[advances in botanical research] advances in botanical research volume 16 volume 16 || lipid...

Lipid Metabolism in Algae

I .

11.

I l l .

IV.

V.

v1.

VII.

VIII.

IX.

JOHN L. HARWOOD and A . LESLEY JONES

Dtpwtmeiit of' Biochemistry, University College CurdiflL

CardiffCF1 I X L , U K

Introduction . . . . . . . . . . . . . . . . . . . .

Lipid Structures

Lipid Composition of Algae. . . . . . . . . . . . . . . A. FattyAcids. . . . . . . . . . . . . . . . . . . B. Lipid Classes . . . . . . . . . . . . . . . . .

General Remarks on Plant Lipid Metabolism . . . . . . . .

Metabolism of Lipids in Cyanobacteria (Blue-Green Algae) . . . .

Studies with Halotolerant and Halophilic Dunuliellu Species . . . .

Metabolism in Marine Algae . . . . . . . . . . . . . . A. Labelling Characteristics . . . . . . . . . . . . . . B. Positional Distribution of Algal Fatty Acids . . . . . . . . C. Effects of the Environment on Algal Lipid Metabolism . . . .

Lipid Metabolism in Other Algal Types . . . . . . . . . . .

Conclusions . . . . . . . . . . . . . . . . . . . .

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5 5 9

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28 28 33 35

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I. INTRODUCTION

Algae are an extremely diverse group of organisms. Not surprisingly, there- Advances in Botanical Research Vol. 16 ISBN 0-12-005916-9

Copyright $2 1989 Academic Press Limited All rights of reproduction on any form reserved.

2 J O H N L. H A R W O O D A N D A . LESLEY J O N E S

fore. their lipid composition and metabolism are exceptionally varied. In this chapter we have concentrated attention on groups of organisms which have been studied in reasonable detail. This selection has, in consequence, neg- lected some interesting, but isolated, studies. Nevertheless, we believe that i t allows readers unfaniiliar with lipids to assimilate more useful information. I n any case. plenty of reviews dealing with specialized aspects are referenced.

In order to lay a basis for further discussion of metabolism, we have begun with sections on lipid structure and occurrence in algae. The metabolic sections then deal with organisms of increasing complexity. from the primitive cyanobacteria to marine macroalgae. Finally. we end with a section devoted mainly to green algae, which can be regarded as the nearest in metabolic characteristics to higher plants.

11. LIPID STRUCTURES

The complex lipids of plants are mainly amphiphilic molecules with a hydrophobic head group and a hydrophilic “tail”, enabling them to form the lipid bilayers of membranes. Algae contain many of the lipids found in higher plants, and also some unusual lipids. The basic structure is a glycerol back- bone derived from glycerol 3-phosphate (from the photosynthetic Calvin cycle, or glycolysis) to which is esterified the hydrophobic head group. Phos- pholipids have phosphate esterified to the sn-3 position with further moieties esterified through this. Glycolipids have sugar moieties as a head group. The structures of the major phospholipids and glycolipids found in algae are given in Fig. 1.

Most of the eukaryotic algae contain a range of phospholipids and glycolipids, many of which are found in higher plants. However, the prokaryotic cyanobacteria have only the lipids of the chloroplast thylakoids of higher plants: namely monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG) (Fig. 2) (Gounaris e f al., 1986).

Tri- and tetragalactosyldiacylglycerols have been found in some higher plants, and Chlorrllu contains trigalactosylglycerol (Benson e f al., 1958). Sugars other than galactose may be found. Some red algae have mannose and rhamnose in their glycolipids (Pettitt, T. R., Jones, A. L. and Harwood, J. L., unpublished). Monoglucosyldiaglycerol (MGlcDG) was found in Nos- toe calcicnla (Feige et al., 1980) and Anuhaena variahilis (Sato and Murata, 1982a). In A . variuhilis, MGlcDG appeared to be a metabolic intermediate, being rapidly converted to MGDG by epimerization of C-4 of glucose (Sec- tion V).

Pham-Quang and Laur (1976a,b,c) found a range of novel glycolipids, phospholipids and sulpholipids in three brown algae, Pelvetia canuliculufn, Fucus vesiculosus and F. serratus. The two Fucus spp. also contain one major

LIPID METABOLISM IN ALGAE 3

unknown lipid (Smith and Harwood, 1984a; Jones and Harwood, 1987). In C. cr isps and P. lunosa, 3SS-labelling revealed a number of sulphur-containing lipids, most of which were minor components (Pettitt, T. R.. Jones, A. L. and Harwood, J. L., unpublished). However. one of these lipids was identified as phosphatidylsulphocholine (PSC), the sulphonium analogue of phosphatidylcholine (PC) (Fig. 3). which has also been identified in diatoms and a Euglena species (Anderson ct ul., 1978a,b; Bisseret L ’ t d., 1984).

A- Llprd

PA

PC

PE

PS

P I

PG

DPG

X

-H

+ -Ct$CH2N(CH3)3

-C H2C $N H3 +

+ 4 H 2 C H N H j

I coo- OH OH

-CH I

C$OC-R I

I 0-

Fig. I . Structures of common phosphoglycerides. PC, phosphatidylcholine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PA, phosphatidic acid. Based on Harwood and Russell (1984).

4 JOHN L. HARWOOD A N D A. LESLEY JONES

Chlorosulpholipids have also been found in some algae and are a major component of Ochromonas danica (Elovson and Vagelos, 1969).

An unusual lipid found in a number of green algae and some lower plants, but not detected in angiosperms, is the homoserine ether lipid 1(3),2-diacyl- glyceryl-(3)-O-4'-(N,N,N-trimethyl)-homoserine (DGTS) (Fig. 4), first identified in 0. danica (Brown and Elovson, 1974). This lipid has since been

Lipid

MGDG

DGDG ?H

X -

SQ DG I CH20H

C H2-S 4- Fig. 2 . Structures of common glycosylglycerides.


0- Fig. 3 . Structure of the sulphonium analoguc ofphosphatidylcholinc

Fig. 4. Diacylglycerol trimethylhomoserine ether lipid.

identified in Chlumydomonus reinhurdtii, Dunuliellu sulina, Codium spp., Ulvu pertusu and Enteromorphu intestinalis (Sato and Furuya, 1984a,b), and is a major lipid of Chlumydomonus reinhurcitii and D. sulinu (Eichenberger, 1982; Norman and Thompson, l985b).

Algae also contain neutral lipids, mostly as triacylglycerols which are, presumably, storage products (Pohl and Zurheide, 1979a,b) with small amounts of mono- and diacylglycerols. They also contain various hydrocarbons as minor constituents and a range of sterols and sterol esters (see Pohl and Zurheide, 1979a). Thus the range of lipid structures in algae varies from the simple constituents of the cyanobacteria to the complex variety in the eukaryotic brown (and other) algae. The structures found vary between the algal divisions and some lipids appear to be characteristic of different algal divisions-for instance, DGTS is found in many Chlorophyceae but has never been detected in any Phaeophyceae. The distribution of lipids within different algae is considered in more detail in the following section.

111. LIPID COMPOSITION OF ALGAE

A. FATTY ACIDS

A large number of algae have now been analysed for their fatty acyl composition. The convenience and sensitivity of gas-liquid chromatographic

6 JOHN L. HARWOOD A N D A . LESLEY JONES

(GLC) methods have made this possible, whereas, in contrast. there has been much less work on the content of individual lipid classes in either marine or fresh-water algae.

Fresh-water algae typically contain similar fatty acids to terrestrial plants. However, the proportions of such acids differ considerably from, for example, higher plant leaves. In general, even-chain acids in the range C,,- C,, account for the bulk of the components. In contrast to plant leaves, fresh-water algae usually have a relatively high proportion of C,, fatty acids and reduced levels of C, unsaturatcd fatty acids, especially r-linolenate (Harwood rt d.. 1989). Of course, the total fatty acid composition of organisms gives little information about the specific acyl contents of individual complex lipids, which are usually very different. For example, it has been noted by several authors that triacylglycerols that can accumulate in certain species of algae are usually very low in polyunsaturated fatty acids (e.g. Tornabene ('I u/.. 1983). in contrast to total algal extracts (see Hitchcock and Nichols, 1971). Furthermore, in an analysis of the individual lipids of two strains of Chlam?.doomotiu.F rc.inhardtii. Eichenberger et ul. (1986) noted that. whereas the glycosylglycerides. PG and phosphatidylinositol (PI) contain mainly or exclusively the n-3 isomer of linolenic acid, phosphatidylethanolamine (PE) and DGTS contained the 11-6 isomer (1'-linolenate). I n addition, it should be remembered that environmental factors such as light, temperature, nitrogen levels. salt stress or pollution can have a marked effect on fatty acid (and lipid) composition.

Representative fatty acid compositions of some fresh-water and salt- tolerant algae are given in Table I . I t will be seen that whereas the C,, fatty acids are absent (or only found in trace amounts) in fresh-water algae, these acids may be significant components of organisms living in high-salt environ- ments such as the Dead Sea. In fact, for marine algae the very long chain polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid (n-3) are usually major components (Pohl and Zurheide. 1979a; Harwood r t al., 1989).

Marine algae in general contain a bewildering array of major fatty acids. Some representative examples of compositions for phytoplankton and macroalgae are shown in Table TI. Palmitate is, invariably, the major saturated fatty acid of phytoplankton with myristate often being found in appreciable quantities. In contrast to higher plants, C,, fatty acids are less important, with palmitoleic acid being the major monoenoic acid component. In some cases, e.g. Monochrysis lathrri (Table 11), total C,, acids represent a very minor proportion of the total acyl constituents. One common theme for all marine algae, with the exception of the Chloro- phyceae, is that C,, polyenoic acids are major constituents. Arachidonic and eicosapentaenoic ( n - 3 ) acids are the main C,, acids and sometimes represent a very high proportion of the total acyl groups of particular lipids. For example, in F. serratus arachidonate accounted for 67% of PC (Smith and

LIPID METABOLISM IN A L G A E 7

F a t t y acid composition (“A, total)

16:O 16: I 16:2 l h : 3 16:4 18: I 18:2 I X : 3 1X:4 3 0 : s 3 2 : h

F r e s h w a t c r spp. Scene~1rsniu.s ohliquus 35 3 t r . t r . 15 9 6 30 2 Clik>r~llti vulguris 26 8 7 2 - 2 34 20 ~

Chumy1onionu.s 20 4 1 4 23 7 6 30 3 reitiliurdt ii

Sa l t - to l e ran t spp. Anki.st,otli~.sriius spp. 13 3 I 1 14 25 2 29 2 1 -

I.socI1 r:v.vis spp. 12 6 ~ ~~ 15 4 6 17 2 13 Nuiinoclrliwi.r spp. 9 2 0 7 9 - 4 1 I - - 27 ~~

Data from Hitchcock and Nichols (19711, Hen-Amotz and Tornabene (198.5) and Eichen- berger el t i / . (1986). Fatty acids are abbreviated with the figure before the colciii indicatinp tlic number of carbon atoms. and the figure alter the colon indicating the number of double bonds contained.

Harwood, 1984a). while in Chondrus crispus eicosapentaenoate represented more than 25% of the total acids of the glycosylglycerides (Pettitt c ~ t ul., 1989). The unicellular marine alga Chnrtonclla untiyuu (Raphidophyccac) was also found to contain rather higher amounts of eicosapentaenoate in all lipid classes (Sato er a/., 1987).

A very unusual fatty acid which is found in photosynthetic tissues From higher plants is truns-A3-hexadecenoate. This acid is locatcd exclusively at the sn-2 position of PG (Harwood, 1980a). Interestingly. in view of the postu- lates concerning its possible role in granal stacking (see Bolton et d., 1978). the same acid is also found in PG in algae with quite different chloroplast morphology. Thus, i t has been reported in brown algae (Smith and Har- wood, 1983), red algae (Pettitt and Harwood, 1986), Raphidophyceae (Sato et a/., 1987) and diatoms (Kawaguchi er al., 1987). Together with all the negative evidence from higher plants (Bolton er d., 1978), it is clear that whatever the role truns-A3-hexadecenoate plays in nature, it is not in granal stacking. More likely explanations concern its possible involvement with chlorophyll-protein complexes (Remy er al. 1982), as suggested by model- building experiments (Foley and Harwood, 1982; see Foley er ul., 1988).

