-ably due to their transient nature.

Glycoprotein transport has beenstudied in more detail and mostlyrequires vesicular structures. Howev-er, the involvement of vesicular pro-cesses in selective lipid transport isstill a matter of debate. In vivo andin vitro approaches have been devel-oped during the past decade to inves-tigate the inrerrnembrane transport ofphospholipids. By using ill vivopulse-chase experiments and mem-brane subfractionation, it has beenpossible to visualize the intracellularmovement of various phospholipidsfrom the ER to the surface of leekcells (5,6,20,21). A typical pulse-chase experiment is shown in Fig-ure I.

The involvement of the Golgiapparatus as an intermediate compart-ment was demonstrated by studyingthe effect of low temperatures andthat of monensin, a drug known 10block the intracellular transport ofproteins at this level (28). Monensinand low-temperature treatmentsshowed that PS transfer was blockedat the level of the Goigi apparatus andto a lesser extent in the ER, whereasPI was still transferred to the cell sur-

95

INFORM. VOl. 9. 00. 1 (January 1998)

96

PLANT LIPIDS

C16fQ8.1'CC16fCIB-FE

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Figure 2. Modal of the transfer pathways of pholphotlpids with various polar h.ad, and 'atty acid chain length to the plalrna membraneof leek call •. PC-VLCFA, PE-VLCFA, and PS: these phoaphollplda (having It ,••• t ona very long chain latty acid of 20 to 24 carbonatomaln the c.ae 01 PC and PE) are routecllhrough the ER·to-Golgi apparalul·!o-pl •• ma membrane pathway, and their transfer to thepI•• ma membrane I, blocked by monenlln and low temperatures (below 12°C). PI, CI61C18-PC and CI6/CI8-PE: these phospholipidscould follow the ER-Goigi plume membrane pathway but, in the presence of monen"n Of at low temperatures, their transport to thepl •• m. membrana'e not blocked. This luggeat. thai other rout .. auch as I direct ER-ta-plasma membraM tranafer and/or a direct cl.medial Golgl-ta-plasma membrane tranaler can occur, or thet other mechen Ism. are Inyoilled. ER, endopla.mlc reticulum, Cis, Med, andnan. corre.pond to the cll,medial and trans cisternae of the Goigi apparatu.; TGN, trans Goigi netwolil: ; PM, pla.ma membrane; M,monensln (the po •• lble location. 01 monansln blocks are Indicated); T", temperatures below 12·C (at 1rC tn. IIIslcles belwHn the ERand the Golgi apparatus .ccumulate, whereas the number of secretory ..... Icle. I. highly dec:r.. Md and the .urface 01 the TGN I.IncreaMd II compared to 24°C).

face. These data revealed a molecularsorting based on the polar head struc-ture. Moreover, for the same polarhead (PC or PE), different fates wereobserved according to the fatty acylchain length. The transport 10 the cellsurface of very-Iong-chain fatty acid-containing PC and PE was blocked,whereas those of short-chain PC andPE were not. Therefore, these experi-ments revealed a selectivity/specificity

in the transport of the various phos-pholipid species according to both thenature of the polar heads and the fattyacyl chain length (21,29). Figure 2summarizes the pathways likely to befollowed by the major phospholipidspecies in leek cells.

While necessary to demonstratethe intracellular trafficking of lipids.the in vivo approach must be comple-memed by an in vitro analysis of the

biochemical events involved in thesecomplex processes. A major break-through was achieved with the devel-opment of methods permitting thereconstitution of these events in cell-free assays (30-32).

By taking advantage of the meth-ods developed in rat liver (33), it hasbeen possible to reconstitute thevesicular transfer of phospholipidsbetween the ER and the Goigi appara-

INFORM. VOl. 9. 00. 1 (January 1998)

97

lipids") (41). Under these conditions,the transfer of PC between the ERand chloroplasts could involve thetransfer (in this case, a partition) oflyso-P'C molecules. Very interesting.ly. it also was shown that purifiedmitochondria from potato tuberformed PC from labeled lyso-PC inthe presence of oleoyl-CoA. Like theplastidial one, this enzyme has a highspecificity toward the acyl acceptor(42). Therefore. as hypothesized forchloroplasts. any acylg\ycerophos-phory1choline transferred from ER tomitochondria may be acylated to PC.

Besides these experiments carriedOUI ill v itro, the fate of lipids from15-day-old leek seedlings also wasstudied tn 1';\'0. In good agreementwith a substrate-product relationshipbetween PC and galnctolipids, a sub-stantial decline of the radioactivityincorporated from labeled acetate

as was hypothesized in yeast andanimal cells (2). Very recently a newhypothesis was proposed for thedelivery of PC to the intracellularorganelles in plants.

Evidence has been proposed of therapid partition of Iyso-PC betweenmembranes and the aqueous environ-ment, and of its free movement toneighboring membranes (for exam-ples. Refs. 38-40). where it may bemetabolized further. Hence. lyso-PC.which is a normal constituent of puri-fied ER from plant cells. could parti-tion and rapidly reach the plastids.where it could be acylated by a highlyactive acyltransferase that we showedto be present in the envelope. Thisplastidial enzyme is specific for Iyso-PC, and the de 1101'0 synthesizedlipids have an 18-carbon atom fattyacid esterified to the sTl-2 position ofthe glycerol backbone ("eukaryotic

rus isolated from spinach leaves (34)and leek seedlings (35). In these sys-tems both the vesicular nature and thespecificity of the transfer of the phos-pholipids were reconstituted (34.35).In typical experiments from leekcells. we observed an ATP-dependent(vesicular) transfer of phospholipids.The transfer of PS between the ERand the Goigi apparatus was highlystimulated by ATP (+120%). thai ofPC and PE also was enhanced (by+79 and +67%, respectively), but noATP-dependent transfer of PI wasobtained (35). These results indicatedthat mechanisms of sorting hadoccurred ill vnro. An ATP-dependent(vesicular) lipid transport from nuclei10 Golgi apparatus of garden pea wasalso reconstituted (36).

