The EMBO Journal vol.15 no.23 pp.6416-6425, 1996

Targeted mutagenesis of acyl-lipid desaturases inSynechocystis: evidence for the important roles ofpolyunsaturated membrane lipids in growth,respiration and photosynthesis

Yasushi Tasakal, Zoltan Gombos1l2,Yoshitaka Nishiyamal, Prasanna Mohantyl,Tetsuhiko Ohba3, Kazuo Ohki3 andNorio Muratal'4'Department of Regulation Biology, National Institute for BasicBiology, Myodaiji, Okazaki 444, Japan, 2Institute of Plant Biology,Biological Research Center of the Hungarian Academy of Sciences,PO Box 521, H-6701 Szeged, Hungary and 3Department of Physics,Graduate School of Science, Tohoku University, Aoba-ku,Sendai 980-77, Japan

4Corresponding author

Acyl-lipid desaturases introduce double bonds (un-saturated bonds) at specifically defined positions infatty acids that are esterified to the glycerol backboneof membrane glycerolipids. The desA, desB and desDgenes of Synechocystis sp. PCC 6803 encode acyl-lipiddesaturases that introduce double bonds at the A12,o3 and A6 positions of C18 fatty acids respectively. Themutation of each of these genes by insertion of anantibiotic resistance gene cartridge completely elimin-ated the corresponding desaturation reaction. Thissystem allowed us to manipulate the number of un-saturated bonds in membrane glycerolipids in thisorganism in a step-wise manner. Comparisons of thevariously mutated cells revealed that the replacementof all polyunsaturated fatty acids by a monounsatur-ated fatty acid suppressed growth of the cells at lowtemperature and, moreover, it decreased the toleranceof the cells to photoinhibition of photosynthesis at lowtemperature by suppressing recovery of the photosys-tem II protein complex from photoinhibitory damage.However, the replacement of tri- and tetraunsaturatedfatty acids by a diunsaturated fatty acid did not havesuch effects. These findings indicate that polyunsatur-ated fatty acids are important in protecting the photo-synthetic machinery from photoinhibition at lowtemperatures.Keywords: acyl-lipid desaturase/membrane glycerolipid/photoinhibition/Synechocystis sp. PCC 6803

IntroductionFatty acid desaturases introduce double bonds (unsaturatedbonds) at specifically defined positions in the alkyl chainof fatty acids (Murata and Wada, 1995) and they catalyzea series of desaturation reactions to ensure appropriatelevels of unsaturation of acyl-glycerolipids (Cossins,1994). Acyl-lipid desaturases, which are a subset of fattyacid desaturases, act on fatty acids that are joined byan ester bond to the glycerol backbone of membraneglycerolipids (Murata and Wada, 1995). It is generallyaccepted that poikilothermic organisms modulate the levels

of unsaturation of their membrane glycerolipids withchanges in ambient temperature (Cossins, 1994). Sinceacyl-lipid desaturases introduce double bonds directly intothe fatty acids of membrane lipids, they are the mostefficient regulators of the extent of unsaturation of mem-brane lipids in response to changes in ambient temperature.

Glycerolipids form bilayers and provide the structuralbackbone for the function of membrane-bound proteins(Doyle and Yu, 1985), the expression of some genes (Loset al., 1993; Vigh et al., 1993) and the insertion of proteinsinto membranes, as well as their translocation acrossmembranes (Van't Hof et al., 1994). The physical andbiochemical properties of membrane glycerolipids dependon the degree of unsaturation of the fatty acids of themembrane lipids (Quinn, 1988). In cellular physiology,the unsaturation of membrane glycerolipids plays a keyrole in the tolerance of living organisms to low temperaturestress (Murata, 1989; Wada et al., 1990; Murata et al.,1992a).The cyanobacterium Synechocystis sp. PCC 6803 is a

useful organism with which the effects of the geneticmanipulation of levels of unsaturation of membrane lipidsand the physiological importance of the unsaturationof membrane lipids can be effectively studied, sincecyanobacterial cells contain four acyl-lipid desaturaseswhich introduce double bonds specifically at the A6, A9,A12 and w3 positions of C18 fatty acids (Wada and Murata,1990). Moreover, all of their genes are present as singlecopies and the cells of this cyanobacterium are easilytransformed by homologous recombination (Williams,1988). We cloned the desA, desB and desC genes for theA12, c3 and A9 acyl-lipid desaturases respectively fromthis strain (Wada et al., 1990; Sakamoto et al., 1994a,b)and Reddy et al. (1993) cloned the desD gene for the A6acyl-lipid desaturase from the same strain.

