Structure and expression of fatty acid desaturases

Biochimica et Biophysica Acta 1394 (1998) 3^15

Review

Structure and expression of fatty acid desaturases Dmitry A. Los a , Norio Murata

b;

*

a

b

Institute of Plant Physiology, Moscow, Russia National Institute for Basic Biology, Okazaki, Japan

Received 17 February 1998; revised 30 June 1998; accepted 16 July 1998

Abstract Fatty acid desaturases are enzymes that introduce double bonds into fatty acyl chains. They are present in all groups of organisms, i.e., bacteria, fungi, plants and animals, and play a key role in the maintenance of the proper structure and functioning of biological membranes. The desaturases are characterized by the presence of three conserved histidine tracks which are presumed to compose the Fe-binding active centers of the enzymes. Recent findings on the structure and expression of different types of fatty acid desaturase in cyanobacteria, plants and animals are reviewed in this article. Roles of individual desaturases in temperature acclimation and principles of regulation of the desaturase genes are discussed. ß 1998 Elsevier Science B.V. All rights reserved. Keywords: Acclimation; Gene expression ; Gene regulation; Fatty acid desaturase; Lipid; Membrane

Contents 1.

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

4

2.

Acyl-CoA desaturases of animals, yeasts and fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5

3.

Acyl-lipid desaturases of cyanobacteria and higher plants . . . . . . . . . . . . . . . . . . . . . . . . .

5

4.

Acyl-ACP desaturases of higher plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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5.

Expression of genes for desaturases . . . . . . . . . . . . . . . . . . . . . . . 5.1. Expression of gene for acyl-CoA desaturases . . . . . . . . . . . . . 5.2. Expression of cyanobacterial genes for acyl-lipid desaturases . 5.3. Expression of genes for fatty acid desaturases in higher plants

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7 8 8 9

6.

Roles of desaturases in acclimation to low temperatures . . . . . . . . . . . . . . . . . . . . . . . . . .

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7.

Physical properties of the membrane lipids and regulation of the expression of genes for desaturases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Fax: +81 (564) 54-4866; E-mail: [email protected] 0005-2760 / 98 / $ ^ see front matter ß 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 5 - 2 7 6 0 ( 9 8 ) 0 0 0 9 1 - 5

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D.A. Los, N. Murata / Biochimica et Biophysica Acta 1394 (1998) 3^15 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Fatty acid desaturases are enzymes that convert a single bond between two carbon atoms (C^C) to a double bond (CNC) in a fatty acyl chain [1,2]. The resultant double bond is often referred to as an unsaturated bond, and the reactions catalyzed by these enzymes are known as desaturation reactions. These reactions require molecular oxygen and occur under aerobic conditions [2,3]. The distribution of fatty acid desaturases is almost universal. The enzyme has been found in all organisms examined, with the exception of some bacteria such as Escherichia coli. The unsaturation of fatty acids in glycerolipids is essential for the proper functioning of biological membranes. At physiological temperatures, polar glycerolipids that contain only saturated fatty acids cannot form the bilayer that is the fundamental structure of biological membranes [4]. The introduction of an appropriate number of unsaturated bonds into the fatty acids of membrane glycerolipids decreases the temperature for the transition from the gel (solid) to the liquid-crystalline phase and provides membranes with the necessary £uidity [5,6]. The £uidity of membranes is, in turn, important for the activation of certain membrane-bound enzymes [7,8]. Unsaturation in the cis con¢guration is more e¡ective than that in the trans con¢guration for changing the physical and biochemical characteristics of membrane lipids, and most unsaturated bonds in glycerolipids of biological membranes are in the cis con¢guration. Fatty acids are synthesized from acetate by dissociable fatty acid synthases in the chloroplasts of plants [1,9] and by complex fatty acid synthases in the cytoplasm of animal, yeast and fungal cells [10,11]. Both types of fatty acid synthase produce saturated fatty acids, which are converted to unsaturated fatty acids by the introduction of unsaturated bonds in reactions catalyzed by desaturases. Saturated fatty acids are synthesized by dissociable fatty acid synthases in prokaryotes such as cyanobacteria [12^14]. However, some bacteria, such as E. coli,