Cyanobacteria, as an evolutionarily less advanced “algal” group, also contain much more simple lipid and fatty acid compositions than other algae. Murata and Nishida (1987) have divided cyanobacteria into four groups based on the original classifications of Kenyon et al. (1972). Examples of each of these four groups are shown in Table I l l . Strains in the first group contain only saturated and mono-unsaturated fatty acids, while those in the other three groups contain linoleate plus polyunsaturated acids characteristic

Fatty acid composition ( "/o total)

14:O 16:O 16:1 16:2 16:3 16:4 18:l 18:2 18:3 18:4 20:4 20:s 22:6

Phytoplankton Monochrysis lutheri (Chrysophyccac) Olisthodiscus spp. (Xanthophyceae) Lauderiu borealis (Bacillariophyceae) Amphiciinium carterue (Dinophyceae) Dunaliellu sulinu (Chlorophyceae) Hemiselmis hrutiescens (Cryptophyceac)

Macroalgae Fucus vesiculosus (Phaeophyceae) Chondrus crispus (Rhodophyceae) U l w luctuca (Chlorophyccae)

10 13 22 5 7 I 3 I t r . 2 1 1 8 7 X 14 10 2 2 I 4 4 6 1 8 2 1 9 2 7 12 21 3 12 1 2 1 tr. ~~ 1 3 - 3 24 1 1 tr. ~ 5 1 2 1 5 - 14 25

tr. 41 IS tr. -- - 1 1 8 1 9 - - - - 1 13 3 3 tr. tr. 2 tr . 9 30 tr. 14 -

- 21 2 tr. ~ tr. 26 10 7 4 I S 8 -

1 18 2 tr. 1 18 9 2 17 24 I 2 tr. - 34 6 tr. - - 9 1 1 4 18 22 -

Fatty acid abbreviations as for Table I


Group Organism Fatty acid composition ('YO total)

1 Anrrcj~.stis nidu1rti.s 46 46 3 0 0 0 0 2 Atiuhicctm vrrriuhilis 32 22 I 1 17 16 0 0 3 Sjnechoiyst is 67 14 28 4 5 17 0 3 i 0 4 To1jpotliri.u tenuis 22 3 16 15 6 13 I I

Data taken from Murata and Nishida (19x7). where rel'crences will he found. €.'ally acid ahhrovations as for Table I .

of each group. Thus, groups 2, 3 and 4 contain a-linolenate, ;j-linolenate and octadecatetraenoate (n-3), respectively (Table I 1 1). Filamentous blue-green algae are distributed throughout the four groups, while the prokaryotic green alga Prochloron has a fatty acyl composition placing i t in the first group (Kenrick er d., 1984).

B. LIPID CLASSES

Just as the cyanobacteria contain a relatively simple fatty acid composition, so is their lipid content confined to four major classes only-monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DCDG), sulphoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). A minor lipid, monoglucosyldiacylglycerol (MClcDG), accounts for a trace amount in some species such as Anucysris vuriahilis but not in others (e.g. Anucystik niduluns). This minor lipid is involved in the biosynthesis of MGDG (see later). The lipid compositions of two cyanobacteria and Prochloron spp. are given in Table IV. When individual membrane fractions were isolated from Anacystis niduluns, the thylakoid, plasma and outer membrane all contained a rather similar amount of the four lipids except that DGDG was somewhat enriched in the outer membrane. However, the lipid content as a percentage of the dry weight was very different, being 57%, 19% and 3% for the plasma, thylakoid and outer membranes, respectively (Murata and Nishida, 1987).

In nitrogen-fixing cyanobacteria unusual glycolipids have been reported. These are found in the heterocysts of the filamentous heterocystous strains as well as in unicellular strains. In A . cylindricu, the glycolipids account for 4.4% of the total lipids. The chemical structures of the four main non- saponifiable glycolipids were determined about 15 years ago (Bryce e t al., 1972; Lambein and Wolk, 1973) and other minor variants have been reported (see Murata and Nishida, 1987).

Eukaryotic algae contain a formidable range of acyl lipids. As a general-

I0 J O H N L. H A R W O O D A N D A. LESLEY JONES

Organism Lipid (YO total)

MGDG MGlcDG DGDG SQDG PG

..I IItrhrrefltr L'oric/hi/i.s 54 I 17 I I 17 A n i i ~ ' j . . \ tis nitluluti.s 57 1i.m. 1 1 I I 21 Proc~/l/ororl spp. _ _ 5 T 3 I I 26 5

i i .ni . = not measured. Data takcn from Muratu and Nishida ( 19x7). where the original references can be found.

ization, the three glycosylglycerides (MGDG, DGDG and SQDG) are major constituents. So far as phosphoglycerides are concerned. all the main classes can usually be detected. Insufficient data are available for generalizations to be made but it appears, thus far, that there are considerable differences between algal divisions in their relative contents of individual phospholipids. Some examples of lipid compositions are given in Table V.

DGTS is an unusual lipid which is found in the Chlorophyta, although i t was first reported in the chromophyte 0. (lunicu (Nichols and Appleby, 1969; Brown and Elovson, 1974). I t represents a major component in green algae such as Ch1rini~~tlot~iotiu.v rrinhurcltii (Eichenberger and Boschetti, 1978; Table V). and a survey of its occurrence in non-vascular green plants has been made (Sato and Furuya, 1985). Apart from 0. dunicu, Chuttonrllu untiyuu (Sato r t

ul., 1987: Table V) is the only chromophyte known to contain DGTS. The lipid has not been reported in brown algae (e.g. Smith and Harwood, 1984a) or diatoms (e.g. Kawaguchi P / ul., 1987).

Many fresh-water algae contain chlorosulpholipids (Mercer and Davies, 1979). Again, it was work with 0. dunicu which first revealed large amounts of these lipids (Haines and Block, 1962). The substances were soon identified as sulphur esters that were present in amounts greater than for most glycolipids or phospholipids in the cell. Many of the sulphur esters also contain chlorine, and are therefore known as chlorosulpholipids. In 0. dunicu they represent about 15% of the total lipids. They have been detected in all fresh- water species but not in any of the marine species examined (Mercer and Davies, 1979). These unusual lipids have been reviewed (Haines, 1973a,b).

Certain diatoms have been found to contain other novel sulphur-containing lipids (Kates, 1987). Characteristically, all species examined contain three sulpholipids in addition to SQDG. They have been identified in Nitzchiu ulhu as sterol sulphate, deoxyceramide sulphonic acid and the PC analogue PSC (Anderson rt ul., 1978a,b; Kates rt ul., 1978). In the non-photosynthetic diatom N. albu, PSC completely replaced PC, but in other diatoms both lipids occurrcd together (Kates, 1987). Overall, the sulpholipids are major constituents of diatoms, representing 30Y0 of the total polar lipids in N. pelliculnsu

TABLE V Tlw acjl lipid conipositiotis of . sonic ulgue

Lipid ( O h ) Chatronella Dutiuliella Aceiahulnriu Chlat?i~.clot?ioti~i.s CIlOt1 dr us Fucus antiqud parvu meditarraticw ‘ reiniiurdiiid crispus ‘’ wsicuIosusJ

PC PE PI PG DPG Other phospholipids MGDG DGDG SQDG Other glycolipids DGTS Lipid X Neutral lipids Free fat ty acids

_ _

29 18 29

6 ~

9 n.d.

2 6

21 1 1 7 1

15

I5 13

-

0.1 I .2 0.6

3

31 20 20

20 ~

5

10 3

~

41 16 7

16

30.1 1 .5

trace 1.1 1.9

16.9 14.8 15.5

.-

n.d.

3.4 2.2

~

4.2’ 6.2 2.9

5.4

15.0 11.3 22.0

n.d. 24. I 4.9

3 7 A_-

~

-

“Sato et ul. (1987); bKates (1987); ‘Eichenberger and Gerber (1987); dEichenberger et ul. (1986); ‘Pettitt el ul. (1988b); ’Unpublished data n.d. = not detected.

12 JOHN L. HARWOOD AND A. LESLEY JONES

and over 74% in N . alba. PSC has also been detected in marine red algae such as Chondrus crispus and Polysiphonia lanosa (Pettitt et al., 1988b).

A review of the lipids of marine algae has been made by Pohl and Zurheide (1979a). There have been sporadic reports of a wide range of unusual structures. However, in many cases these have not been confirmed and may, in at least some cases, be the products of lipid degradation or modification during analysis. Such processes are well known in plant tissues, and special pre- cautions must be taken to prevent their occurrence (see Harwood, 1980a).

IV. GENERAL REMARKS ON PLANT LIPID METABOLISM

A detailed discussion of plant lipid synthesis is beyond the scope of this chapter, and, moreover, specific algal aspects will be dealt with later. How- ever, a few very general remarks together with comprehensive references will be made here.

Fatty acid metabolism has been reviewed very recently (Harwood, 1988). De novo synthesis involves the concerted action of acetyl-CoA carboxylase and a type I1 (dissociable) fatty acid synthetase. The end-product of such synthesis is palmitoyl-ACP, which can be specifically chain-lengthened to stearoyl-ACP using a special condensing enzyme. Thus C,, and C,, saturated fatty acids are the products of de novo synthesis.

The first unsaturated bond is usually introduced at the A9-position by a desaturase which probably uses acyl-ACP substrates in most instances. Palmitoleic and oleic acids result from this action. Further desaturations are thought to involve complex lipid substrates such as PC and MGDG (Harwood, 1988). These may take place within choroplasts (prokaryotic pathways) or include extra-chloroplastic (eukaryotic pathway) enzymes.

Fatty acids can also be elongated, and such reactions are clearly of great importance to many algae where C,, fatty acids are major constituents. Nothing is known about the substrates for such reactions in algae, although acyl-CoAs are used in a number of higher plant tissues (Harwood, 1988). Obviously, where both chain elongation and desaturation are involved in the formation of a given fatty acid, there will be more than one possible route. Thus, arachidonate has been shown in some algae to be formed by a desaturation-elongation-desaturation route from linoleate (Nichols and Appleby, 1969):

18 : 2 (n-6)+18 : 3 (n-6)+20 : 3 (n-6)+20 : 4 (n-6)

In contrast, in a euglenoid (Hulanicka et al., 1964) it appears to be formed thus:

18 : 2 (n-6)+20 : 2 (n-6)+20 : 3 (n-6)+20 : 4 (n-6)


So far as complex lipids are concerned, the formation of MGDG and DGDG (Joyard and Douce, 1987), SQDG (Harwood, 1980b; Mudd and Kleppinger-Sparace, 1987) and phospholipids (Moore, 1982; Harwood 1989) has been reviewed. The metabolism of unusual lipids such as the sulpholipids of diatoms (see Kates, 1987). chlorosulpholipids (Mercer et d., 1374) and DGTS (see Schlapfer and Eichenberger, 1983) has also been discussed. In addition, the arsenic lipid (arsenoribosylphosphatidylglycerol) which appears to mediate arsenic excretion in marine organisms has been discussed (Benson, 1987). Other general aspects of plant lipid metabolism will be found in the excellent volumes edited by Stumpf (Stumpf and Conn, 1980, 1987).

V. METABOLISM OF LIPIDS IN CYANOBACTERIA (BLUE- GREEN ALGAE)

The cyanobacteria, like the eukaryotic algae, have an oxygen-evolving photosynthetic mechanism, yet their prokaryotic morphology makes study of their metabolism much simpler. Their lipid composition and metabolism have been recently covered in an excellent review (Murata and Nishida, 1987), so only a few aspects will be dealt with here.

The plasma and thylakoid membranes of cyanobacteria contain glycerolipids-almost exclusively MGDG, DGDG, SQDG and PG. The outer membrane contains lipopolysaccharides and hydrocarbons in addition to glycerolipids. In addition, some nitrogen-fixing species of filamentous cyanobacteria may contain vegetative cells which change into heterocysts (Hasel- korn, 1978). These heterocysts contain a refractile multilayered heterocyst envelope which contains unique glycolipids (glycosidic glycolipids and glycosyl ester glycolipids) (Lambein and Wolk, 1973).

The glycerolipid composition of several species of cyanobacteria is shown in Table IV. I t is noteworthy that the only glycerolipids present in appreciable amounts are those which are regarded traditionally as typical chloroplast thylakoid lipids (Harwood, 1980a; Gounaris rt d., 1986). In addition, i t will be seen that for Anucysfis nzduluns, where more detailed analyses have been undertaken, the lipid compositions of different membranes in the cyanobacterium are broadly similar. However, the lipid contents (YO dry weight) are quite different, being 19%, 57% and 3% for the thykaloid, plasma and outer membranes, respectively (Murata and Nishida, 1987). In addition to the four main lipid classes present in cyanobacterial membranes, MGlcDG seems to be a ubiquitous, though minor, component (Feige, 1978).

When the fatty acids of cyanobacterial lipids were first studied, it was real- ized that although these were broadly similar to those of higher plant chloroplasts, there were some notable differences. These include the increased


importance of C , (especially palmitoleic) acids, the relative reduced preval- ence of polyunsaturated fatty acids (including the absence of linoleate and a- linolenate from some organisms), the presence of y-linolenate in some cyanobacteria and an absence of trans-A3-hexadecenoate from PG (Hitchcock and Nichols, 1971; Murata and Nishida, 1987). Some efforts have been made to classify cyanobacteria into four subgroups, according to their fatty acyl composition (Kenyon, 1972; Kenyon et al., 1972) (see Section 111). Examples of fatty acid compositions for different organisms are given in Table 111.

The individual glycerolipids differ from one another in their acyl compositions. In general, MGDG and DGDG contain the highest ratios of unsaturated to saturated acids. In those cyanobacteria which contain polyunsaturated fatty acids, the latter are also enriched in the glycosylglycerides. The two acidic glycerolipids, SQDG and PG, tend to contain very large amounts of palmitate with only small quantities of palmitoleate. Naturally, the fatty acyl compositions of cyanobacteria and their glycerolipids are very dependent on growth temperature (see later). All glycerolipids of cyanobacteria display the typical "prokaryotic" positional concentration of C,, acids at the sn-1 position and C, , acids at the sn-2 position (Zepke et al., 1978).