In a previous study we mutagenized Synechocystis sp.PCC 6803 by treatment with ethyl methanesulfonate andisolated the Fad6 mutant, which is defective in thedesaturation of fatty acids at the A6 position when cellsare grown at high temperatures (Wada and Murata, 1989).We further mutagenized the Fad6 mutant by inserting akanamycin resistance gene cartridge into the desA gene(Wada et al., 1992). The resultant Fad6/desA- mutant cellswere defective in desaturation at the A6 and A12 positionsof fatty acids of membrane glycerolipids when they weregrown at high temperature, such as 34°C. However, in achemically induced mutation in the Fad6 and Fad6/desA-cells, the A6 desaturase was active at low temperatures,such as 25°C. Therefore, we attempted to produce strainsthat completely lacked activity of the A6 desaturase,regardless of the growth temperature.

In this study, we mutated the desA, desB and desDgenes of Synechocystis sp. PCC 6803 by targeted muta-genesis. These genes were mutated by insertion of anti-

6416 K Oxford University Press

Targeted mutagenesis of acyl-lipid desaturases

A pdesA::(Kmr+Bler)Kmr 795 bp Bier 381 bp

|H/ndMm ,

desA 1056 bp

B pdesD::Cmr Cmr 660 bp

desD 1080 bp

C pdesB::KmrKmr 795 bp

desB 1080 bp

D pdesC::KmrKmr 813 bp

EcoR

desC 957 bp

500 bp

Fig. 1. Details of construction of plasmids used for the disruptionalmutation of Svnechocvstis sp. PCC 6803. (A) pdesA::(Kmr+Bler) inpBluescript II KS(+). (B) pdesD::Cmr from pCRII. (C) pdesB::Kmr inpBluescript II KS(+). (D) pdesC::Kmr in pBluescript II KS(+). Smallarrows indicate the primers used for PCR for evaluation of the state ofthe genes for the various desaturases.

biotic resistance cartridges. In the resultant strains, thecorresponding desaturation reactions were completelyeliminated at both high and low temperatures. As partof our efforts to understand the physiological roles ofunsaturation of membrane glycerolipids, we studied theeffects of replacement of polyunsaturated fatty acids witha monounsaturated fatty acid in membrane glycerolipidson the physical phase of thylakoid membranes, growth,photosynthetic activity and tolerance to low temperaturephotoinhibition of the cells.

ResultsTargeted mutagenesis of acyl-lipid desaturases inSynechocystis sp. PCC 6803The desA gene was mutated by transforming wild-typecells of SYnechocystis sp. PCC 6803 by homologousrecombination with pdesA::(Kmr+Bler) (Figure lA) and

the resultant mutant strain was designated desA-, asdescribed previously (Wada et al., 1990).

The desB and desD genes were also mutated by trans-forming wild-type cells with pdesB::Kmr and pdesD::Cmr(Figure lB and C) and the resultant mutant strains weredesignated desB7 and desD- respectively. To obtain adoubly mutated strain, desA-IdesD-, the desA- cells (Wadaet al., 1990) were transformed with pdesD::Cmr in thesame way. To obtain another doubly mutated strain, desBldesD-, the desD- mutant cells were transformed withpdesB::Kmr. We attempted to mutate the desC gene inwild-type cells with pdesC::Kmr (Figure ID) but we wereunable to obtain a mutant strain that completely lacked afunctional desC gene.

Since about six to eight copies of the chromosome arepresent in each cell of the cyanobacterium (Williams,1988), production of mutants requires mutation of thegene on all copies of the chromosome. The completereplacement of native genes by mutated genes was con-firmed by PCR (Sakamoto et al., 1994a). The primers forPCR are indicated by arrows in Figure 1. The resultindicated that on every copy of the chromosome inthe mutants the native genes had been replaced by theinsertionally mutated genes.

Changes in fatty acid and lipid class compositionsof membrane lipidsTable I shows the fatty acid composition of total glycero-lipids from the wild-type and mutant cells of Synechocystissp. PCC 6803 after growth at 25°C. The most abundantfatty acid was saturated 16:0, which is mostly joined byan ester bond to the snz-2 position of the glycerol backbone(Murata et al., 1992b). The level of this fatty acid wasunaffected by mutations in the acyl-lipid desaturases,whereas the unsaturation of C18 fatty acids was directlyaffected by the mutations. The wild-type cells contained18:3(6,9,12), 18:3(9,12,15) and 18:4(6,9,12,15). The desD-cells did not contain any fatty acids with a double bondat the A6 position, namely 18:3(6,9,12), 18:4(6,9,12,15)and 18:2(6,9), but they did contain 18:2(9,12) and18:3(9,12,15) at levels that were twice those in the wild-type cells. Inactivation of the desB gene by insertionalmutagenesis resulted in complete elimination of18:3(9,12,15) and 18:4(6,9,12,15). The desB/desD- cellscontained 18:1(9) and 18:2(9,12), but no 18:2(6,9),18:3(6,9,12), 18:3(9,12,15) or 18:4(6,9,12,15). The desAldesD- cells had completely lost all fatty acids with doublebonds at the A6 and A 12 positions. Consequently, thesecells did not contain any polyunsaturated fatty acids and18:1(9) was a major component of the monounsaturatedfatty acids. These results indicate that each insertionalmutagenesis completely eliminated the correspondingdesaturation reaction.The Fad6 mutant cells, which had been produced