contain a unique fatty acid synthase that can introduce a double bond during the synthesis of fatty acids [15]. Living organisms, in particular poikilothermic organisms, respond to a downward shift in temperature by desaturating the fatty acids of their membrane lipids [16,17]. This acclimative response re£ects the ability to maintain the £uidity of biological membranes over a certain range of temperatures. The phenomenon has been designated homeoviscous acclimation or, alternatively, homeophasic acclimation [18,19]. The ability of cells to modulate the physical characteristics of their membrane lipids is determined mainly by the actions of fatty acid desaturases, which introduce double bonds into fatty acids [20,21]. There are three types of fatty acid desaturase: acyl-CoA, acyl-ACP, and acyl-lipid desaturases [21]. In plants and cyanobacteria, most desaturation reactions are catalyzed by acyl-lipid desaturases, which introduce unsaturated bonds into fatty acids that are in a lipid-bound form [14,21]. Acyl-ACP desaturases are present in the plastids of plant cells and introduce the ¢rst double bond into fatty acids that are bound to acyl carrier protein (ACP) [14,21,22]. Acyl-CoA desaturases are present in animal, yeast and fungal cells, and they introduce unsaturated bonds into fatty acids that are bound to coenzyme A (CoA) [23]. Although the desaturation of fatty acids is, in the end, an oxidation reaction, it requires two electrons in addition to one molecule of oxygen. Ferredoxin is the electron donor in the desaturation reactions catalyzed by acyl-ACP desaturases, by acyl-lipid desaturases of cyanobacteria, and by acyl-lipid desaturases in the plastids of plants [24,25]. By contrast, the acyl-lipid desaturases of plants, localized in the cytoplasm, and the acyl-CoA desaturases of animals and fungi, use cytochrome b5 as the electron donor [3,23,26]. Each fatty acid desaturase introduces an unsaturated bond at a speci¢c position in a fatty acyl chain, for example, at the v9, v12 or v6 position. Some acyl-lipid desaturases recognize speci¢c polar head groups, as well as the sn-position of the glycerol

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backbone to which the fatty acid is esteri¢ed [21]. However, most acyl-lipid desaturases are insensitive to head groups and to sn-positions. 2. Acyl-CoA desaturases of animals, yeasts and fungi Most acyl-CoA desaturases consist of 300^350 amino acid residues [27,28]. Acyl-CoA desaturases are hydrophobic proteins and modeling suggests that they each span the lipid bilayer of membranes four times [29,30]. They accept electrons from an electron-transport system that is composed of cytochrome b5 and NADH-dependent cytochrome b5 reductase [31^33]. The activities of v5, v6 and v9 acyl-CoA desaturases have been described [34^37]. However, the only genes that have been cloned, with characterization of the corresponding proteins, are genes for stearoylCoA desaturase. cDNAs and genes for stearoylCoA desaturases have been cloned from more than 10 species [27,28,36,39^43,45]. This group of desaturases has a unique structure, with three conserved motifs that contain histidine residues (Table 1). These histidine residues are considered to provide ligands to ferric iron at the catalytic center [30]. Two genes, encoding isoforms of stearoyl-CoA desaturase, have been found in both the mouse and the rat [46^48]. The isoforms are distributed in a tissue-

5

speci¢c manner and their expression can be manipulated by changes in the animals' diet [46,49,50]. The gene for stearoyl-CoA desaturase from Saccharomyces cerevisiae encodes a protein of 510 amino acid residues [40], which is an unusually large number for an acyl-CoA desaturase. The sequence of the carboxy-terminal portion of the protein is homologous to that of cytochrome b5 , and this portion functions as the electron donor for the desaturase in a mutant that is defective in cytochrome b5 [32]. 3. Acyl-lipid desaturases of cyanobacteria and higher plants Acyl-lipid desaturases introduce double bonds into fatty acids that are bound to the glycerol moiety of polar glycerolipids, and they are distributed in cyanobacteria and plants [21]. Most acyl-lipid desaturases consist of 300^350 amino acid residues, being hydrophobic proteins that appear to span membranes four times [21]. Such enzymes in cyanobacterial cells and chloroplasts use ferredoxin as the electron donor [25,51], whereas those in the cytoplasm of plant cells utilize a system composed of cytochrome b5 and NADH:cytochrome b5 oxidoreductase [69]. The genes for four speci¢c desaturases have been cloned from the cyanobacterium Synechocystis sp. PCC 6803. These desaturases introduce double