Studies using ['"C] acetate first established the rapid labelling of all major glycerolipid classes (Nichols, 1968). When H14C0, was used for pulse- labelling of 30 species of cyanobacteria, the first lipid highly labelled was MGlcDG. Later, radioactivity appeared in MGDG, and it was proposed that these lipids had a precursor-product relationship (Feige et al., 1980). Moreover, analysis of the glucose and galactose moieties of these two lipids in Anacystis nidulans showed a similar relationship (Fig. 5) and indicated that the sugar moiety was not lost during this interconversion. As a result of these experiments, Sat0 and Murata (l982a) proposed that the mechanism of formation of MGDG involved epimerization at the C-4 atom of the precursor glucose unit. In addition, experiments with cerulenin (see below) suggested that DGDG was formed by galactosylation of MGDG rather than via a glycolipid : glycolipid transferase (see Joyard and Douce, 1987).

Cerulenin, a known inhibitor of fatty acid biosynthesis, was tested with Anacystis nidulans. Its inclusion reduced severely the incorporation of radioactivity from H1"C03 into all lipid classes except DGDG (Sato and Murata, 1982a). This was in keeping with the idea that newly synthesized acyl chains are needed for the overall formation of glycerolipids, but because DGDG can be synthesized by galactosylation of MGDG, its labelling is less affected. Indeed, analysis of the sugar residues of DGDG confirmed that a high proportion of the total radioactivity of this lipid was present in the galactose moieties (Sato and Murata, 1982a).

A membrane-bound UDP-glucose : diacylglycerol glucosyl transferase was detected in A . variubilis (Sato and Murata, 1982a). This enzyme seems to be present in both thylakoid and plasma membranes (Omata and Murata, 1986) and it is interesting that, in higher plants, the equivalent galactosyl

I LIPID METABOLISM IN ALGAE

r 15

( a )

Time ( h ) after labelling

( b )

Fig. 5. Changes in the radioactivity of monoglucosyl- and monogalactosyldiacylglycerol during pulse-labelling from HI4CO, in A . niduluns and subsequent chase. Gal, galactose; Glc, glucose. Taken from Sato and Murata (1982a) with permission.

transferase is found in the chloroplast envelope and, in some cases, also in prothylakoids (see Murata and Nishida, 1987).

Fatty acid synthesis was followed in A . vuriubilis by labelling from H14C03 (Sato and Murata, 1982b). Radioactivity was incorporated initially into palmitate, stearate and oleate. In fact, it was suggested that saturated fatty acids were initially incorporated into complex lipids such as MGlcDG and that subsequent desaturations occurred while the acyl groups remained attached to complex lipids. This was in agreement with proposals for higher plants (see Harwood, 1988). Examination of molecular species labelling of individual lipid classes led to proposals that in MGlcDG, stearate could be desaturated to oleate and linoleate but hardly at all to linolenate. In contrast, MGDG was an efficient substrate for successive desaturation of stearate through to linolenate as well as for that of palmitate to hexadecadienoate. DGDG did not appear to be used for desaturation, whereas successive desaturation of stearate, but not of palmitate, appeared to occur on PG and SQDG (Fig. 6).

Direct demonstration of lipid-linked desaturation of palmitate in MGDG was made by the use of isotopic labelling. Incubation of cells with H13C03 caused the formation of MGDG enriched in its acyl groups. After 2.5 h, 19% of the palmitate but virtually none of the palmitoleate at the sn-2 positions were enriched in 13C. During a subsequent incubation for 7.5 h in the presence of unlabelled CO, and the fatty acid synthesis inhibitor cerulenin,


38°C 22°C

1 1 18:l 18:2 16:l --+ 1611

[Ga l E Gal

GalDG

18:O 18:l 18:2 18:3 16:O --+ 16:O - 16:O - 16:O

[Gal [ G a l [ G a l 1 Gal

1 18:3 [g;: -[ 16:l Gal

L 18:Z

18:3 16:2 Gal

I 1

1 I I J

Fig. 6. Pathways for the desaturation of fatty acids esterified to the acyl lipids of A . niduluns. Taken from Sato and Murata (1982b) with permission.

[I3C]palmitate was desaturated to [13C]palmitoleate. Mass spectrometric analysis of the 2-acylglycerol moiety showed that [L3C]palmitoyl- [ I 3C]glycerol was converted to [ 3C]palmitoleoyl-['3C]glycerol, and ['ZC]palmitoyl-['zC]glycerol to ['ZC]palmitoleoyl-['ZC]glycerol. If a pathway involving deacylation, desaturation and reacylation had been involved, then this would have been expected to yield products containing partial enrichment (Fig. 7) and this was not found. The results were fully in support of an MGDG-linked desaturation of palmitate (Sato et al., 1986).

The idea that desaturation of fatty acids in cyanobacteria required lipid- linked substrates was in agreement with data for A . variahilis reported by Lem and Stumpf (1984a). They showed that cell-free extracts were able to synthesize palmitate and stearate but not oleate from [14C]malonyl-CoA. In addition, although [14C]palmitoyl-ACP could be elongated to ['4C]stearoyl- ACP, n o desaturation was detected. When ['4C]stearoyl-ACP was used as


Lipid- linked desaturation

Gly ( U 1-16; 0 ( U)

Gly (E)-16:0 ( E l Gly(U )-16 : 1(U 1

Gly( E 1-16 : 1 ( U ) Deacylat ion desaturation and reacylatton

Fig. 7. Principle of isotopic species analysis to discriminate between the two possible mechanisms of dcsaturation of palmitate. Combinations of the glycerol back- bone and the C16 acids at the sn-2 positions of MGDG are presented as Gly-16:O and Gly-16:I. U, unenriched; E. enriched with I T . Redrawn from Sato rt ul. (1986) with permission.

substrate in a A9-desaturase assay, activity was detected in green algae such as C. pyrenoidom, Scenedesmus ohliquus and Chlamydomonas moewensii, but not in A. variahilis or Nostoc spp. Data from other laboratories agree with the above conclusions (Stapleton and Jaworski, 1984b; Al Araji and Walton, 1980).

The fatty acid synthetase of A. variahilis is of the type I1 non-associated type (see Harwood, 1988). The malonyl-CoA : ACP transacylase has been purified and found to have rather similar properties to that from spinach chloroplasts (Stapleton and Jaworski, 1984a). The elongation of palmitate to stearate uses palmitoyl-ACP and NADPH and not palmitoyl-CoA or NADH (Lem and Stumpf, 1984a; Stapleton and Jaworski, 1984a).

Radioactivity from ['4C]acyl-ACPs was rapidly transferred into complex lipids, especially MGDG, by crude cell extracts of A . variahilis. The first intermediate detected was diacylglycerol (Lem and Stumpf, 1984b), but one presumes that the incorporation into this compound was due to glycerol 3-phosphate acylation and phosphatidate phosphatase. Acyl-CoAs could not act as substrates. Although [14C]oleoyl-ACP was effective for acylation, the probable lack of formation of this compound in vivo (see above) would prevent the esterification of this moiety which would have to be formed by lipid-linked desaturation (Sato and Murata, 1982b).

Compositional studies on cyanobacteria have demonstrated that shifts in growth temperature lead to several types of changes in lipids. In Anacystis

18 JOHN 1.. HARWOOD A N D A . LESLEY JONES

TABLE VI Chunge~ in the moleculur .species composition qf’glycerolipids 0f’Anabaena variabilis

cuustd by growth ietnperuture

Lipid Growth temp. Molecular species (YO total) ( C)

C-l 18.0 1 8 . 1 1 8 . 2 1 8 : 3 1 8 . 1 1 8 : 2 1 8 . 3 1 8 . 3 C-2 16 .0 16 .0 16.0 16:O 16.1 16.1 16 .1 16 .2

GlcDG 38 22

MGDG 38 22

DGDG 38 22

PG 38 ...- 7 7

SQDG 38 22

24 60 10 22 40 24

1 25 23 2 2 12

1 16 24 1 4 20

1 56 41 0 10 26

10 48 26 2 10 16

0 6

1 34

0 19

0 61

0 sx

0 0 0 0 0 0 0 0

1 1 35 0 0 0 3 32 12

16 38 0 0 I 9 3 1 4

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

Fatty acid abbreviations as in Table I . Taken from Murata (1987). with perinission

niciuluns, lowering growth temperature led to an increase in unsaturation and a shortening of chain length (Holton et a/., 1964). Similar changes were found with S~~nec.hococcu.s cedrorum (Sherman, 1979), and in S. lividus low- temperature growth led to a decrease in palmitate and oleate and an increase of palmitoleate in MGDG and DGDG while stearate decreased and palmitoleate and oleate increased in PG and SQDG (Fork et ul., 1979). In Anucjxtis nidulrms, lowering growth temperature led to a decrease in chain length of mono-unsaturated acids at the sn-I position of all lipids but increased desaturation of palmitate at the sn-2 position of MGDG and DGDG (Sato et d., 1979).

In contrast to the above, the unicellular cyanobacterium A . variahilis contains polyunsaturated fatty acids. Lowered growth temperatures led to an increase in a-linolenate at the sn-l position of all lipids, while the composition of C, acids at the sn-2 position remained nearly constant apart from a slight increase in hexadecadienoate in MGDG and DGDG (Sato et al., 1979; Sat0 and Murata, I980b). These experiments are summarized in Table VT, where the molecular species patterns for different lipid classes are shown.

In temperature-shift experiments it has been found that A . variahilis rapidly alters its fatty acid composition as well as the positional distribution of such acids on individual glycerolipids. For example, for 10 h after a change in growth temperature from 38 ’C to 28 ‘C, the total amounts of lipids

LIP113 METABOLISM IN ALGAE 19

stayed constant but a desaturation of palmitate at the .~n-2 position of M G D G took place (Sato and Murata, 1980a). Molccular oxygen was required for the desaturation, which was prevented by chloraniphenicol or rifanipicin, suggesting that the specific A9-desaturase activity was induced by the downward shift in temperature (Sato and Murata, 1981). Slower decreases in the amounts of oleate and linoleate and commensurate increases in 3-linolenate which occur in M G D G and SQDG and PG were also prevented by protein synthesis or RNA synthesis inhibitors (Sato and Murata. 1981).

Conversely, sudden increases in growth temperature transiently increase de novo fatty acid synthesis but suppress desaturation of existing lipids in A . vuriuhilis (Sato and Murata, 1980a). The rapid changes in unsaturation levels of C, , acids only occurred in MGDG, in keeping with its role in palmitate desaturation (above). Slower changes in the unsaturation of C , fatty acids occurred in all major lipid classes (Murata, 1987).

The changes which were found in the lipids of cyanobacterial membranes have been related to an adaptive response to prcvcnt damage caused by low temperatures. The results have been fully discussed (Murata. 1987: MuraLa and Nishida, 1987). I t appears that the plasma membrane (rather than the thylakoids) is particularly susceptible to low-temperature stress in both A nucysl is n iduluns and A nuhuenu vur iuhilis.

Apart from temperature, a number of other environmental factors have been shown to alter lipid composition in cyanobacteria. These eKects are summarized in Table V11.

Mention was made of the unusual heterocyst glycolipids a t the beginning of this section. These compounds have the structures shown in Fig. 8. The biosynthesis of these glycolipids was first studied by [I4C]acetate incorporation (Abreu-Grobois c t d . , 1977). I t was suggested that hydroxylation of the aliphatic chain at the C-3 and C-25 positions took place after the fatty alco- hol was linked to the glycosides. Krespki and Walton ( 1 983) compared the formation of heterocyst glycolipids and glycerolipids during heterocyst formation. They concluded that biosynthesis of the hydrocarbon moieties of heterocyst glycolipids was regulated independently of fatty acid synthesis. In addition, they suggested that an enzyme system for the hydroxylation of aliphatic hydrocarbon chains of the glycolipids was activated transiently during heterocyst formation. This process and heterocyst glycolipid synthesis are both increased by 7-azatryptophan (Krepski and Walton, 1983). Mohy-Ud- Dhin el ul. (1982) suggested that the latter increased one or more steps of primary alkanol synthesis, making the (2-25 hydroxylation rate-limiting for the formation of the glycoside containing 1,3,-hexacosanetriol.

VI. STUDIES WITH HALOTOLERANT AND HALOPHILIC DUNALIELLA SPECIES

Dunuliclla spp. possess some special features which make them attractive ex-


Environmental Organism Effect Reference change

Nitrate increase ilnciej~s1i.r nidu1un.s 16:01 I 6 : I f Piorreck ei nl. (1984) .Spirul/nu pltrtc~I1.si.s 18:2t 18:31 Piorreck P I a/ . (1984) Microc,j,stis uerugiiioscr No change Piorreck ei ul. ( 1 984) 0.5 cillur oriu ru hrscens Piorreck P I uI. ( 1984) N o change

Increasing Agnienrlluni 16: 1. 18: It 1 8 : 2 , Olsonandingram culture age yuurlruplicuruni 1 8 : 3 J ( 1975)

Anuh~rrnrr wriuhilis 18: l t16 :O. 1 8 : 2 1 Gusevrru/.(1980)

Apl i~ t io~l i rc~c~ hulophj~ticu 18 : 21 Anaerobic growth O.sc,iNtrtoriu linineticu No change

Light presence Various No change Kenyon e l ul. ( 1972)

Light intensity Anuc.j.sti.7 riidulnns 16:0, 1 8 : I f 1 6 : I J DohlerandDatz

Oren ei a/. ( 1985)

with increased light (1980)

Light quality Anrrc~j~sti.~ nit1ulnn.s GI ycerolipid Datz and Dohler changes (1981)

Temperature Various drop

Chain length1 Unsaturationt See text Molecular species

changes

Fatty acid abbreviations as i n Table I

perimental models. They grow rapidly under axenic conditions to yield popu- lations of very homogeneous cells. Since they are without cell walls, they are easily disrupted and can therefore be separated into relatively pure subcellular fractions. Their lipid composition is rather similar to that of higher plants and is typical of green algae. Moreover, their ability to tolerate a wide range of temperatures and salinities (Brown and Borowitzka, 1979) allows the effects of these environmental stresses on metabolism to be studied.