by chemical mutagenesis with ethyl methanesulfonate,contained low levels of 18:3(6,9,12) and 18:4(6,9,12,15)and Fad6ldesA- mutant cells also contained a low levelof 18:2(6,9) (Table I). These results suggest that the Fad6mutation did not completely block A6 desaturation at 25°C,in clear contrast to the previous finding that desaturation atthe A6 position was eliminated in Fad6 cells when theywere grown at 34°C (Wada et al., 1992).We examined the effect of targeted mutagenesis on lipid

6417

Y.Tasaka et al

Table I. Fatty acid composition of the total glycerolipids from wild-type and mutant cells of Synechocystis sp. PCC 6803

Strain Fatty acid (mole %)

16:0 16:1 18:0 18:1 18:1 18:2 18:2 18:3 18:3 18:4(9) (9) (11) (6,9) (9,12) (9,12,15) (6,9,12) (6,9,12,15)

Wild-type 53 4 1 9 tr tr 10 5 14 4desA- 55 4 1 28 tr 12 0 0 0 0desB- 51 5 2 6 tr tr 13 0 22 0desD- 52 6 1 8 tr 0 23 11 0 0desBjdesD- 53 6 1 12 tr 0 29 0 0 0desA-ldesD- 51 7 1 41 tr 0 0 0 0 0Fad6 54 4 1 8 tr tr 8 11 2 2Fad6/desA- 53 5 1 36 2 3 0 0 0 0

Cells were grown at 25°C.tr, trace amount (<0.5%). Experiments were repeated twice and deviations were within ±3% of individual values. Numbers in parentheses indicateA positions of double bonds in the cis configuration.

class composition. In the wild-type cells, monogalactosyldiacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyldiacylglycerol and phosphatidylglycerol contributed 59 ±2, 15 ± 3, 16 ± 3 and 10 ± 2% of the total glycerolipidsrespectively. The lipid class compositions of the desD-,desB7/desD- and desA-/desD- cells were the same asin wild-type cells. This result indicates that targetedmutagenesis in the genes for desaturases did not affectthe lipid class composition.

Changes in proteins in thylakoid membranesWe examined the effects of targeted mutagenesis of the A6and A12 desaturases and of replacement of polyunsaturatedfatty acids by a monounsaturated fatty acid, namely18:1(9), in membrane glycerolipids on the composition ofmembrane proteins and the ratio of protein to lipid inthylakoid membranes. SDS-PAGE of thylakoid mem-branes from wild-type and desA-/desD- cells revealed thatthe polypeptide patterns were not significantly changedby targeted mutagenesis. Only one difference was foundin a minor peptide of ~15 kDa, which was present inwild-type cells but was absent from the desA-/desD- cells.However, we did not identify this peptide.We determined the ratios of protein to lipid in isolated

thylakoid membranes. Average ratios from three independ-ent experiments were 2.3 ± 0.2 and 2.0 ± 0.2 forthe membranes from wild-type and desA-IdesD- cellsrespectively. These results indicate that neither targetedmutagenesis of desaturases nor replacement of polyun-saturated fatty acids by a monounsaturated fatty acidin the membrane lipids had any effect on the proteincomposition and the ratio of protein to lipid in thylakoidmembranes.

The phase behavior of membrane lipidsEffects of unsaturation of fatty acids on the thermotropicbehavior of thylakoid membranes from wild-type andmutant cells that had been grown at 25°C were examinedby differential scanning calorimetry (DSC). In the lowtemperature range (0-300C), one major endothermic peakwas observed reproducibly, whereas several major endo-thermic peaks appeared in the high temperature range(60-90°C) in the first temperature scan (data not shown).The thermograms of the second temperature scan ofthylakoid membranes isolated from wild-type, desD-,desB-/desD- and desA-/desD- cells are shown in Figure

12°C dosB-1desD-8

desAB/desD-

5 J K-1 (g Chlorophyll)-1

0 1 0 20 30 40 50 60 70

Temperature (°C)

Fig. 2. Differential scanning calorimetry of thylakoid membranesisolated from wild-type, desD-, desB/desD- and desA-ldesD- cellsgrown at 25°C. Arrowheads indicate temperatures of phase transition.