Table 1 Conservative histidine clusters in acyl-CoA and acyl-lipid desaturases Desaturase Organism

v9 Acyl-CoA Animal, yeast v9 Acyl-lipid Cyanobacteria Higher plants v12 Acyl-lipid Cyanobacteria Higher plants g3 Acyl-lipid Cyanobacteria Higher plants v6 Acyl-lipid Cyanobacteria Higher plants a

Histidine clustera

Reference

1st

2nd

3rd

HxxxxH

HxxHH

ExxHxxHH

[27,40^42,46,47]

HxxxxH HxxxxH

ExxxxHRxHH ExxxxHRxHH

EGWHNNHH EGWHNNHH

[52,53] [54,55]

HDCGH HxCGH

HxxxxxHxxHH ExxxxxHxxHH

HxxHH HxxHH

[56,57] [58^60]

HDCGH HDCGH

HxxxxxHRTHH HxxxxxHRTHH

HHxxxxHVAHH HHxxxxHVIHH

[53,61] [62^66]

HDxNH HDxGH

HxxxHH NxxxHH

QxxxHH QxxxHH

[67] [68]

Amino acids are shown in the single-letter code. The symbol x refers to any amino acid except histidine.

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bonds at the v6 [67], v9 [52,53], v12 [53,57] and g3 [61] positions of fatty acids. The speci¢c site of desaturation by these enzymes is de¢ned by reference to the carboxyl terminus (v-position) or the methyl terminus (g-position) of the fatty acid [70,71]. The order in which desaturases operate is very strictly determined (Fig. 1): the ¢rst double bond is introduced by the v9 desaturase into stearic acid; and the v12 and v6 desaturases introduce a double bond into fatty acids that have a double bond at the v9 position [21,70]. The g3 desaturase introduces a double bond into fatty acids that have a double bond at the v12 position [70]. All known desaturases are characterized by the presence of three histidine clusters, which are localized at strongly conserved positions in the amino acid sequence of each protein [21]. It has been suggested that these clusters might be involved in the formation of the active site of each desaturase, as has been demonstrated in other di-iron enzymes [29,30,72]. Table 1 shows details of the conserved histidine clusters in acyl-lipid desaturases. A comparison of the acyl-lipid desaturases of cyanobacteria with the corresponding desaturases of higher plants reveals the presence of similarly positioned histidine clusters in the enzymes from all types of organism [21,30]. It is of considerable interest that the histidine clusters in the v9 acyl-lipid desaturases from cyanobacteria are identical to those in v9 acyl-CoA desaturases from animal, yeast and fungal cells (Table 1). Moreover, the details of the histidine clusters are speci¢c to the type of desaturase, when the desaturases are classi¢ed with respect to the position in the acyl chain at which each introduces an unsaturated bond.

Fig. 1. The pathway of fatty acid desaturation by acyl-lipid desaturases in Synechocystis sp. PCC 6803. Fatty acids are represented by X:Y (Z) in which X and Y indicate, respectively, the number of X carbon atoms and the number Y double bonds in the cis con¢guration at the Z position counted from the carboxy terminus.

Fig. 2. The predicted positioning of the v12 acyl-lipid desaturase in the membrane : a computer prediction based on the localization of the conserved histidine clusters and iron atoms, which are assumed to constitute the catalytic center, at the cytoplasmic side.

Fig. 2 shows the predicted positioning of the v12 acyl-lipid desaturase in relation to the membrane. The enzyme spans the membrane four times and exposes the three histidine clusters to the cytoplasmic side. It is assumed that the histidine clusters and iron ions constitute the catalytic centre of the desaturase. Site-directed mutagenesis of the stearoyl-CoA desaturase of the rat [29,30] and of the v12 acyl-lipid desaturase of Synechocystis sp. PCC 6803 [73] revealed that substitution by another amino acid of any of the conserved histidine residues leads to the loss of enzymatic activity. Such loss of activity is probably due to the inability of each mutant enzyme to bind ferric iron at the necessary sites [73,74]. All four desaturases in the cells of Synechocystis sp. PCC 6803 have been localized by immunogold labeling [75] and Western blotting [76]. The acyl-lipid desaturases of Synechocystis are present in both thylakoid and plasma membranes, a ¢nding that indicates that the desaturation of fatty acids occurs both in cytoplasmic membranes and in thylakoid membranes in cyanobacterial cells [75]. These desaturases do not have any recognizable signaling sequences that might target the proteins to speci¢c cellular compartments. It is likely, therefore, that the acyllipid desaturases of cyanobacteria enter cell membranes as a result of certain features of their secondary structure. Homologs of a v9 acyl-lipid desaturase that is not localized in plastids have been cloned from rose petals [54], and from Arabidopsis [55]. The exact biochemical reaction catalyzed by this type of desaturase remains to be clari¢ed. However, the presence of a v9 acyl-lipid desaturase in plant cells suggests that