The lipids of two halotolerant species of Dunuliellu, ( D . purva and D. ter- riolectu) (Evons et ul., 1982a) and six halophilic species (D. viridis from the Dead Sea and various unidentified species from the Sinai) (Evans and Kates, 1984) have been examined. All these species were found to contain high proportions (40 mol %) of glycosylglycerides and low proportions (20 mol YO) of phosphoglycerides. The main glycolipids were MGDG, DGDG and SQDG. The main phospholipids were PG and PC. Analyses for the two halotolerant and two halophilic species are shown in Table VIII.


Glycosyl ester glycobpids Glycosd~c glycolipi@

a-0-Glucopyranos yl 25-hydroxyhexacosa~te

3,2 5-Dihydroxyhexacosanyl-0-D-glycopyranoside

YY no no 0- a n d

OH on noon on no ou ( 9 0 -1. ) ( 1 0 ' I. )

0-D-Glucop yranos yl 3,25.27-Trihydroxyoctacosanyl-a-Dglycopyranoside 25.27-dihydroxyoctacosaMte

Fig. 8. Structures of heterocyst glycolipids of cyanobacteria

TABLE VIII Lipid composit ion of h u b tolerant and haloph ilic Dun a I iel la species

Lipid D. parva D. tertiolecta C, D, , Phosphatidylcholine 9 4 1 2 Phosphatid ylethanolamine n.d. 2 1 1 Phosphatidylinositol 2 3 tr. tr. Phosphatid ylglycerol 6 8 4 3

tr. n.d Phosphatidic acid n.d 2 Total phospholipid 17 19 6 6

- - - -

Monogalactosyldiacy lglycerol Digalactosyldiacylgl ycerol Sulphoquinovosyldiacylgl ycerol Other glycolipids Total glycolipids

Diacylglycerol-0-( N,N,N-trimethyl)- homoserine

Non-esterified fatty acid Neutral lipids

21 22 I I 21 7 10 I tr.

40 53 - -

15 8

13 7 15 15

22 24 19 15 14 14 tr. tr. 55 5 3 - -

3 9

14 7 21 25

Data expressed as mol YO and taken from Kates (1987) with permission n.d. = not detected.

22 J O H N L. H A R W O O D A N D A . LESLEY JONES

In general, halophilic species had lower levels of phospholipids and pro- portionally higher amounts of glycosylglycerides than halotolerant species. Moreover, PI, which was found in appreciable amounts in halotolerant species, was only found in trace amounts in the halophilic species. PE was not detected in D. parva.

Significant amounts of neutral lipids, principally triacylglycerols and non- esterified fatty acids, were found in all species. These represented 15-25 mol YO and 7-14 niol ‘4, respectively, of the total lipid contents. Interestingly, diacylglycerol-O-4’-( N.N,N-trimethyl)-homoserine was identified as a major component (3 ~ 1 4 mol YO) in all species examined (Evans et al.. 1982b). As mentioned in Section 111, this zwitterionic non-phospholipid has been found in many algal species (Eichenberger, 1982; Sato and Furuya, 1985). including D. hrirckau~il (Fried Ct a/ . , 1982). I t is common also in lower plants (Sato and Furuya, 1984a,b) but has not been detected in any angiosperms or gymno- sperms examined.

The fatty acids of individual lipid fractions from all Dunalirlla species showed characteristics typical of other plant types, including angiosperms. Thus, palmitate was the major saturated fatty acid and this was enriched in SQDG of the glycolipids. The two galactosylglycerides were enriched in polyunsaturated fatty acids. MGDG was enriched in a-linoleic and hexadecatetraenoic acids. while DGDG contained high amounts of linoleic and x-linolenic acids. DGTS also contained a similar fatty acid content to DGDG. As in higher plants, PG contained high amounts of trans-A3- hexadecenoate as well as appreciable amounts of palmitate, linoleate and a- linolenate.

D. .salina has been extensively studied at the subcellular level by Thomp- son’s group. Firstly, studies were made of phospholipid metabolism during growth at 30 C or 12 C or after temperature shifts. Generation times were found to correspond approximately to a Q,, of 2. with times of 20 h at 30 C and 80 h at 12 ‘C. Both cultures reached the same cell density but had rather different lipid and protein contents. In general, cells cultured at 12 C had a higher protein and acyl lipid content but a lower chlorophyll content than those grown at 30 C. Interestingly, the chlorophyll ujh ratio was 2.77 at 30 C but 4.09 at 12 C (Lynch and Thompson, 1982). The relative changes in acyl lipid and chlorophyll contents could be correlated with a proportional decrease in thylakoid membranes but an overall increase in cell volume at 12 C. When cells were shifted from 30°C to 12”C, no division occurred for about 96 h, after which division resumed, with a generation time of 80 h.

The major particulate subcellular fractions were the chloroplast and microsomal. Whereas the chloroplast fraction was enriched at least four-fold in glycolipids compared to phospholipids, the microsomal fraction contained about twice as much phospholipid as glycolipid. Moreover, the relative proportion of cell phospholipid contained in chloroplasts was significantly reduced when cells were shifted to, or grown at, 12°C. This reflected the relat-


ive increase in microsomal menibrancs compared to thylakoids for cclls grown at lower temperatures (Lynch and Thompson, 1982).

When the proportions of individual phospholipids of chloroplast or micro- soma1 membranes were analysed, they were found to change little with growth temperature. For cells grown at either 30 C or 12 C, the major phospholipids of chloroplasts were PG and PC with smaller amounts of PE and PI. Microsomal menibranes contained PE and PC as the main phospholipids. In contrast, the relative proportions of glycolipids responded to temperature change. In particular, the relative content o f D G D G increased and that of M G D G decreased at 12 C. The ratio of M G D G to DGDG therefore changed from 3.4 in chloroplasts from 30 C cells to 2.1 for 12 C cells.

Because poikilotherms need to maintain membrane fluidity at different growth temperatures, it was not surprising that phospholipid unsaturation was increased at 12 C for both chloroplast and microsomal membranes. In temperature-shift studies. the majority of these changes occurred after more than 60 h for chloroplast fatty acids. In the microsomal fraction, significant decreases in palmitate and increases in octadecatrienoate contents were seen within 12 h of temperature shift. Similarly, changes in the fatty acyl content of DGTS were also greater in the microsonial membranes. In contrast, the fatty acid patterns of the chloroplastic glycolipids changed little in response to growth temperature (Lynch and Thompson, 1982).

The rather slow response of fatty acid unsaturation to shifts in growth temperature--especially for the chloroplast membranes-raised the obvious question as to how poikilotherms are able to withstand sudden changes in environmental conditions. Following observations with cyanobacteria (Section V), where an “emergency response” seemed to be the retailoring of individual molecular species (Sato and Murata, 198Oa,b), the phospholipids of D. .sulinu membranes were examined in more detail. The individual phospholipids were isolated following thin-layer chromatography (TLC). and converted to diacylglycerols by digestion with phospholipase C; the diacylglycerols were separated by G L C of trimethylsilyl derivatives. Such pro- cedures showed that there were significant changes in the molecular species of PE and PG. For PE there was a decrease in C,, species and co,ncomitant increases in C,, and C,, species. Changes associated with PG included the detection of a new molecular species, dioleoyl, not found at 30 C. Although there were some, limited, changes in fatty acid unsaturation detected during the first 12 h of low-temperature acclimation, it was concluded that the initial alterations in response to low temperatures involved discrete changes in certain molecular species. Such changes in molecular species composition would then augment the effects of acyl chain unsaturation in modifying membrane fluidity (Lynch and Thompson, 1984a).

As mentioned above, changes in the fatty acyl composition of chloroplast phospholipids were much slower than for microsomal components. Thus only minor alterations in phospholipid acyl chain compositions were evident


after 36 h of shifting cells from 30 to 12 C. Between 36 h and 60 h, increases i n the percentage of palmitate and a-linolenate were observed in PG. These alterations were accompanied by decreases in trans-A3-hexadecenoate and linoleate (Lynch and Thompson, 1984b). In contrast, the fatty acyl composi- :ion of the other main chloroplast phospholipid, PC, did not alter very much over the 60-h period.

Changes in the molecular species distribution for PC and PG were also seen after 60-h acclimation. The molecular species changes for PG correlated well with the overall fatty acyl changes. Thus, the 18 : 2/16 : I species was particularly reduced, in keeping with the decrease in both linoleate and rrans-A3- hexadecenoate. Significant increases in molecular species containing palmitate and r-linolenate were seen-again in keeping with the fatty acid analysis (Lynch and Thompson. I984b).

The change in molecular species of PG which occurred between 36 h and 60 h, following a shift in growth temperature, coincided with a large change in the threshold temperature of thermal desaturation of the photosynthetic apparatus. This was measured by chlorophyll fluorescence and, since lipid compositional changes other than those associated with PG were negligible during this period, suggested that a correlation existed between the molecular species of composition of PG and the thermal stability of the photosynthetic membrane. Taken together with the data on microsomal membrane compositional changes, the experiments suggested strongly that the initial steps in cellular acclimation to low temperatures involved molecular species retailoring. These changes then augmented the effects of increased acyl chain unsaturation as a means of restoring appropriate membrane properties (Lynch and Thompson, 1984~).

Further studies on the subtle changes in acyl lipid molecular species used HPLC as the separation technique. Because of the poor absorbance of lipids in the UV. initial quantitation used the laborious procedure of effluent split- ting and measurement by GLC of fatty acid methyl esters (Lynch and Thompson, 1983). However, the development of a flame ionization detector allowed more rapid quantitation and the procedure was applied to the analysis of D. sdina PG and galactosylglycerides (Smith et a/., 1985; Cho and Thompson, 1987).

In Section IV, mention was made of the so-called prokaryotic and eukaryotic pathways for fatty acid and acyl lipid synthesis in eukaryotic organisms. The relationship between these two pathways in D. salznu was investigated in detail through radiolabelling with [I4C]palmitate, [I4C]oleate and [14C]laurate as precursors. Since only [I4C]laurate could be elongated in D. salina, palmitate gave rise to C,, acids only and oleate to C, , acids (Norman eta/. , 1985). After a 2-min incubation with 4 pCi of [I-14C]palmitate, about 3% of the isotope was taken up per 5 x lo8 cells. This allowed experiments to be conducted with a 2-min labelling period followed by a chase period. During the total incubation a gradual movement of radioactivity was


seen from the microsomal membranes to the chloroplasts. This movement was associated with a decrease in phospholipid labelling and an increase in that of glycolipids. When the individual acyl groups were analysed, i t was found that phospholipids only contained [I4C]palmitate. In contrast, glycolipids contained unsaturated C,, acids. After a 16-h chase, over 30% of the glycolipid radioactivity was accounted for by [14C]hexadecatetraenoate.

Similarly, when [14C]oleate was used in labelling studies, the radioactivity was incorporated initially into phospholipids of the microsomal membranes. After only a 20-min chase more than 50% of this microsomal phospholipid labelling was in the form of [*4C]linoleate, showing the presence of an active extra-chloroplastic A1 2-desaturase. By 16 h, chloroplast phospholipids contained 58% and 12% of their radioactivity in linoleate and sr-linolenate, respectively. In contrast, chloroplast glycolipids contained over 70% of their radioactivity as a-linolenate. These results confirmed that in D. salina, as in higher plants (Harwood, 1988), desaturation of oleate occurs mainly outside the chloroplasts, followed by transfer of the resultant linoleate back into plastids for its desaturation to a-linolenate in association with glycosylglycerides.

When [I4C]laurate was used as precursor, the acid entered the chloroplast quite rapidly, where it was used by the de n o w synthesizing enzymes and gave rise to radiolabelled C, , and C, , fatty acids. Moreover, in contrast to [ 14C]palmitate labelling, [‘4C]laurate incorporation resulted in the accumulation of appreciable [’4C]trans-A3-hexadecenoate.