2. The endothermic peaks in the high temperature rangewere absent from the second scan, a result that suggeststhat these peaks might have originated from irreversibledenaturation of proteins. The reversible endothermic peaksin the low temperature range corresponded to phasetransition of membrane lipids. The temperatures for theonset of phase transition were 14, 13, 12 and 21°C forthylakoid membranes isolated respectively from wild-type,desD-, desB/desD- and desA-IdesD- cells that had beengrown at 25°C. These results indicate that the mostsignificant difference in the temperature for onset of phasetransition was that between the membranes from the desB:/desD- cells, which contained 18:1(9) and 18:2(9,12) as

the major C18 acids, and the membranes from the desA/desD- cells, which contained 18:1(9) as the major C18acid. Replacement of diunsaturated fatty acid by a mono-

unsaturated fatty acid in membrane lipids increased thetemperature for phase transition of the lipids in thethylakoid membranes, while the replacement of tri- andtetraunsaturated fatty acids, 18:3(6,9,12) and 18:4(6,9,

6418

14°C_~ V

_>,_~~~~Wl tp

Targeted mutagenesis of acyl-lipid desaturases

B

40 120

Duration of incubation (h)

Fig. 3. Growth of wild-type and mutant cells at various temperatures.Cells were cultivated at 35°C and then cultures were transferred to theindicated temperatures. (A) Wild-type cells; (B) desD- cells;(C) desB-desD- cells; (D) desA-ldesD- cells. Ol, 35°C; 0, 30°C;A, 25°C; V, 20°C. The values are the averages from two independentexperiments.

12,15), by a diunsaturated fatty acid, 18:2(9,12), hadpractically no effect on phase transition.

The growth of cellsThe effects of unsaturation of membrane lipids on thegrowth of cells were examined at various temperatures(Figure 3). There was no significant difference in termsof growth profile among the wild-type, desD- and desB7/desD- cells which contained polyunsaturated fatty acids(Table I). Maximum growth of these cells was observedat 30°C and the cells were able to grow even at 20°C. Incontrast, the desA-/desD- mutant cells, which contained18:1(9) but no polyunsaturated fatty acids, yielded agrowth profile that was very different from those of theother strains. Although growth at 35 and at 30°C washardly affected, the growth at lower temperatures wasmarkedly depressed (Figure 3D). At 25°C, the cells grewslowly after a long lag period and at 20°C they failed togrow at all.

Figure 4 shows the dependence on temperature of thegrowth rate of the wild-type, desD-, desB7/desD- anddesA-IdeslD cells. There were no significant differencesin growth rate at 35°C among the various types of mutantcell. While the wild-type, deslY and desB-/desD- cellshad similar growth rates at all temperatures, the growthrate of the desAldesD- cells was dramatically lower at20 and 25°C than that of the wild-type and the othermutant cells. Thus, the optimum temperature for growthapparently shifted from 30°C for the wild-type, desD- anddesB/IdesD- cells to 35°C for the desA-/desD- mutant cells.These results indicate that replacement of polyunsaturatedfatty acids by a monounsaturated species had a significanteffect on the growth of cells, whereas replacementsamong polyunsaturated fatty acids had no apparent effectson growth.

Photosynthesis and respirationIn order to study the biochemical basis for the suppressiveeffect of unsaturation of membrane lipids on growth of

20 25 30 35

Temperature of growth (OC)

Fig. 4. Dependence on temperature of growth rates of wild-type andmutant cells during the exponential phase of growth. El, Wild type;0, desD-; A, desB/desD-; V, desA-ldesD-.

500

400

2L000~~~

0.C

aI0

BXjZuOl15 20 25 30 35Temperature of measurement (°C)

Fig. 5. Dependence on temperature of the photosynthetic oxygen-evolving activities of wild-type and desA/ldesD- cells. Cells weregrown at 25°C. Oxygen-evolving activities were measured with CO2as the terminal acceptor of electrons. 0, Wild-type cells; *, desA1desD- cells. The values are the averages from three independentexperiments.

cells at low temperatures, we compared the photosyntheticand respiratory activities of wild-type and desA/desD-cells at various temperatures. When cells were grown atthe same temperature, 25°C in this case, no differencesin photosynthetic activities were observed when they weremeasured over a wide range of temperatures from 18 to33°C (Figure 5). No differences in respiratory activitieswere observed over a wide range of temperatures from15 to 35°C (Figure 6). These results indicate that replace-ment of polyunsaturated fatty acids by a monounsaturatedspecies had no effects on photosynthesis and respiration.

Photoinhibition of photosynthesisSince the photosynthetic activity measured over a shortperiod of time was not affected by unsaturation of mem-brane lipids, we suspected that unsaturation might affectthe photosynthetic activity after a long period of illumina-

6419

1.5

1.0

05

04

1.5 -A

a.0

80ah c,C1.5 -

0= 1.0-C,---

Y.Tasaka et al.