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the entire process of fatty acid desaturation in plant cells might occur with fatty acids exclusively in the lipid-bound form. It is possible that the acyl-ACP desaturase in plastids is not the only enzyme that can introduce the ¢rst double bond. The amino acid sequences of a desaturase and cytochrome b5 have been identi¢ed in the v6 acyl-lipid desaturase of borage [68] and in a putative desaturase of sun£ower [78]. The cytochrome region is located in the amino-terminal portion of the protein in each case. Some animals and yeast appear to have acyl-lipid desaturases. Previously, the activity of v12 desaturase have been assigned to microsomes in yeast [77]. This enzyme used lipid-bound oleic acid as a substrate, rather than oleoyl-CoA. Also, in a protozoan Acanthamoeba, the substrate for microsomal v12 desaturase was oleoyl phosphatidylcholine [38]. Recently, an g3 desaturase was cloned from Caenorhabditis elegans [44]. It is likely that this fatty acid desaturase from C. elegans may be an acyl-lipid desaturase. 4. Acyl-ACP desaturases of higher plants According to a currently accepted scheme, the desaturation of stearic acid to yield oleic acid in the ACP-bound form is catalyzed by stearoyl-ACP desaturase (v9 acyl-ACP desaturase) in the stroma of chloroplasts (or plastids) [2,79]. The oleic acid produced is transported to the thylakoid membrane or into the cytoplasm for further desaturation in the lipid-bound form [80,81]. The activity of a soluble v6 acyl-ACP desaturase was detected in the endosperm of the seeds of Thunbergia alata [82,83]. Cloning of the corresponding gene revealed that the product of the gene was homologous to other acyl-ACP desaturases, such as the v9 acyl-ACP desaturases (18:0) of Spinacia oleracea, Arabidopsis thaliana and Oryza sativum and the v4 desaturase (16:0) of Coriandrum sativum. These observations imply that the ¢rst double bond in the fatty acids might be generated at the v4 or v6 position, as well as at the v9 position. It has been suggested that each acyl-ACP desaturase binds two atoms of iron to form a reactive complex with oxygen (Fe-O-Fe) [30,72]. This complex

7

converts the C^C single bond to a CNC double bond in the carbon chain of the fatty acid substrate. Crystallographic analysis of stearoyl-ACP desaturase from castor seeds [84] demonstrated that this desaturase forms a di-iron active center. The two iron atoms are bound in a highly symmetric environment, with one of them interacting with the side chains of E196 and H232 and the other interacting with the side chains of E105 and H146 [84]. In the crystal structure, there is a deep channel that extends from the surface into the interior of the enzyme. This channel might be the site to which the fatty acyl chain binds. The replacement of ¢ve amino acid residues in a plant v6 palmitoyl-ACP desaturase converted this enzyme into an enzyme functioned as a v9 stearoyl-ACP desaturase [85]. In addition, a v9 stearoylACP desaturase was converted to a v9 palmitoylACP desaturase by replacement of two amino acid residues at the putative active site [85]. The crystallographic analysis of the stearoyl-ACP desaturase [84] and the speci¢city of engineered desaturases to substrates [85] suggest that the recognition of the fatty acid chain length (C18 or C16) is determined mainly by properties of the amino acid residues that are located at the bottom of the cavity which accommodates the acyl-ACP substrate. The substitution of alanine-188 and tryptophan-189 for glycine and phenylalanine, respectively, in the v6 -16:0-ACP desaturase enlarges the cavity, and the resultant enzyme can accommodate two additional methyl groups in the cavity. Thus, it can desaturate both C16 and C18 fatty acids with the same e¤ciency [85]. These ¢ndings provide clues to the mechanisms of desaturation, as well as to the mechanisms whereby speci¢c sites of desaturation are determined. 5. Expression of genes for desaturases The expression of genes for desaturase is very important since it provides the molecular basis for the acclimation of organisms to changing environmental temperatures, in particular in the case of plants, cyanobacteria, yeasts and poikilothermic animals [21,86]. Cloning of genes for desaturases from a variety of organisms over the past 10 years has allowed