These studies were extended to a consideration of the molecular species of PG which were radiolabelled during temperature stress. When[14C]palmitate was used as precursor for a 2-min pulse, the specific radioactivity of PG was higher initially than that of any other microsomal phospholipid. The major molecular species of microsomal PG (80%) was 16 : 0/18 : 2, with about 4- 5% of the 16 : O/ 16 : 0 species. Smaller amounts of 16 : O/ 14 : 2 species were also present (Lynch and Thompson, 1984a). In spite of the heavy concentration of 16 : 0 at the sn-l position, analysis of the radiolabelled PG by phospholipase A, digestion revealed that 30% of the [I4C]palmitate wgs present initially at the sn-2 position. In fact, immediately after labelling, the specific radioactivity of the palmitate at the sn-2 position was 10 times that at the sn- 1 position. Molecular species analysis by HPLC confirmed that [I4C]palmitate was preferentially incorporated into the 16 : 0/16 : 0 species.

During the cold-chase period a changing pattern of incorporation of [L4C]palmitate into either microsomal or chloroplast PG was seen. Thus, whereas microsomal PG always contained more radioactivity at the sn-l position, chloroplast PG initially contained radioactivity almost exclusively at the sn-2 position. With time, both positions of chloroplast PG became equally labelled. These results indicate the “prokaryotic” nature of chloroplast PG synthesis initially, and it is only later that molecular species of pre- sumed microsomal origin appear in the plastid (Norman and Thompson,


I985a). I n fact, further analysis of the chloroplastic molecular species showed that 18: 2/16: 0 was labelled initially, followed only later by 16: Ojl8 : 2 species.

When [I"C]laurate was used as precursor, the acid was rapidly taken up by chloroplasts, where i t was used by fatty acid synthetase to produce [14C]palmitate and [14C]stearate, and the latter was desaturated to [14C]oleate. These products were rapidly exported to the endoplasmic reticu- lum, where PG was labelled more rapidly than other microsomal phospholipids. I n marked contrast, chloroplast PG contained no radiolabelled C, fatty acids. Immediately after the 2-min labelling period, this phospholipid contained large amounts of ['4C]laurate, but within 10 min of chase, [I"C]palmitate was the major radiolabelled moiety. In keeping with the pro- posal that 1 -acyl,2-palmitoyl-PG is the substrate for the A3-desaturase (Harwood and James, 1975), the decline in radioactive palmitate during the 20-60-min chase period was matched by an equivalent rise in the labelling of truns-A3-hexadecenoate.

As mentioned above. the proportions of dilrerent cellular membranes in Dutitrlir//rr changes with low-temperature growth. There were also significant changes in the metabolism of chilled cells. Thus, for example. there was no delay in the labelling of the sn-l position of chloroplast PG from [lSC]palmitate in chilled cells. This was due to the increased contribution of "eukaryotic" (microsomal) metabolism at lower temperatures. Moreover, this was in keeping with the previous observations that the molecular species composition of microsomal PG responded more quickly to temperature stress than that of chloroplasts (Lynch and Thompson, 1984a.b).

I f niicrosomal phospholipids are retailored initially in response to temperature stress. then there should be appropriate enzymes present to remove and re-esterify the different acyl groups (Lynch and Thompson, 1984~). The first enzyme needed would be a phospholipase A and, indeed, such an enzyme has been reported in D. .su/inu (Norman and Thompson, 1986). Microsomes, but not chloroplasts. contained a fatty acyl hydrolase with high activity towards endogenous or exogenous PG and PE. The enzyme had little activity towards other phospholipids or MGDG. Because no monoacyl products could be detected, it was not possible to analyse independently the activities of any specific enzymes, such as phospholipase A , or A,, or lysophospholipase which might be present. Lipolysis was most active in the presence of 10 mM Ca2+ , and was enhanced by calmodulin and inhibited by calmodulin antagonists, such as W-7 or 48/80. Most interestingly, the acyl hydrolase activity of 30 C-grown cells was low when measured in vitro at 12 C. How- ever, when cells were chilled to 12'C, activity as measured at 12 C in vitro rapidly increased, thereby providing the mechanism for retailoring phospholipids during low-temperature acclimation. The low acyl hydrolase activity in chloroplasts from similarly treated cells emphasized the key role of micro-

LIPID METABOLISM IN ALGAE

18:1/16:0 MGDG

27

18:2/16:0 DGDG 18 1/16 1 MGDG

18 1/16 1 DGDG

18 3/16 0 DGDG D' s

18 3/16 4 MGDG >18 3/16 4DGDG

l i g . 9. Pathways for galactosylglyccridc synthesis in Diititrlidltr .srr/inti chloroplasts. Redrawn from Cho and Thompson (1987) with permission.

soma1 metabolism in temperature adaptation (Norman and Thompson, 1986).

Although the labelling of galactosylglycerides in D. siilinu has not been studied in as much detail as that of the phospholipids. a recent report de- scribes labelling of molecular species from I4C-labelled fatty acids (Cho and Thompson, 1987), and a fatty acyl hydrolase preferentially attacking MGDG has been reported (Cho and Thompson, 1986). The results of the labelling studies are summarized in Fig. 9. They showed that, as expected (Harwood, 1988), the initial molecular species of MGDG labelled by &novo synthesis was the 18 : 1/16 : 0. This molecular species could then be further desaturated at both positions to eventually produce the highly unsaturated 18: 3/16:4 species. However, after the initial desaturation to 18: 1/16: I , this species (like others) of MGDG was a substrate for galactosylation. The DGDG so produced was itself a substrate for continued desaturation. The 18 : 1/16 : 0 species of MGDG could be galactosylated but the DGDG so produced could only be desaturated at the sn-l position to yield a-linolenate. No further metabolism of palmitate at the sn-2 position took place (Cho and Thompson, 1987). These observations are particularly interesting because not only do they provide further evidence for the involvement of complex (galactosylglycerides) lipids as substrates for desaturations, but they also confirm that ( I ) lipid-linked acyl chains can be subject to a whole series of desaturations and ( 2 ) that several lipid types (in this case DGDG as well as MGDG) may serve as substrates for an individual desaturation. These topics are reviewed more fully in Harwood ( 1 988).


VII. METABOLISM IN MARINE ALGAE

A. LABELLING CHARACTERISTICS

There have been relatively few studies on the lipid metabolism of marine macroalgae. There is now, however, a considerable body of work on their lipid and fatty acid compositions. Earlier work in this area has been summarized by Pohl and Zurheide (1979a). Most of the lipids which are found in marine algae occur in higher plants, although there are exceptions, and marked contrasts are seen in the lipid patterns of different algal divisions (Table IX). Marine algae have a characteristic pattern of polyunsaturated fatty acids which is quite distinct from that of higher plants (see Section I l l ) , and again reflects differences among the algal divisions (Pohl and Zurheide, 1979a). These observed differences in marine algal lipids and fatty acid content are presumably a reflection of differing metabolism, but there is very little detailed information available.

The marine algae typically contain high proportions of polyunsaturated fatty acids (e.g. Jamieson and Reid, 1972; Pohl and Zurheide, 1979a). The brown and red algae contain arachidonic (20 : 4. w-6,9,12,15) and eicosapentaenoic (20 : 5 , w-3,6,9,12,15) acids as major fatty acids, with the brown algae tending to have more of the former and the red algae more of the latter. The green algae have only small amounts of C,, fatty acids, but do contain C,, and C, , fatty acids, which are more unsaturated than those of higher plants. Entrromorphu intrstinalis, a marine green macroalga, has among its major fatty acids a-linolenic acid ( 18 : 3, w-6,9,12), which is typical of higher plant photosynthetic tissue, and also octadecatetraenoic (18 : 4, w-3,6,9,12) and hexadecatetraenoic ( 1 6 : 4, w-3,6,9,12) acids (Jones and Harwood, 1987). Octadecatetraenoate is found in some higher plants, e.g. borage (Stymne rt ul., 1987). In this species, octadecatetraenoate is formed from cz-linolenate while esterified to MGDG (Griffiths et al., 1988).

Labelling studies on the fatty acids of algae using [I4C]acetate show that the major fatty acids labelled are generally palmitate and oleate. Incorpora- tion of radioactivity into palmitoleate, stearate and the more polyunsaturated C, , fatty acids with long-chain C,,, C,, and occasionally C,, saturated fatty acids (Table X), is usually seen also. This indicates that the initial pathway of fatty acid synthesis is similar to that of higher plants, but that additional desaturase and elongase enzymes must be present for the production of the complete marine algal fatty acid pattern. The long-chain polyunsaturated fatty acids are themselves synthesized slowly, as indicated by time-course studies for up to 24 h with [I4C]acetate, when n o radiolabel accumulates in arachidonate or eicosapentaenoate (Jones, A. L. and Har- wood, J. L., unpublished). A preliminary report for Porphyra yezoensis sug- gests a route for chain elongation and desaturation in this alga, but further studies are needed to confirm this (Kayama et al., 1986).

TABLE IX The lipid composition of marine algae representing different algal divisions

Lipid Phaeophyta Rhodophyta Chlorophyta

F. serratus F. vesiculosus Ascophyllum nodosum Chondrus crispus Polysiphonia lanosa E. intestinalis

MGDG DGDG SQDG X

18.1 23.1 32.9

7.5

15.0 11.3 22.0 24.1

19.7 16.6 19.4 17.2

17.6 12.8 14.7 n.d.

17.6 24.6 11.9 n.d.

45.9 14.8 14.8 n.d.

PC PE PG PI DPG DGTS

4.3 5.7 2.5 2.1 4.2 -

4.2 6.2 2.2 2.9 5.4 -

3. I 9.8 1.5 2.5 3.1 -

31.6" 1.8 8.2

18.3" 1.5 5.0

-

1.5 -

-

13.7 2.6 1.5

NL 1.6 4.9 5.0 7.6 15.0

Rest 1.8 2.1 1.8 2.7 3.3

Data as means n = 3-7; X, unidentified glycolipid; n.d., none detected. Results expressed as YO total lipid "PC + PSC

30 J O H N L. H A R W O O D A N D A. LESLEY J O N E S

TABLE X Rudioluhelling (?f:firttj' acids f rom [ ' 4C]uce1ate in various marine algae

Fatty acid (YO total fatty acids)

1 4 : 0 16:0 16.1 18:O 1 8 : l 1 8 : 2 20:0 22:O Others

Phaeophyta F. si~rrutiis 5 42.0 I 17 13 2 6 10 4 F. i~e.cicu1osu.c 3.4 16.0 1.8 12.1 41.1 3.8 5.3 8.9 7.7 A.vc~op/ivlluni norlosion 2.6 19.9 t r 11.6 43.3 4.8 4.0 9.2 4.1

Rhodophyta C'honclrus c~ i~p t i . s I .5 5.7 12.0 3.8 62.6 4.3 3.5 4.6 2.8 Polj~siphoniu Itrrro.srr 6.5 40.9 10.9 7.2 21.5 ~ 4.8 ~ ~

Chlorophyta E. intc.stinu1i.s 1.7 9.8 2.8 2.2 42.1 9.2 2.9 8.7 16.5"

" ~ 1 8 : 3 + IX.4: tr = <0.5"%. Labclling X h in the light a t 15 C. F. .serru/u.s at 20 C . Fatty acid abbreviations as for Table I . Data for F. .scrrLrfu.s from Smith and Harwood (1984a). Othcr da t a from Jones. A. L. and Har-

wood. J . L. (unpublishcd).

The major lipids of most of the marine macroalgae that have been studied are the glycolipids (Table IX). In the fucoids, Fucus vesiculosus, F. serriitus and Ascophylluni nodosuni, they account for 70- 80% of the total acyl lipids (Smith and Harwood, 1984a; Jones and Harwood, 1987). Similarly high proportions are found in the red and green algae (Pettitt et d., 1988b; Jones and Harwood, 1987) as well as in cyanobacteria and some fresh-water green algae (Section 111). The leaf tissues of higher plants have comparable proportions of glycolipids (Harwood, 1980a), but higher plants generally have more MGDG (25-45%) and less SQDG (10%). All the marine algae so far examined have significantly more SQDG (Table IX). E. intestinalis has the glycolipid distribution most similar to that of higher plant leaf tissue, with 46% MGDG. It has been postulated that the high levels of glycolipids in algae indicate their location in membrane systems other than the chloroplast thylakoids (Smith et ul., 1982). This idea is partially supported by work on Polj,siphonia lunosa, which indicated that the chloroplast lipid content was similar to that of the whole tissue (Pettitt, T. P. and Harwood, J. L., unpublished results).

In most of the algae studied, the glycolipids appear to be similar to those of higher plants. However, some unusual glycolipids have been detected. Pham- Quang and Laur (1976a,b,c) reported unusual glycolipid structures in several fucoids, and other sugars (such as mannose and rhamnose) have been detected which may partly replace the galactose of MGDG and SQDG in the red algae Chondrus crispus and Polysiphonia lanosa (Pettitt and Harwood, 1986). Pham-Quang and Laur (1976b) found unusual sulpholipids in some


Ascoplij~lluni tiodosuni F. ws icdosus F. sewatus

Lipid "/o I4C Lipids "h I4C Lipids YO I4C Lipids YO 3 2 P Lipids

M G D G 5.8 8. I 9.2" D G D G 1.4 2.1 4 . P SQDG 13.2 12.5 9.0 X 16.3 28.4 19.2

PC PE PG DPG PI

1 .9 6.3 4.4 I .4 6.9

2.3 2.4 5.4 6.8 4.0 50.0 3.9 7.0 2.6 12.2 3.7 2.3 15.4

Neutral 17.4 23. I 11.3 lipids

Rest 25.3 6.5 4.0 10.0

"MGDG includes DPG. *DGDG includes PG. Data as means. I I = 2 or 3 . Jones, A. L. and Harwood, J. L. (unpublished) and Smith P I cil . (1982)

fucoids, and Chondrus crispus and Polysiphoniu lunosu contained lipids other than SQDG which were detected by 35S-labelling. In both these red algae one of these sulpholipids was probably PSC (Pettitt et ul., 1988b), the sulphur- containing analogue of PC first noted in the diatom N . ulhu (Anderson et ul., 1978a,b).