180-

60-

40-

20

15 20 25 30 35Temperature of measurement (°0)

Fig. 6. Dependence on temperature of the respiratory activity of wild-type and desA-/desD- cells. The cells were grown at 25°C. Oxygenuptake in darkness was monitored after the cells had been incubated at25°C in fresh BG-1 medium in darkness for 60 min. 0, Wild-typecells; 0, desA-IdesD- cells. The values are the averages from threeindependent experiments.

tion. Therefore, we examined inhibition of activity of thephotosystem II complex by monitoring photosystem II-mediated transport of electrons from H20 to 1,4-benzo-quinone. Measurements were made during strong illumina-tion at 20 and 30°C of wild-type and desA-IdesD- cells bylight at 1.5 mmol/m2/s (Figure 7A and B). Photoinhibitionoccurred in both wild-type and desA-1desD- cells at both20 and 30°C. However, desA-/desD- cells were moresensitive to light than wild-type cells at both temperaturesand both types of cell were more sensitive to light at 20than at 30°C.To investigate the contribution of protein synthesis to

photoinhibition, we performed similar experiments inthe presence of chloramphenicol, an inhibitor of proteinsynthesis (Figure 7C and D). Both the wild-type anddesA-IdesD- cells were preincubated in the presence ofchloramphenicol for 20 min at 25°C in darkness and thenincubated in light at 1.5 mmol/m2/s at 20 and at 30°C.Under these conditions, there were no significant differ-ences in the extent of photoinhibition between wild-typeand desA-IdesD- cells and the effect of temperature onphotoinhibition apparently disappeared. These findingsindicate that light-dependent inactivation of the photosys-tem II complex was unaffected by unsaturation of mem-brane lipids and they suggest that recovery of thephotosystem II complex from photoinhibition, whichinvolves protein synthesis (Aro et al., 1993), might havebeen suppressed by replacement of polyunsaturated fattyacids by a monounsaturated fatty acid.

Incubation in darkness at 20 or at 30°C for 60 min didnot affect electron transport activity in the absence ofchloramphenicol (Figure 7A and B). The presence ofchloramphenicol in darkness slightly decreased electrontransport activity (Figure 7C and D), presumably as a

result of a direct inhibitory effect of chloramphenicol.

Recovery of the photosystem 11 complex fromphotoinhibitionSince the above analysis suggested that recovery fromphotoinhibition was influenced by the unsaturation of

at 100

c 80

60

O 40

20c

. O

z1000

40

@!20

o

100

80

60

40

20

0

0 20 40 60 0 20 40

Duration of Incubation In lightat 1.5 mmol/m2/s (min)

60

Fig. 7. Photoinhibition of photosynthesis in wild-type and desA-ldesD-cells. Cells were incubated at 20 or 30°C in the presence or absence ofchloramphenicol (Cm) in light at 1.5 mmol/m2/s for designated times.Photosynthetic oxygen-evolving activity was measured at 30°C bymonitoring oxygen concentration. The absolute oxygen-evolvingactivities, corresponding to 100%, for wild-type and desA-/desD- cellswere 570 ± 30 and 550 ± 20 gmol 02/mg chlorophyll/h respectively.(A) 20, (B) 30°C, both in the absence of chloramphenicol. (C) 20,(D) 30°C, both in the presence of 0.2 mg chloramphenicollml. 0,

Wild-type in light; A, desA-ldesDl in light; 0, wild-type in darkness;A, desA-IdesD- in darkness. The values are the averages from threeindependent experiments.

membrane lipids, we made a direct comparison of recoveryof the photosystem II complex from photoinhibitionbetween wild-type and desA-/desD- cells by monitoringoxygen-evolving activity with 1,4-benzoquinone as theelectron acceptor. The cells were preincubated in light at3.0 mmollm2/s at 20°C for 40 min, which resulted in~80% photoinhibition. Then the cells were incubated at20 or 30°C in light at 0.07 mmollm2/s. At 20°C, the wild-type cells regained 80% of the original activity afterincubation for 120 min, whereas the desA-/desD- cellsscarcely regained any activity (Figure 8A). At 300C, therestoration of photosystem II activity was almost completeafter incubation for 120 min in both wild-type and desAldesD- cells, even though the rate of restoration was higherin the wild-type cells than in the desA-ldesD- cells(Figure 8B).The rates of restoration of activity in both types of cell

in darkness were much lower than those in the light, butwild-type cells regained activity more rapidly than desA1desD- cells in darkness. In the presence of chlorampheni-col, the restoration of activity was completely blocked(Figure 8C and D) at 20 and at 30°C, indicating thatrecovery of the photosystem II complex depended on

protein synthesis. These findings indicate that recovery ofthe photosystem II complex from photoinhibition at 20,but not that at 300C, was suppressed by the replacementof polyunsaturated fatty acids by a monounsaturatedfatty acid.