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an examination of the expression of these genes and the roles of their protein products in acclimation. 5.1. Expression of gene for acyl-CoA desaturases The genes for stearoyl-CoA desaturases of yeast and the rat provide useful models with which the expression of genes for this type of desaturase can be studied [87,88]. Addition of saturated fatty acids to the growth medium of yeast stimulated the activity of this desaturase [89]. Conversely, exogenous unsaturated fatty acids reduced the activity of stearoylCoA desaturase [90]. Such alterations in the activity of stearoyl-CoA desaturase were related to regulation of the expression of the corresponding gene [91]. An unsaturated fatty acid (20:4) had a similar inhibitory e¡ect on the expression of the gene for stearoyl-CoA desaturase in rat lymphocytes [47,92]. Thus, feed-back regulation by the substrate appears to be the basis for regulation of expression of the gene for the desaturase in animal cells. The expression of the gene for stearoyl-CoA desaturase in carp was upregulated upon a downward shift in temperature, as well as by feeding with a diet that did not contain unsaturated fatty acids [37,93]. In carp liver, the level of the mRNA for the v9 acyl-CoA desaturase increased 10-fold during cold acclimation. However, increases in the activity of the desaturase depended on posttranslational regulation [37,42]. The activities of the v9 and v6 desaturases in animals appear to be controlled by hormones [94]. For example, a tight correlation has been found between the expression of genes for desaturases and changes in levels of insulin [95,96]. 5.2. Expression of cyanobacterial genes for acyl-lipid desaturases The fatty acids of membrane glycerolipids in cyanobacterial cells are desaturated after a decrease in the ambient temperature from high to low, for example, from 35³C to 25³C [21,86]. Some models have been proposed to explain the low-temperature-induced desaturation of the fatty acids of membrane lipids. In one model, low temperature activates desaturases in membranes [97,98]. In another model, the accelerated desaturation at low temperatures is explained by the reduced synthesis of saturated fatty

Fig. 3. Temperature-dependent changes in mRNA levels of four genes for desaturases in Synechocystis sp. PCC 6803. Open bars represent the mRNA levels in cells which were grown at 34³C; solid bars represent the mRNA levels in cells which were grown at 34³C and then incubated at 22³C for 1 h. The mRNA level of the desC gene in cells which were grown at 34³C was taken as 100%. The desC, desA, desD and desB genes encode the v9, v12, v6 and g3 desaturases, respectively.

acids, while the activity of desaturases is a¡ected to a lesser extent with a resultant shift in the balance between saturated and unsaturated fatty acids [99]. In yet another model, desaturases are synthesized de novo at low temperatures. This model is based on the ¢nding that low-temperature-induced desaturation is inhibited by rifampicin and chloramphenicol, inhibitors of transcription and translation, respectively, in prokaryotes [100]. The expression of genes for the v6, v12 and g3 desaturases is upregulated upon a downward shift in temperature (Fig. 3) [101,102]. A study with a reporter construct, which consisted of the promoter of the gene for the v12 or g3 desaturase fused to a reporter gene for luciferase, con¢rmed that the promoter of the desaturase gene responded to a downward shift in temperature by regulating transcription. Moreover, the stability of the transcripts of the genes for desaturases increased at low temperatures [101^103]. In a recent study of the v12 desaturase, which was overexpressed in E. coli, we found that the dependence on temperature of the activity of this enzyme was the same as that of other enzymes: the higher the temperature, the higher the activity of the enzyme, over a range of temperatures at which the enzyme was not denatured [104]. These ¢ndings demonstrate clearly that the low-temperature-induced synthesis of polyunsaturated fatty acids is deter-