The glycolipids of the brown algae F. serrutus, F. vesiculosus and Ascophyl- lum nodosum tend to be poorly labelled from [I4C]acetate in relation to their abundance (Table XI). However, SQDG is well labelled, particularly in Ascophyflum notlosum. This may be a reflection of the higher proportion of C , , and C, , saturated and mono-unsaturated fatty acids in SQDG, because such acids are more rapidly labelled than the polyunsaturated C,, fatty acids which constitute a major proportion of the MCDG and DGDG acyl chains. MGDG has a higher proportion of label at all incubation times than DGDG, and this may partly reflect a possible role as substrate for the desaturation of linoleate to linolenate, by analogy with higher plants (Roughan e f ul., 1979; Wharfe and Harwood, 1978). However, any discussion of desaturation- elongation reactions in these marine algae is complicated by the presence of a much greater variety of polyunsaturated fatty acids than occurs in higher plants.

Another notable feature of lipid labelling in these algae is the high propor-


T A B L E XI1 Unsaturrctron indices of red and brown algal glycoliprdJ

Lipid F. vesiculosus Ascophyllum Chondrus Polysiphoniu Porphyrtr nodosum crispus lanasa . 13rzoensis

M G D G 3.5 3.4 4.6(1) 4.0(1) 4.8 ( I ) 2.1 (2) 2.4 (2) 2.3 (2)

DGDG 3.1 2.8 2.1 2.4 3.6

SQDG 1.5 0.8 3.4 ( I ) I .4 2.5 1.9 (2)