6420

A 20°C, Cm--------

0 20 40 60

C 20°C, +Cm

A

Targeted mutagenesis of acyl-lipid desaturases

0 40 80 120D 3000, +CmD 30°C, +Cm

100

80

60

40

20 -----

O _-0 40 80 120

Duration of Incubation (min)

Fig. 8. Recovery of the photosystem II complex from photoinhibitionin wild-type and desAldesD- cells. Cells were preincubated at 20°Cfor 40 min in light at 3.0 mmol/m2/s to induce 80% photoinhibition ofthe photosystem II complex. Then recovery of the complex fromphotoinhibition was followed by monitoring oxygen-evolving activitywith 1,4-benzoquinone as the electron acceptor after incubation at 20or 30°C in light at an intensity of 0.07 mmol/m2/s in the presence of0.2 mg chloramphenicol/ml (Cm) or in its absence. At designatedtimes, portions of the cell suspension were withdrawn and, afteraddition of 1.0 mM 1,4-benzoquinone (final concentration), transportof electrons from H2O to 1,4-benzoquinone was examined at 30°C bymonitoring oxygen concentration. The absolute oxygen-evolvingactivities, corresponding to 100%, for wild-type and desA/desD- cellswere 570 + 30 and 550 + 20 Itmol 02/mg chlorophyll/h respectively.(A) 20, (B) 30°C, both in the absence of chloramphenicol. (C) 20,(D) 30°C, both in the presence of 0.2 mg chloramphenicol/ml. 0,

Wild-type in light; A, desA-ldesD- in light; 0, wild-type in darkness;A, desA-IdesD- in darkness. The values are the averages from threeindependent experiments.

Discussion

Mutation of the genes for acyl-lipid desaturasesIn the present study, we successfully mutated Svynechocystissp. PCC 6803 cells by disrupting the desA, desB and desDgenes for the A12, W3 and A6 desaturases respectivelywith antibiotic resistance gene cartridges. Our attemptsto disrupt the desC gene for the A9 desaturase were

unsuccessful, suggesting that mutation of the A9 desaturasethat resulted in elimination of all unsaturated fatty acidsfrom membrane lipids might have been lethal.

Using the technique of insertional mutagenesis, we were

able to manipulate the level of unsaturation of C18 fattyacids to introduce one, two, three or four double bondsinto fatty acids and to produce mutant strains in whichthe specific fatty acids in membrane lipids were welldefined. This is the first successful example, to our

knowledge, of totally effective manipulation of the unsatur-ation of fatty acids by genetic engineering. The mutatedstrains that we produced are useful tools to examine thedirect effects of unsaturation of fatty acids of membranelipids on membrane functions.

Changes in lipid molecular speciesThe physicochemical characteristics of glycerolipids inbiological membranes are related to the molecular species

composition, but not directly to the fatty acid composition(Quinn et al., 1989). Therefore, in order to understandthe direct relationship between glycerolipids and theirbiological functions in membranes it is essential to knowthe molecular species composition of glycerolipids in cellsof both wild-type and mutant strains of Synechocystis sp.PCC 6803.

In Synechocystis sp. PCC 6803, the molecular speciescomposition can be calculated from the fatty acid composi-tion, since the sn-2 position of the backbone glycerol isexclusively esterified with 16:0 (Murata et al., 1992b).Table II shows the calculated molecular species composi-tions of cells of the wild-type and mutant strains. Mutationof the desD gene for A6 desaturase (desD-) completelyeliminated the sn- 1-18:3(6,9,12)Isn-2- 16:0 and sn-i-18:4(6,9,12,15)/sn-2-16:0 species. Mutation of both thedesD and desB genes, which encode the wo3 desaturase(i.e. desB-IdesD-), additionally eliminated the stn-l-18:3(9,12,15)/sn-2-16:0 species. As a result, desB-IdesD-cells contained monounsaturated and diunsaturatedspecies. Mutation of both the desD and desA genes forA12 desaturase (i.e. desAldesD-) further eliminated thesn-1-18:2(9,1 2)1sn-2- 16:0 species. As a result, desAldesD- cells contained almost exclusively monounsaturatedspecies, with only a very minimal level of saturated species.

Advantages of insertional mutagenesis ofdesaturasesIn order to examine the effects of unsaturation of mem-brane lipids on the responses of cyanobacterial cells tolow temperature, we previously used the Fad6 and Fad6/desA- mutant strains (Gombos et al., 1992, 1994; Wadaet al., 1992). Since the Fad6 mutant was leaky at lowtemperatures, we were unable to examine whether ornot unsaturation of membrane lipids affected growthtemperature-dependent alterations in physiologicalactivity. In the genetically well-defined mutant strains thatwe obtained in the present study, all the individualdesaturases were completely inactivated at low temper-atures. Therefore, we were able to study the effects ofspecific unsaturated molecular species of membrane lipidson the dependence on growth temperature of variousphysiological activities.