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mined by the upregulation of the expression of the genes for desaturases. 5.3. Expression of genes for fatty acid desaturases in higher plants The fad2 gene of Arabidopsis thaliana, which encodes the microsomal v12 desaturase, does not respond to a shift in temperature, at least, at the level of its transcript [60]. By contrast to A. thaliana, soybean has two fad2 genes [105]. One of the genes is expressed in developing seeds exclusively, whereas the other gene is expressed constitutively in both vegetative and reproductive tissues. The former gene might be responsible for the synthesis of polyunsaturated fatty acids in storage lipids [105]. The promoter of the fad7 gene of A. thaliana, which encodes one of the two g3 desaturases of chloroplasts, includes sequences that are homologous to the cis elements of certain light-regulated genes, namely, the box II and the G-box [64,106]. The pro-

9

moter is activated by light. By contrast, while low temperatures do not a¡ect the level of the transcript of the fad7 gene [64], the promoter of the fad7 gene can be activated by wounding in a tissue-speci¢c manner [107]. The inhibitors of the octadecanoid pathway strongly suppress the wound-induced activation of the promoter of the fad7 gene in roots but not in leaves or stems. In intact plants, exogenous methyl jasmonate activates the promoter of the fad7 gene in roots, whereas it inactivates this promoter in leaves. It seems reasonable to suggest that the wound-responsive expression of the fad7 gene in roots might be mediated via the octadecanoid pathway. In leaves, by contrast, jasmonate-independent wound signals might induce activation of the fad7 gene [107]. In parsley (Petroselinum crispum), the transcription of the gene for the plastid-speci¢c g3 desaturase (a homolog of the fad7 gene) is strongly and rapidly induced by fungal infection or by the peptide elicitor Pep25 [108]. The addition of Pep25 to parsley cells

Fig. 4. The molecular species composition of glycerolipids in cells grown at 25³C and the temperature dependence of growth of the wild-type (A) and desA3 /desD3 mutant (B) cells of Synechocystis sp. PCC 6803. In the growth pro¢le, cells which had been grown at 35³C were incubated at 30³C (a), 25³C (O), or 20³C (P). Numbers in the circular graphs represent numbers of double bonds in the individual molecular species of lipids. The data are reproduced from Tasaka et al. [113].

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also induces a rapid and massive increase in the production of jasmonate [109,110]. It is possible that the end product of the reaction catalyzed by the g3 desaturase, K-linolenic acid, might serve as an important precursor to speci¢c cellular signal molecules, such as jasmonate [108,111,112]. The gene for stearoyl-ACP desaturase is controlled in a temporal and a tissue-speci¢c manner. The promoter of this gene is most active in developing tissues, such as young leaves and seedlings, and its activation is induced by abscisic acid [79]. 6. Roles of desaturases in acclimation to low temperatures The importance of the polyunsaturated fatty acids of membrane lipids and fatty acid desaturases in the acclimation of various organisms to changes in environmental temperatures has been well reviewed [21,23,86]. The roles of individual desaturases in regulation of the levels of unsaturation of membrane lipids during acclimation to low temperatures can be studied by genetic manipulation of the genes for desaturases in cyanobacteria. A series of mutants of Synechocystis sp. PCC 6803, which were defective in the unsaturation of membrane lipids in a stepwise manner, was generated by targeted mutagenesis of individual desaturases [113]. Using another approach we transformed Synechococcus sp. PCC 7942 with genes for foreign desaturases in order to produce strains that synthesized abnormal polyunsaturated fatty acids in their membrane lipids [57,61,114]. These approaches allowed us to study the roles of the individual desaturases and polyunsaturated fatty acids in the acclimation of these microorganisms to various ambient temperatures. Attempts at the inactivation of the gene for the v9 desaturase in Synechocystis sp. PCC 6803 by targeted mutagenesis have failed [113], and such mutations are probably lethal to the cells. Thus, the presence at high levels of unsaturated fatty acids in membrane lipids appears to be essential for the organism's survival [113], as predicted by physico-chemical studies of model membranes. These studies have demonstrated that v9 unsaturation (introduction of the ¢rst double bond in the fatty acids of membrane lipids) is essential for conversion of the physical state of a