( % ratty acid x no. double bonds) I00

~~~ ~~~~ Unsaturation index = ~- ~

Where twc: bands were detected by TLC. the raster running band is designated ( I ) . Porphyrci ~ ~ ( ~ : o ~ m i . s data from Araki (I! ul. (1986).

tion of label found in lipid X (Table XI). This unknown lipid is clearly an important constituent of the fucoids, but its role in metabolism is difficult to assess, as i t remains unidentified (Smith and Harwood, 1984a; Jones and Harwood, 1987). I t is an acyl lipid with no net charge which stains positively with Dragendorf reagent, indicating the possible presence of a quarternary amino group. I t does not contain phosphorus or sulphur.

In Chondrus crispus the glycolipids are all poorly labelled from [I4C]acetate, but in Polysiphoniu lanosa the proportion of label in these lipids is much higher (Pettitt, T. R. and Harwood, J. L., unpublished). In E . intestinalis, SQDG is poorly labelled but MGDG and DGDG are fairly well labelled. As noted previously, the fatty acid and lipid pattern of E. intestinulis is different from that of the brown and red algae and, therefore, differences in lipid labelling are not unexpected (Jones and Harwood, 1987).

In all these algae, as in higher plants, MGDG is the most unsaturated lipid (Smith and Harwood, 1984a; Jones and Harwood, 1987; Pettitt and Harwood, 1986). The relative unsaturation of the glycolipids of brown and red algae is shown in Table XII. Double bands occur on TLC for DGDG of F. vesiculosus and Ascophyllum nodosum (Jones and Harwood, 1987), for MGDG and SQDG of Chondrus crispus (Pettitt et al., 1988a; Jones and Har- wood, 1987), and for MGDG of Porphyra yezoensis (Araki et d., 1986) and Polysiphoniu lanosa. The faster running band (band 1) has a higher unsaturation index in all cases (Table XII). Separation of double bands is caused by binding materials in commercial silica gel plates. The more saturated band is generally more highly labelled (Pettitt et al., 1988a).

A similar range of phospholipids is found in marine algae to that of higher plants. All the eukaryotic marine algae so far examined have PG with trans-


A3-hexadecenoate, typical of higher plant chloroplasts (see Gounaris et al., 1986). As granal stacking is limited in the brown algae and does not occur in the red algae, this is further evidence that this is not the role of trans-A3- hexadecenoate (e.g. Roughan, 1984). Caron et al. (1985) reported PG containing trans-A3-hexadecenoate to be preferentially associated with the light- harvesting complexes of F. serratus, but point out that its role may not be the same in algae as in higher plants. PE is the major phospholipid of the brown algae and PC of the red algae. PE and PC in the brown algae and PC in the red algae are highly unsaturated and tend to be only poorly labelled from [14C]acetate (Table XI). Arachidonate and eicosapentaenoate tend to be major components of these lipids (Section 111).

The only detailed study of phospholipid metabolism in marine algae is that of Smith et al. (1982). [32P]Orthophosphate labelling in F. serratus showed phosphatidate to be labelled at short time intervals, with labelling decreasing as the incubation time increased, a result which would be expected if phosphatidate is an intermediate in phospholipid biosynthesis (Moore, 1982; Harwood, 1989). A concomitant rise in 32P-labelling in PI and PE was seen, although PC was poorly labelled at all time intervals. As PC was well labelled from [14C]acetate in this alga, such a result may reflect the role of PC as a substrate for desaturation of oleate to linoleate, as in higher plants (see Harwood, 1988).

In E. intestinalis no PC was detected and only a small amount of PE. How- ever, this alga contains DGTS, which was first reported in 0. danicu (Brown and Elovson, 1974). It has been found in various single-celled green algae (Eichenberger, 1982; Evans et al., 1982b), bryophytes and pteridophytes (Sato and Furuya, 1984b). As discussed in Section 111, various marine Chlorophyceae contain this lipid, including Ulva lucruca (Sato and Furuya, 1984b) and E. intestinalis (Jones and Harwood, 1987). In E intestinalis this lipid is a significant component (Table IX). It too is a Dragendorf-positive lipid with no net charge, but it does not have the same characteristics on TLC as the unknown lipid X of brown algae (Smith and Harwood, 1984a; Jones and Harwood, 1987). Eichenberger (1982) analysed several algal ‘species for the presence of DGTS and detected none in F. vesiculosus either.

I t has previously been noted that DGTS is often present in algae with little or no PC (Sato and Furuya, 1984a,b). In Chlamydomonas reinhardtii, which contains no PC, DGTS may be the substrate for the desaturation of oleate to linoleate, a role attributed to PC in higher plants (Schlapfer and Eichen- berger, 1983; Section VIII). In D . salina, PC and DGTS are both present, and have similar fatty acid distributions. Metabolic studies using 14C-labelled fatty acid traces also indicate a close metabolic relationship between these lipids (Norman and Thompson, 1985b; Section VI). In E. intestinalis we have carried out labelling studies with [14C]acetate and found DGTS to be only poorly labelled compared with labelling of the glycolipids MGDG and DGDG and the phospholipid PG.


TABLE XI11 Percentage MGDG derived from prokaryotic (chloroplastic) or eukaryotic (cyto-

plasmic) pathways of lipid synthesis

Prokaryotic Eukaryotic Reference 16- [18+20 or 16.[16 1 8 + 2 ~ - r 1 8 + 2 0 or

1 8 + 2 0 - r 1 6

Entcromorpha spp. 95

Ulva spp. 90

C. vulgaris 60

Phaeodacrylum 90 tricornutum

Porphyra yezoensis 20

F. vesiculosus 10

5 Heinz ( 1 977)

10 Rullkotter et al. (1975)

40 Roughan and Slack ( I 984)

10 Arao et al. (1987)

80 Araki et al. ( 1 987)

90 Jones and Harwood (unpublished)

B. POSITIONAL DISTRIBUTION OF ALGAL FATTY ACIDS

In higher plants there are two pathways producing the diacylglycerol moieties for incorporation into glycolipids (e.g. Roughan and Slack, 1984; Frent- Zen, 1986; Joyard and Douce, 1987). The chloroplastic pathway produces lipids with a typically prokaryotic arrangement of fatty acids, i.e. with C , , fatty acids at the sn-2 position as in the cyanobacteria (Zepke et al., 1978). The PG of the photosynthetic membranes is of the prokaryotic type in all plants (Murata et al., 1982). The cytoplasmic pathway produces lipids with C,, fatty acids at the sn-2 position. Marine green algae generally show a prokaryotic fatty acid distribution in.their MGDG (Table XIII) (Rullkotter et al., 1975; Heinz, 1977). Phaeodactylis tricornutum (a marine diatom) also shows this typically prokaryotic MGDG (Arao et al., 1987). In F. vesiculosus, however, 10% of the C,, fatty acids are esterified to the sn-2 position of MGDG (Jones and Harwood, unpublished), and this is similar to published data for Chattonella antiqua (Sato et al., 1987), a raphidophyte and a member of the Chromophyta (organisms with chlorophylls a and c). More unusual is the situation in Porphyra yezoensis. In this alga only 20% of the MGDG appears to be derived from the prokaryotic pathway, yet the DGDG appears to be almost entirely chloroplastic in origin (Araki et al., 1987). On the basis of our current understanding of the synthesis of DGDG from MGDG in higher plants, it is difficult to see how this occurs (e.g. Joyard and Douce, 1987).


Sato et al. (1987) also noted positional specificity in the distribution of other fatty acids between the sn-1 and sn-2 positions in Chattonella antiqua. Eicosapentaenoate is enriched at the sn-1 position in MGDG, DGDG and SQDG, but at the sn-2 position in DGTS and PE. In Phueoductylis tricornutum, this acid was located at the sn-1 position in all the polar lipids except PC (Arao et al., 1987; Kawaguchi et al., 1987). In F. vesiculosus eicosapentaenoate is located (almost exlusively) at the sn-1 position in MGDG and DGDG, and mostly at the sn-1 position in SQDG (Fig. 10; Jones and Har- wood, unpublished results), and preliminary results indicate that in PC and PE eicosapentaenoate occurs at both sn-1 and sn-2 positions. In F. vesiculosus MGDG and DGDG and Chattonella antiqua MGDG, octadecatetraenoate occurs at the sn-2 position, as i t does in Borago (Stymne et al., 1987). These results from a range of algal species indicate some positional specificity in the transacylation (and, possibly, elongation) reactions for fatty acyl chains, and this specificity differs in both nature and extent in different algae. Generally, however, it appears that in the galactolipids of chromophytes, eicosapentaenoate is esterified to the sn-1 position (Fig. 10). Although C , , acids are not common membrane components of most terrestrial plants, they have been detected at the sn-2 position in some species (Auling et al., 1971).

C.

1. Light Light has a marked effect on the lipid composition of photosynthetic tissues in higher plants, synthesis of the typical chloroplast lipids MGDG, DGDG, SQDG and PG being stimulated in the light, as is the desaturation of their acyl chains (see Harwood, 1983).

In Chondrus crispus the total amount of [' 4C]acetate incorporated in the light was higher than in the dark, and a marked increase was noted in PG labelling (Table XIV); this also occurs in Polysiphonia lanosa (Pettitt and Harwood, unpublished results). However, in F. serratus labelling of PG from [14C]acetate did not increase significantly in the light (Table XIV), although a small but significant increase in PG labelling from [32P]orthophosphate did occur (Smith et ul., 1982).

In Chondrus crispus, Polysiphonia lanosa (Pettitt, T. R. and Harwood, J. L., unpublished) and Bryopis maxima (a green marine alga) (Ohnishi and Yamada, 1977), light increased the proportion of oleate and linoleate labelled from [14C]acetate, with decreases in the amount of saturated fatty acids (Table XV). Algal metabolism in this respect appears similar to that of higher plants. The importance of the polyunsaturated C,, fatty acids is difficult to assess as they are labelled only slowly from [14C]acetate. This makes it difficult to study the effect of altered environmental parameters on these important fatty acids in vitro, although some conclusions can be drawn from a

EFFECTS OF THE ENVIRONMENT ON ALGAL LIPID METABOLISM


F. vesiculosus

MGOG

18: 18:4 2 65% 8%~(~, 18: 3 7%

( 3 )

i? vezoensis

MGDG

18: 1 8% 18:2 5%

OGDG

16%

18:4 5S0/ (2) 18 :3 14% 18:2 12% 1

DGDG

16:O 76% 18:l 9% 18: 2 9%

-20:5 86% 16:O 9%

(2 I

-Gal.Gal

Fig. 10. Positional distribution of fatty acids in the galactosylglycerides of two marine algae. See text for details.

study of endogenous fatty acid content when algae are harvested under different environmental conditions. No results have so far been reported of the effect of light on the synthesis and occurrence of C,, fatty acids in marine algae.

2. Temperature Temperature is known to affect the lipid composition of many organisms, and these effects are thought to be a response of the organism to maintain fluidity in, and hence function of, membranes. The alterations that occur in


TABLE XIV Effect of light on radiolabelling from [32P]orthophosphate or [I4C]acetate in the acyl

lipids of F. serratus and Chondrus crispus respectively

Lipid F. serratus' Chondrus crispusb

Dark Light Dark Light

MGDG DGDG SQDG

PE PG PC DPG PA PI

10.3 f 0.2 1.9 f 0.4 9.3 f 0.6

14.3 f 2.6' 6.9 f 0.6

13.0 f 5.0

54.3 f 5.9 52.1 f 8.6 10.3 f 1.0 11.6 f 0.8' 5.4 f 2.0 18.5 f 1.7' 4.9 f 1.9 19.3 f 2.9 27.0 f 1.1 17.6 f 2.9' 9.8 f 0.6 1.4 f 0.4

19.3 f 2.9

10.8 f 2.4 1.5 f 0.3

18.9 f 3.3

"Data from Smith er at. (1982). bData from Pettitt and Harwood (1987). 'P < 0.05 Labelling in F. serratus was with [32P]orthophosphate and in Chondrus crispus with (I4C]ace-

tate. Results are % of total acyl lipid labelled.

TABLE XV Effect of light on the distribution of radioactivity from [I4C]acetate amongst acyl chains

in F. serratus and Chondrus crispus

Distribution of label (YO 14C-labelled fatty acids)

14:O 16:O 16:l 18:O 18: 1 18:2 Others

F. serratus' Dark 3.4 f 0.5 47.7 f 7.8 - 12.9 f 2.4 29.6 f 6.3 0.9 f 0.7 5.5 f 1.4 Light 3.2 f 0.4 31.5 f 5.4' - 9.9 f 0.5 42.6 f 6.0' 9.4 f 1.6' 3.5 f 1.4

Chondrus crispusb Dark 6 f l 3 1 f l 2 f l 7 & 3 4 0 f 6 3 f t r I l f l Light 1 f 1 19 f 2 3 f 2 tr' 6 4 f 6 ' I O f T 3 f 5

'Data from Smith and Hanvood (1984b). bData from Pettitt and Hanvood (1987). 'P<0.05 Incubation for F. serratus was carried out for 7 h at 20°C. and for Chondrus crispus for 24 h at

Fatty acid abbreviations as for Table I. 15°C.


plant membranes in response to decreased temperatures lower the phase transition temperature of the membrane lipids, and these effects have been best studied in the single-celled marine green alga D. salina (Section VI).

Major strategies which maintain membrane fluidity despite lowered temperature are a reduction in fatty acid chain length, an increase in unsaturation and changes in positional distribution of acids. These changes have been seen in higher plants (Harwood, 1983) and Tables XVI and XVII show that they also occur in some marine algae. Some, but by no means all, higher plants also alter the levels of their complex acyl lipids (Clarkson er al., 1980), though this may also reflect a change in the subcellular morphology of re- sponsive cells (see Harwood, 1989).

The relatively constant endogenous fatty acid pattern of Ascophyllum nodosum in winter and summer has been noted before (Jamieson and Reid, 1972). However, many other algae do show increased unsaturation of their fatty acids in winter-the saturation indices of F. vesiculosus, Polysiphonia lanosa and Chondrus crispus are higher in February than in June (Table XVI). Pohl and Zurheide (1979b) reported increased unsaturation in F. vesiculosus and Phycodrys sinuosa from the Baltic Sea in the winter. Changes in unsaturation indices in F. vesiculosus follow very closely changes in sea-water temperature (Pohl and Zurheide, 1979b). In Polysiphonia lanosa the level of eicosapentaenoate-the most unsaturated fatty acid in this alga-drops significantly in the summer (Table XVI).

In both Ascophyllum nodosum and Polysiphonia lanosa, labelling from [I4C]acetate shows increased unsaturation of fatty acids at 5°C compared with 15°C. In particular, labelling of the long-chain saturated fatty acids 20 : 0 and 22 : 0 is lower at 5"C, indicating less elongation of saturated fatty acids at lower temperatures. In Chondrus crispus there is increased labelling of C I 4 + C l 6 fatty acids at 15°C and decreased labelling of C,, fatty acids, although a higher proportion of unsaturated fatty acids are labelled at 15°C (Table XVII).

Another seasonal alteration in lipid metabolism is the distribution of label from [14C]acetate amongst the neutral lipids from algae collected in summer compared with those collected in winter. F. vesiculosus from the Baltic Sea was reported to have reduced triacylglycerol levels in winter (Pohl and Zurheide, 19796) and labelling patterns in F. serratus also change in a similar way. F. serratus collected in summer accumulates [14C]acetate into triacylglycerol, whereas in the winter, label was incorporated into unesterified fatty acids. This may, therefore, be related to an accumulation of triacylglycerol as a storage product in the summer with subsequent breakdown in the winter.

In Chondrus crispus, increased labelling of SQDG and DGDG was seen at higher temperatures, and it has been suggested that a similar mechanism to that seen in wheat is in operation (Pettitt, T. R., Jones, A. L. and Harwood, J. L., unpublished). In wheat, modification of SQDG fatty acids occurs during temperature acclimation (Vigh et al., 1985). However, there is no

TABLE XVI Eflect of season on the fat ty acid composition of red and brown algae

Alga Fatty acid (% total fatty acid) Saturation index

16:O 16: l 1 8 : O 18.1 18:2 18:3 18:4 20:4 20:5 Others

Ascophyllum nodosum Feb. June

F. vesiculosus Feb. June

Chondrus crispus Feb. June

Polysiphonia Ianosa Feb. June

14 2 tr. 28 9 6 5 16 9 12 15 2 1 27 11 5 4 18 8 9

16 1 tr. 1 1 10 10 9 15 16 11 21 2 1 25 10 7 4 15 8 6

24 5 I 12 2 1 - 21 29 5 34 6 2 9 1 1 4 18 22 tr.

- 29 8 tr. 12 3 - 6 38 4 32 8 1 1 5 4 2 1 6 26 5

1.95 1.98

2.38 1.84

2.53 2.18

2.40 1.95

~

Data as means n = 2.3; tr. = <0.5%. Fatty acid abbreviations as for Table I. Jones, A. L. and Harwood, J. L. (unpublished)


TABLE XVII Effect of temperature on labelling of fatty acids from [14C]acetate

Alga Temperature Distribution of label (YO 14C-labelled fatty acids) (-C)

14:O 16:O 16:l 18:O 18: l 18:2 18:3 20:O 22:O Others

Ascophyllum 5 nodosum 15

F. vesiculosus 5 15

Chondrus 5 crispus 15

Polysiphonia 5 lanosa 15

2 14 - 1 1 59 10 - tr tr 4 4 2 4 1 1 0 3 7 8 t r 4 4 9

3 13 t r 9 75 tr tr - - tr 4 1 8 4 1 2 4 0 7 1 5 5 4

1 2 8 6 12 5 3 - - - - - - 1 10 37 5 11 34 - - - - 3

3 23 15 3 36 7 - 2 - 2 8 36 1 1 10 29 tr - 4 2 tr

Labelling for 24 h in the light: tr = <0.5%. Results from Jones, A. L. and Harwood. J. L. (unpublished).

indication in any alga so far studied of the alteration in ratio of PE to PC seen in frost-resistant rye (Clarkson et al., 1980).

There are several possible molecular mechanisms for altering membrane fluidity in plants (see Harwood, 1989), most of which may apply to marine algae. The details of these mechanisms are generally unclear, although there are suggestions, for example, that desaturation rates may be linked to oxygen concentrations which are higher at low temperatures (Rebeille et al., 1980). The response of marine algal acyl lipids to changes in temperature is particularly complex in view of the relatively large range of their polyunsaturated fatty acids.

3. Heavy Metals The heavy metal concentrations to which marine algae are exposed vary with the consumption of the underlying rock and with the amount of pollution. The effects of industrial pollution may raise levels of heavy metals in sea water many-fold, especially for transient periods. The marine algae cannot prevent the uptake of metals, and accumulate them, often to 100~10,000- fold the concentration in sea water (Bryan and Hummerstone, 1973; Bryan, 1980). The metals are probably sequestered in cell walls or physodes of brown algae (Lignell et a f . , 1982; Smith et al., 1986), thus reducing potential toxicity. However, heavy metal exposure has been found to affect lipid metabolism, and a selection of green, red and brown algae have been studied.

In E. intestinalis and Chondrus crispus, although Cu2 ' and Cd2 + are taken up by the algae, no effect on lipid metabolism was seen using either an acute or chronic incubation with the metals. The fatty acid labelling from


T A B L E XVIII EHect of Cu2+ on the ratio of counts found in the aqueous and organic phases of'a

Garbus extraction

Alga c u * + ( p g 1 - 1 ) Ratio d.p.m. organic/aqueous

F. serratus 0 30

300

2.6 + 0.6 4.4 f 1.7 4.0 f 0.2"