Several research groups have studied the effects ofunsaturation of membrane lipids on the temperature-dependent properties of cells by modifying the degree ofunsaturation of membrane lipids with changes in growthtemperature. For example, in early studies, an increase ingrowth temperature resulted in a decrease in the degreeof unsaturation of membrane glycerolipids and in adecrease in the tolerance of photosynthesis to low tempera-ture (Pearcy, 1978; Raison et al., 1982). However, thisapproach failed to clarify the direct relationship betweenthe degree of unsaturation of glycerolipids and the stabilityof the photosynthetic machinery at high and low tempera-tures, because not only the unsaturation of membranelipids but also a number of other metabolic factors areaffected by changes in growth temperature (Guy et al.,1985; Mohapatra et al., 1987; Cooper and Ort, 1988).The physiological importance of unsaturated fatty acids

of membrane lipids was studied by modifying the fattyacid composition of membrane lipids of an unsaturatedfatty acid auxotroph of Escherichia coli with supplement-

6421

A 20C, -Cm

- - ~ ~ -

I- -

B 30°C, -Cm100

80

60-

40-0

200

100

i 80

660

° 40

c 200

0B 1000* 80-0

in 600Z 40a 20Is

0 40 80 120

0 40 80 120

iI

c

I(D,--

20°C, +Cm

Y.Tasaka et aL

Table II. Molecular species composition of total glycerolipids from wild-type and mutant cells of Synechocystis sp. PCC 6803

Strain Molecular species (mole %)

sn-I 16:0 16: la 18:0 18: lb 18: c 18:2d 18:2e 18:3f 18:39 18:4hsn-2 16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:0 16:0

Wild-type 6 8 2 18 tr tr 20 10 28 8desA- 10 8 2 56 tr 24 0 0 0 0desB7 2 10 4 12 tr tr 26 0 44 0desD- 4 12 2 16 tr 0 46 22 0 0desB7/desD- 6 12 2 24 tr 0 58 0 0 0desA-IdesD- 2 14 2 82 tr 0 0 0 0 0Fad6 8 8 2 16 tr tr 16 22 4 4Fad6/desA- 6 10 2 72 4 6 0 0 0 0

Cells were grown at 25°C.Values were calculated from the data in Table I on the assumption that the sn-2 position is esterified exclusively with 16:0 (Murata et al., 1992b). Asa consequence, the range of experimental error for the molecular species sn-1-16:0-sn-2-16:0 is ±6%, whereas that for the other molecular species is±43% of the values.a16: I(9), b18:1(9), c18:1(I 1), d18:2(6,9), e18:2(9,12), f18:3(9,12,15), 918:3(6,9,12) h18:4(6,9,12,15).

ation of variously unsaturated fatty acids to the culturemedium (Silbert et al., 1968). Changes in unsaturation ofmembrane lipids by this method affected the physicalphase of the plasma membrane (Baldassare et al., 1976;Kato and Bito, 1980) and growth of cells (Esfahani et al.,1969; Broekman and Steenbakkers 1973). Similar studieswere performed in Acholeplasma laidlawii (Silvius andMcElhaney 1978; Christiansson and Wieslander, 1980).However, there are some disadvantages of this method,e.g. the fatty acid composition cannot be precisely con-trolled and exogenously supplemented fatty acids affectnot only membrane lipids but also carbon metabolism.Moreover, free fatty acids are toxic to living organisms.Therefore, a strict relationship between unsaturation ofmembrane lipids and the physiological activities of cellscannot be fully understood.

Physicochemical characterization of thylakoidmembranesThe study by DSC of isolated thylakoid membranesdemonstrated that changes in membrane fluidity, monitoredin terms of the phase transition, reflected alterations inunsaturation of membrane lipids in cells of the geneticallymodified strains. Replacement of polyunsaturated speciesof membrane lipids by monounsaturated species markedlyincreased the temperature for physical phase transition ofthylakoid membranes (Figure 2). In contrast, replacementof tri- and tetraunsaturated species of membrane lipids bydiunsaturated species had no significant effect on thetemperature of phase transition (Figure 2).

Between 15 and 21 °C, the thylakoid membranes isolatedfrom desA-/desD- cells were in the phase-separated state,whereas those from wild-type, desDY and desB7/deslYcells were all in the liquid crystalline state (Figure 2).Studies on model membranes (Coolbear et al., 1983),prepared from synthetic glycerolipids with different levelsof unsaturation, have indicated that the most significantdifference in phase behavior is between saturated andmonounsaturated molecular species, that the next mostsignificant difference is between monounsaturated anddiunsaturated species and that further unsaturation to tri-and tetraunsaturated species does not significantly alterthe physicochemical characteristics of the membrane. Theresults in the present study are consistent with those

obtained with model membranes and provide the firstdemonstration of the effects of various levels of unsatur-ation on the phase behavior of membrane lipids in bio-logical membranes.