Vesicular intermediates (transportvesicles) have been isolated from ani-mal cells (32) and yeast (31). In plantcells. few attempts have been made attheir isolation. ER-derived transportvesicles have been isolated and partlypurified from etiolated seedlings ofgarden pea (37) but have not beencharacterized further. Recently. goodcandidates for ER-derived transportvesicles have been isolated and partlypurified from leek seedlings. In thiscase. the lipid composition of thetransport vesicles revealed a high PSenrichment over PI. The PS/P! ratio is0.5 in ER and reaches 2.! in the ER-derived vesicles (Sturbcis-Balcerzcket al .. submitted for publication).

Transfer to organelles: the Iyso-PC hypothesis. It remains unclearhow and in what form the lipids syn-thesized in plant ER are exported tochloroplasts (and to mitochondria)and used us precursors for the biosyn-thesis of plastid gulactolipids. whichare the most abundant lipids on earth.As yet. neither the mechanisms oftransfer nor the nature of the lipidsimported into plastids or into mito-chondria have been elucidated.

The movement of phospholipidsto the organelles has been found notto be mediated by vesicles, andrecently the involvement of phospho-lipid transfer protein was questioned(for a review. see Ref. 22). Thismovement could depend on mem-brane contacts between a specificregion of the ER and the organelle.

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lowed by a discussion of the solubility of lipids in stlpcrcriticallluids. Otherchapters discuss the sn: of oilseeds and lipids from natural products. Thesupercritical fractionation of lipids is discussed liS well as oilseed solubilitylind extraction modeling. The design and cconomtcs of oilseed SFE extrac-tion processes nTCdiscussed. Other chapters deal with SFE lind exrrnortonof fish oil, cocoa and (.'0(.'011 products, eggs, meat. nntrnnl fats, and extractionof algae. Supcrcritica! lIuid chromatography (SFC) represents an alliedtechnique for the lipid chemist. Several chapters on deterrrnnation of tracecomponents in fats nnd oils and the usc of SFE for oh.-ochcmlcals arc Includ-ed as well as an overview of the applications of SFC to lipid analysis. Achapter on the usc of supcrcriticailluids for enzymatic synthesis is includedand the effects of SFC on enzymes, proteins. and amino acids are discussed.

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INFORM. \til. 9. no. 1 (January 1998)

98

PLANT LIPIDS

into PC (but not in other phospho-lipids) was accompanied by anincrease in the label found in galac-telipids during the chase. The analy-sis of the positional distribution oflabeled fauy acids in PC and galac-tolipids as a function of the chaselime clearly showed that the increasein galactolipid radioactivity mainlyconcerned the SII- t position whereasPC was preferentially labeled at theslI-2 position (43), These resultsstrongly suggested that the diacyl-glycerol moiety of PC might not beintegrmed as a whole into the galac-tolipid and that a deacylation/reacyla-tion process was involved in thePC-galactolipid retanonsblp. There-fore, both in vivo and in vitro experi-ments suggested that a dencylationcould induce the synthesis of Iyso-PCin the ER. the resulting I-acylglyc-erophosphocholine molecules being"exported" to the chloroplast. thenacylated by (chiefly) unlabeled fauyacids in the plastids during the chase.The resulting plastidial PC. labeledmainly in .511-1 position. could furtherbe used for the synthesis of galee-tolipids.

Membrane lipid domains:future prospectsIt has been fairly well established thatcell membranes have different andspecific phospholipid compositions.How can such differences be estab-lished and maintained? Membranebiogenesis requires successive andcooperative processes by which theindividual components of the variouscell membranes are synthesized. sort-ed. and transferred/targeted to theirfinal location. Although the mecha-nisms of biosynthesis of lipids arefairly well understood. the regulationof their biosynthesis should be stud-ied more closely. particularly the reg-ulation of the expression of the genescorresponding to the enzymesinvolved in their metabolism. Anotherlevel of investigation. directly relatedto the previous one. concerns the tar-geting of these enzymes. since cellmembranes are not similar in relationto their capabilities to synthesizeand/or to degrade lipids. Therefore.sorting and correct targeting of theseenzymes must be tightly controlled.

as is true for many proteins (31.32).In addition. not only the proteins butalso the lipids can be sorted andspecifically addressed through differ-ent mechanisms of lipid transport.The concept of lipid sorting first camefrom experiments with polarized ani-mal cells where a glycosphingoJipidsorting was demonstrated clearly (I).This suggested the existence of spe-cific lipid rafts in cell membranes(44). Similar structures also couldexplain the sorting and, consequently.the selective transport of phospho-lipids with longer fatty acyl chainsand different polar heads (3.21). Thelipid domains of the ER membranesalso might be at the origin of a selec-tive synthesis of phospholipids andtriacylglycerol due to the existence ofseparate precursor pools (45.46).

AcknowledgmentsThe work of the authors was support-ed by the CNRS. the "Universite Vic-tor Segalen Bordeaux 2" and the"Conseil Regional d' Aquitaine.t'Wealso thank Prof. Thomas J. Bach forcritically reading the manuscript.

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