F. vesiculosus 0 3.5 f 1 . 1 1000 7.1 f 1.2"

"Significantly different ( P i 0 . l ) Results are means f S.D. (n = 3). The extraction method is described in Smith and Harwood (1984) and results are taken from

that reference and Jones, A. L. and Hanvood, J. L. (unpublished).

[14C]acetate was unaffected, as was the labelling of complex lipids (Jones and Harwood, unpublished).

The best-studied marine alga with regard to the effects of heavy metals on lipid metabolism is F. serratus. Cd2 +, Pb2 + and ZnZ + caused a decrease in the incorporation of [14C]acetate into complex lipids, but few changes in the distribution of label amongst lipid classes (Smith and Harwood, 1984b). In this study the most notable change was that seen in the ratio of radioactivity between the organic and aqueous phases of a high-saltxhloroform-mefha- nol-water (Garbus) extraction. Pb2 +, Cd2 +, Zn2+ and Cu2 + all increased the ratio of counts in the organic phase of the extraction, but this was most marked for Cu2+ (Smith et al., 1984, 1985). In vitro results for F. vesiculosus and F. serratus (Table XVIII) show this quite markedly.

Further work has shown that lipid metabolism in F. serratus is more susceptible to Cuz + than that of Ascophyllum nodosum or F. vesiculosus (Jones and Harwood, 1989, unpublished). However, the pattern of fatty acid labelling from [14C]acetate after incubation with Cu2+ in these brown algae is altered in a rather similar fashion (Smith et al., 1984; Jones and Harwood, 1988). Generally, labelled oleate levels are raised and palmitate levels lowered (Table XIX). Similar differences in endogenous fatty acids were seen in algae collected from sites heavily polluted with Cuz+ (Smith et al., 1985). If the pathways of fatty acid synthesis are similar to those of higher plants then it is possible that the activity of the acyl-ACP and/or acyl-CoA transacylases is being affected by Cu2+ and other metals. These enzymes transfer acyl chains from thioesters into complex lipids. Palmitoyl-ACP is the initial product of the fatty acid synthetase enzyme system, and the condensing enzyme which adds the C, unit to give 18 : 0 is specific for this reaction (Shimakata and Stumpf, 1982). The desaturation of both palmitoyl- and stearoyl-ACP is carried out by a A9-desaturase. Thus, inhibition of palmitoyl-ACP acyl transfer would allow a higher flux of radioactivity through to oleate, while at


TABLE XIX Effect ofCu2 + on the incorporation of [I4C]acetate into fatty acids

Alga Treatment YO Radiolabelled fatty acid

14:O 16:O 16:l 18:O 18:l 18:2Others

F. serratus Control 7.5 74.2 1.5 1.6 7.2 7.8 0.2 300pgCu2+ I - ' 4.3" 58.6" 3.1" 2.7" 15.6" 10.9" 4.8

( 1 hour)

A scophyllum Control 2.5 21.1 12.8 62.0 1.6 -

nodosum 30pgCu2+ I - ' tr.' 17.1 - 6.0b 71.6" 3.7 0.9 (8 days)

F. vesiculosus Control 4.0 28.7 - 15.6 28.0 1.6 22.0 120pgCu2+ I - ' 3.4 26.7 12.7 34.1b 2.3 20.8

"P< 0.0 I 'P < 0.05 Fatty acid abbreviations as for Table 1. Data for F. serratus is from Smith el a/. (1984) and for the other algae from Jones, A. L. and

Harwood. J . L. (unpublished).

the same time reducing palmitate labelling. At present, there is no experimental evidence to support such a hypothesis, but we are currently evaluat- ing this possible mechanism.

VIII. LIPID METABOLISM IN OTHER ALGAL TYPES

Although D. salina and the cyanobacteria are probably the best studied algae, work with some other algae, notably species of Chlorella, merits de- scription here.

Important work on the features of fatty acid desaturation was carried on C . vulgaris. The alga failed to desaturate added palmitate or stearate except when denied any carbon source. This was demonstrated by feeding [I4C]acetate under anaerobic conditions. Labelled palmitate and stearate accumulated but there was no desaturation under anaerobic conditions. However, on changing to aerobic conditions, [14C]stearate was converted rapidly to [ 4C]oleate, demonstrating the aerobic nature of the A9-desaturase (Harris et al., 1967). The substrate specificity of the desaturase system form- ing oleate was investigated with a series of radiolabelled acids from myristate (14 : 0) to nonadecanoate (19 : 0) (Howling et al., 1968). Each was desaturated to the corresponding A9-monoenoate, but, in addition, myristate, pen- tadecanoate and palmitate yielded the A7-monoenoates. These results were interpreted as possibly indicating the presence of a A7-desaturase in addition to the main A9-desaturase, and, in addition, to show that the enzymes held


Substrate Product

I I I I I

I I I

,I I I I

I I I

I I il (D) I

I I

DC

DC

D-C-H

D-C-H _____) II ( A )

i ( 6 ) H

HC

H-C-D ,

H-C-D -

HC

DC

H-C-D

D-C-H - I 1 (C)

DC

HC

D-C-H

H-C-D

Principle of experiment to demonstrate the reaction mechanism for stearate desaturation in Chlorella vulgaris. No isotope effect: twice as many monodeuterated products [(C) + (D)] as dideutey- ated (A). Isotope effect at one position: equal numbers of mono- and dideuterated products. Isotope effect at both positions: product (A) formed preferentially. See Morris (1970) for details.

Fig. 11 .

the substrate bound through the carboxyl group, probably as a thiol ester. The desaturation is highly stereospecific. Morris et al. (1967) incubated C . vulgaris with [erythro-9, 10-*H,]stearate, [threo-9,1 O-ZH,]stearate, [ ~ - 9 - 3H]stearate and [~-9-~H]stearate. Analysis of the oleate produced demonstrated that the desaturation involved the loss of the A9 hydrogen atom and of a pair of hydrogen atoms of cis relative configuration. Moreover, because there was an isotope effect at both positions, a stepwise reaction mechanism seemed unlikely and it was concluded that desaturation involves the simul- taneous concerted removal of hydrogens (Morris, 1970; Fig. 11).

Harris et al. (1965) showed that photosynthetically grown cells of C. vulgaris rapidly formed linoleate and a-linolenate by an aerobic process. Exo- genous [1-l4C]stearate was converted to [l-14C]oleate and then [ l - 14C]linoleate, showing the sequential nature of the desaturation pathway at the C,, level. Cell-free homogenates from such cells retained the ability to desaturate [l-14C]oleate in the presence of oxygen and NADH or NADPH (Harris and James, 1965). This activity was lost when the homogenate was separated into particulate and supernatant fractions. However, the particu-


late fraction regained activity if the substrate was added as [I-14C]oleoyl- CoA.

Labelling experiments with C. vulgaris showed that PC became very highly labelled with regard to linoleate (Harris et al., 1967). This led to the sugges- tion that PC could be involved in oleate desaturation. In addition, further experiments suggested that other complex lipids could be involved in the different fatty acyl desaturations (Nichols et al., 1967). These were the first suggestions of a mechanism for desaturation involving non-thioester substrates, now accepted for, at least, the major pathways of polyenoate formation (Harwood, 1988). Direct involvement of PC in oleate desaturation was shown for Chlorella particulate fractions by Gurr et al. (1969).

The chain length specificity of desaturases in C. vulgaris has been studied by Howling et al. (1968). They found that each of the A9-monoenates formed by the first desaturation could be desaturated further to form A9,12-die- noates (with the exception of A-tetradecenoate). Oleate was, however, the most active substrate. Further experiments were carried out with 8-hepta- decenoate, 9-octadecenoate and 1 0-nonadecenoate, which yielded, respectively, A8,11-18: 2, A9,12-18: 2 and A10,13-18: 2 (where 18 : 2 stands for octadecadienoate). If the monoenoates were incorporated into a complex lipid before desaturation, then the concept of a desaturase having a specificity relating to the methyl end of the acyl chain is logical. Thus, it was concluded that C. vulgaris might possess two desaturases which were responsible for dienoic fatty acid synthesis. The first was a A12 desaturase which accepted A9-monoenoates, while a second 6-desaturase used 9-monoenates. Oleate would be a substrate for both enzymes. The stereochemistry of oleate and linoleate desaturation was demonstrated to be identical to that for stearate desaturation (Morris et al., 1967), i.e. cis elimination of hydrogen from the 12,13 or 15,16 positions involving the vic-D atoms took place.

The presence of a very unusual fatty acid, trans-A3-hexadecenoate, exclusively located at the sn-2 position of leaf PG (Harwood, 1980a), was mentioned in Section 111. Similarly, C. vulgaris cells grown photosynthetically in the light accumulate the acid in this position exclusively. When organic nutri- ent is available, the acid is almost completely absent from cells, whether grown in the light or dark (Nichols, 1965). The biosynthesis of trans-A3- hexadecenoate was investigated, and palmitate established as the direct precursor (Nichols et al., 1965). Desaturation was reduced in the absence of exogenous oxygen, indicating either a substrate requirement for the reaction or the need for oxygen in the supply of enzymes or cofactors involved. When [14C]trans-A3-hexadecenoate was added to C. vulgaris cells it was acylated to all lipids, showing that the specific location of the acid in PG in vivo was not due to a specific acyl transferase. The experiments showed that the most likely substrate for the desaturase was another complex lipid (I-acyl,2- palmitoyl-phosphatidylglycerol), and this accounted for the specific location of the trans-A3-hexadecenoate product (Bartels et al., 1967). In addition, a


reductase system was found in Chlorella which could convert the trans- hexadecenoate to palmitate; such a system has been suggested to be involved in the function of trans-A3-hexadecenoate in thylakoid membranes (Harwood and James, 1975; Gounaris et al., 1986).

Several workers have carried out general metabolic studies with green algae. Frequently, 14C0, and [I4C]acetate have been used. These have the advantage that radioactivity can be incorporated into all fatty acids (acyl groups) and, hence, into all classes of acyl lipids. However, the former, in particular, has the disadvantage that other parts of the lipid molecule are also well labelled (e.g. glycerol, sugars), leading to problems in interpretation. For this reason, [I4C]acetate is often a preferable precursor (Hitchock and Nichols, 1971). Optimal conditions for the incorporation of label from [I4C]acetate with C. pyrenoidosa have been worked out (Yung and Mudd, 1966). Using the same alga and 14C0,, Ferrari and Benson (1961) had earlier established the high metabolic activity of PG and MGDG during steady- state photsynthesis. Similar studies using [I4C]acetate and C. vulgaris showed that the rate of labelling of acyl groups of individual lipids was in the order:

PG > MGDG > PC > SQDG > PI > DGDG > PI

This showed again, as with C. pyrenoidosa, that PG and MGDG were very rapidly labelled (Nichols et al., 1967). The rapid labelling of PG was also seen with [3ZP]glycerophosphate labelling (Sastry and Kates, 1965).

In a comparative study on lipid metabolism in C. vulgaris and two cyanobacteria, Nichols (1968) noted that the fatty acids of DGDG and SQDG were labelled much better in the blue-green algae. However, in all three organisms, MGDG was the best labelled of the glycosylglycerides.

An important additional result from the general studies of lipid labelling with C. vulgaris was the demonstration that major changes in the fatty acid composition of individual lipids could take place after their de novo synthesis (Nichols et al., 1967; Nichols, 1968; Nichols and Moorhouse, 1969; Gurr et al., 1969; Safford and Nichols, 1970). This in turn led to the idea of a dynamic movement of acyl moieties between lipids and, more especially, to the concept that complex lipids can act as substrates for desaturation. These ideas have now been extended to all types of plants and were discussed briefly in Section IV. Fuller reviews of these aspects have been made recently (Stumpf and Conn, 1987; Harwood, 1988,1989).

Chlamydomonas reinhardtii has been used as a useful experimental organism by a number of laboratories. Janero and Barrnett (1981a,b) reported on the detailed lipid composition of strain 137'. However, their results differed somewhat from the recently analysed arg-2 mt + strain (Eichenberger et al., 1986). The latter workers also noted the very similar lipid composition of a chlorophyll b-deficient mutant. Several workers have studied biogenesis of photosynthetic membranes. Beck and Levine (1977) used cultures which had


been synchronized by a 12-h light/l2-h dark regime. They then followed incorporation of radioactivity into lipids using 35SO:-, 32PO:- and H14CO; as precursors. They concluded that while lipid synthesis occurred predomin- antly during the light part of the cycle, different (chloroplast) lipids were synthesized at different times. For example, PG was synthesized between 3 h and 4 h in the light, but SQDG was labelled between 7 h and 9 h. Galactolipid synthesis appeared to reach maximal rates at two times-immediately after the lights were switched on and after 7 h. All the lipids (and chlorophyll) were made and inserted into chloroplast membranes before any major increases in photosynthetic capacity were seen. The sequential nature of lipid insertion during membrane biogenesis was notable.

These results were extended somewhat by Janero and Barrnett (1 982), who looked at cell-cycle variations in the synthesis of PE and PC in non- photosynthetic membranes. Peak incorporation of radioactivity from [14C]acetate was found around 7 h in the light. Thus, synthesis of thylakoid and non-thylakoid lipids in Chlamydomonas reinhardtii is confined essentially to the light period and occurs in mid-to-late GI.

De novo synthesis of acyl lipids relies on the successive acylation of glycerol 3-phosphate to form the key intermediate phosphatidate. The two acylations concerned have been studied in Chlamydomonas reinhardtii (Jelsema et al., 1982). Both enzymes were very much increased upon light induction, the lysophosphatidate acyltransferase particularly so. The peak activities of the enzymes preceded thylakoid membrane biogenesis during the cell cycle. The enzymes were localized by cytochemical techniques, and activity was concluded to be associated with the chloroplast envelope (and in the pyrenoid tubules of the chloroplast and the Golgi apparatus and associated vesicles (Michaels et al., 1983)). The results are in keeping with a role for acyltrans- ferases in chloroplast biogenesis as discussed for plants in general (Joyard and Douce, 1987; Hanvood, 1989).

Mention has already been made of the involvement of various complex lipids in fatty acid desaturation. As described in Section IV, PC is thought to be the major substrate for oleate‘desaturation in plants (Harwood, 1988), including C . vulgaris (Gurr et al., 1969). However, in Chlamydomonas reinhardtii, PC is either absent (Eichenberger et al., 1986) or is present in very small amounts in non-thylakoid membranes (Janero and Barrnett, 198 1 b). When labelling studies were made with [ 1 -14C]oleate, DGTS was very rapidly labelled (Schlapfer and Eichenberger, 1983). Within 3 h about 80% of the labelled oleate incorporated into DGTS was desaturated to linoleate and linolenate. At the same time, label present in the 16 : 0/18 : 1 molecular species moved into 16 : 0/18 : 2 and 16 : 0/18 : 3 species. These data implied that DGTS acted as a substrate for the desaturation of oleate and linoleate. As further evidence for this role, radiolabelled di- 18 : 1 -DGTS was used. The labelled lipid was di-([l-14C]oleoyl)-glyceryl-(N,”-tri-[3H]methyl)- homoserine. Appreciable quantities of the intact lipid were taken up by the


alga and, in part, its oleoyl moieties were desaturated as shown by the successive appearance of 18 : 1 / 18 : 2 and 18 : 1 / 18 : 3 molecular species.

Interestingly, in relation to the possible role of DGTS in fatty acid desaturation, this lipid does not appear to be confined to one subcellular com- partment. In Chlamydomonas, 15% (Mendiola-Morgenthaler er al., 1985) or 40% (Janero and Barrnett, 1982) of total DGTS is present in the thylakoids. In Dunaliella, 90% was located in the thylakoids (Norman and Thompson, 1985b). A careful subcellular fraction of Acetabularia mediterranea showed that the bulk of DGTS was found in microsomal fractions with a similar distribution to NADH<ytochrome c reductase (Eichenberger and Gerber, 1987). Almost all of the DGTS found in the chloroplast fraction could be accounted for by microsomal contamination.

Several examples of environmental effects on algal lipid metabolism have been described in Sections V, VI, and VII. When the green alga Neochloris oleoabundans was starved of nitrogen it accumulated 35-54% of cell dry weight as lipid, of which 80% was triacylglycerol (Tornabene er al., 1983). These results were extended to a whole series of algae, all of which, with the exception of D . salina, accumulated increased lipid under nitrogen deficiency. However, the nature of the accumulating lipid varied from organism to organism. In some algae (but not all) nitrogen deficiency led to less desaturation at the C,, level (Ben-Amotz and Tornabene, 1985). Salt stress seemed to have a similar effect.

Another environmental stress which has been shown to lead to lipid accumulation is silicon deficiency in diatoms (Coombs et al., 1967). These results were confirmed in metabolic experiments with three species (Roessler, 1987). In Cyclotella cryptica (which was studied in most detail) lipid accumulation occurred in two phases. First, there was an increase in the percentage of newly assimilated carbon which partitioned into lipids. Second, a redistri- bution of previously fixed carbon from other cellular materials into lipids occurred.

IX. CONCLUSIONS

It should be clear from the foregoing sections that our knowledge of lipids, and especially their metabolism in algae, is poor. Only a few organisms have been studied in any detail, and in most algae which have been examined unknown lipids still remain to be identified. Considering the tremendous variety of different algae, it will not be surprising to uncover many novel pathways for lipid synthesis in the future. Conceivably some of these metabolic differences may have important applications in other areas. Undoubt- edly, the biotechnological exploitation of algae will become more prevalent in the next decade. However, it seems likely to us that, as in the past, the most


significant discoveries will be made by scientists, inspired by a sense of curi- osity, looking “where the light shines brightest”.

ACKNOWLEDGEMENT

Work on algae in the authors’ laboratory has been supported financially by the SERC and the NERC, for which we are very grateful.

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