membrane from the gel state to the liquid-crystalline state at physiological temperatures [90,91]. Subsequently introduced unsaturated bonds have progressively smaller e¡ects than the ¢rst double bond [4,18]. Targeted mutagenesis of the gene for the v12 desaturase resulted in dramatic changes in the fatty acid composition of Synechocystis sp. PCC 6803 [21,113] and Synechococcus sp. PCC 7002 [115], with a considerable increase in the level of oleic acid at the expense of the polyunsaturated fatty acids in the membrane lipids (Fig. 4). The resultant strains were characterized by strong sensitivity to low temperatures. The desA3 /desD3 strain of Synechocystis sp. PCC 6803, in which the genes for v12 and v6 desaturases had been inactivated, contained no polyunsaturated fatty acids. This strain grew as well as the wild-type strain at 35³C, but there was severe retardation of growth at 25³C, and cells were unable to propagate at 20³C (Fig. 4) [113]. Moreover, the cells were unable to recover from the photo-induced damage to the photosystem II complex at low temperatures as a consequence of their inability to process the precursor to the D1 protein of the photosystem II complex [116]. Since only genes for fatty acid desaturases were disrupted in the desA3 /desD3 strain, we can conclude that the ability of Synechocystis to tolerate low temperatures is determined by its ability to synthesize polyunsaturated fatty acids. This conclusion is in agreement with the results of another set of experiments that involved transformation of Synechococcus sp. PCC 7942, which contains saturated and monounsaturated but no polyunsaturated fatty acids, with the gene for the v12 desaturase from Synechocystis sp. PCC 6803. The transformed cells synthesize diunsaturated fatty acids at the expense of monounsaturated fatty acids, and they were able to tolerate lower temperatures than the wild-type cells [57]. Inactivation of the g3 desaturase in Synechocystis sp. PCC 6803 by targeted mutagenesis had no marked e¡ect on the ability of the cells to tolerate low temperatures [113]. However, it was recently demonstrated that, under conditions of nutrient limitation, both growth and survival of Synechococcus sp. PCC 7002 at a low temperature, such as 15³C, are repressed when the g3 desaturase has been inactivated [117]. The mutant cells failed to grow at

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11

15³C when nitrate was supplied to the medium as the nitrogen source, but replacement of nitrate by urea allowed the propagation of the cells at 15³C [117]. In higher plants, the ability to survive at low temperatures is also correlated with the presence of polyunsaturated fatty acids. The fad5 and fad6 mutants of A. thaliana have defects in the sn-2-palmitoyl-desaturase and the v12 desaturase, respectively. Both of these enzymes are localized in chloroplasts and the mutant plants are characterized by leaf chlorosis, growth retardation, and changes in the chloroplast morphology at low temperatures [118]. Another mutant of A. thaliana, the fad2 mutant, which has a defect in the microsomal v12 desaturase, exhibits decreased stem elongation at 16³C, and the plants die at 6³C [119,120].

a decrease in temperature was demonstrated by the catalytic hydrogenation of fatty acids of plasma membrane lipids in Synechocystis sp. PCC 6803. This chemical intervention caused a decrease in membrane £uidity and enhanced the transcription of the desA gene [121]. The e¡ects of catalytic hydrogenation and of low temperature, both of which results in a decrease in membrane £uidity, were additive in terms of the induction of expression of the desA gene [101,121,122]. This observation leads to the hypothesis that, at least in cyanobacterial cells, a novel type of regulatory control exists that is based on a feedback link between the physical state of the membrane and the level of expression of the genes for desaturases.

7. Physical properties of the membrane lipids and regulation of the expression of genes for desaturases

This work was supported, in part, by a grant from the Russian Foundation for Basic Research (97-0450200) to D.A.L. and by a Grant-in-Aid for Specially Promoted Research (08102011) from the Ministry of Education, Science and Culture, Japan, to N.M.

Two questions must be answered with respect to the molecular mechanism of the regulation of expression of genes for desaturases in response to changes in temperature: how does an organism sense a change in temperature and how is this signal transmitted to the regulatory regions of genes for desaturases to induce their activation at low temperatures? A scheme for the mechanism of acclimation of cyanobacterial cells to low temperatures was proposed by Murata and Wada [21]. Upon a downward shift in temperature, the £uidity of the membrane decreases. This signal is detected by an as yet unidenti¢ed sensor of low temperature and is then transmitted to regulatory molecules that directly or indirectly interact with the regulatory regions of the genes for desaturases, leading to their activation. As a result, the level of desaturases increases and fatty acids are desaturated. Finally, the accelerated accumulation of unsaturated fatty acids leads to the recovery of membrane £uidity and to restoration of the activity of membrane-associated enzymes [21,86]. In this model, membranes play a key role in the perception and transduction of the temperature signal to the regulatory regions of the genes for the desaturases. The role of the membranes in sensing

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