Changes in proteins in thylakoid membranesThe fluidity of glycerolipids in intact thylakoid membranesis lower than that of glycerolipids that have been extractedfrom thylakoid membranes, purified and used to reconstit-ute lipid bilayers (Ford and Barber, 1983). These observ-ations suggest that proteins in thylakoid membranes mightplay an important role in modulating the fluidity ofglycerolipids in the thylakoid membranes. Therefore, weexamined the polypeptide compositions and ratio of lipidto protein in thylakoid membranes isolated from wild-type and desA-/desDl cells. The results indicated thatmutation of the desA and desD genes did not significantlyalter the polypeptide composition or ratio of lipid toprotein in thylakoid membranes. These findings indicatethat the changes in the physicochemical and physiologicalproperties of thylakoid membranes due to mutation ofdesaturases are attributable solely to alterations in theunsaturation of membrane lipids.

Dependence on temperature of cell growthThe growth profiles of cyanobacterial cells (Figures 3 and4) indicated that wild-type, desD- and desBrldesD cellsgrew at moderately low temperatures, such as 20 and25°C, whereas desA-/deslD cells failed to grow at 20°Cand their growth rate was much lower than that of cellsof the other strains at 25 and at 30°C. These observationssuggest that replacement of di-, tri- and tetraunsaturatedmolecular species by monounsaturated molecular speciessignificantly depressed cell growth at low temperatures,whereas replacement of tri- and tetraunsaturated speciesby diunsaturated species had practically no effect ongrowth. These findings further emphasize the importanceof polyunsaturated molecular species of membrane lipidsfor growth at low temperatures. This conclusion is in goodagreement with the data obtained with Synechococcus sp.PCC 7942 in which a genetically increased level ofdiunsaturated fatty acids, due to transformation with thedesA gene from Synechocystis sp. PCC 6803, markedly

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enhanced the tolerance of cells to low temperature (Wadaet al., 1990, 1994).

Changes in stability of the photosyntheticmachineryPhotosynthetic activity in desA-IdesD- cells, which lackedpolyunsaturated molecular species of membrane lipids,was the same as wild-type cells at various temperatures(Figure 5). It should be emphasized that replacement ofpolyunsaturated lipid molecules by a monounsaturatedspecies in thylakoid membranes had no effect on photosyn-thetic activity when such activity was measured over ashort time period. However, the photosynthetic activityover a long period of time, which depends on stability ofthe photosynthetic machinery under specific environmentalconditions, could be affected by the level of unsaturationof membrane lipids and this phenomenon might have beenresponsible for the decrease in growth rate of desAde'sD-cells at low temperatures (Figure 4).We compared photoinhibition at low temperature of

wild-type and desAldesD- cells. The results of theseexperiments (Figures 7 and 8) revealed that the extent ofphotoinhibition was higher in desA-IdesD- cells than inwild-type cells, indicating that replacement of polyunsatur-ated molecular species by monounsaturated molecularspecies decreased the capacity for protection of the photo-system IL complex against low temperature photoinhibi-tion. The details of recovery of the photosystem II complexfrom the photoinhibited state (Figure 8) indicate thatreplacement of polyunsaturated molecular species by amonounsaturated species decreased the capacity forrecovery.

Recent research on photoinhibition and recovery (Aroet al., 1993) has demonstrated that these processes arerelated to degradation of D 1 protein and to reincorporationof newly synthesized DI protein into the photosystem IIcomplex. Light-induced inactivation of photosystem II-mediated transport of electrons corresponds to damage tothe DI protein, and restoration of electron transportcorresponds to a series of reaction steps, such as degrada-tion of damaged Dl protein, de novo synthesis of theprecursor of D1 protein, incorporation of the precursorinto the photosystem II complex and processing of theprecursor to yield mature DI protein. Our recent researchdemonstrated that replacement of polyunsaturated molecu-lar species by a monounsaturated species strongly sup-pressed processing of the precursor to DI protein atlow temperature (E.Kanervo, Y.Tasaka, N.Murata andE.-M.Aro, unpublished result).

RespirationThe oxidative transport of electrons associated with res-piration in cyanobacterial cells occurs on the thylakoidmembranes (Omata and Murata, 1985; Molitor andPeschek, 1986). It has been argued for many years thatoxidative electron transport activity is enhanced when anorganism is exposed to low temperature and that thisincrease is caused by desaturation of fatty acids of therespiratory membranes at low temperature (Miller et al.,1974). However, a direct relationship between respiratoryactivity and unsaturation of membrane lipids could not bedemonstrated with the previously examined acclimatiza-tion systems because growth temperature affects not only

desaturation of membrane lipids but also numerous othermetabolic processes.

Replacement of polyunsaturated species of glycerolipidsby a monounsaturated species in Synechocystis sp. PCC6803 in the present study did not alter respiratory activityof cells at various temperatures. This finding suggests thatunsaturation of membrane glycerolipids does not playa key role in oxidative electron transport reactions ofcyanobacterial respiration.

Materials and methodsOrganisms and culture conditionsThe wild-type strain of Synechocystis sp. PCC 6803 was originallyprovided by Dr J.G.K.Williams (DuPont de Nemours and Co..Wilmington, DE). The Fad6 mutant (Wada and Murata, 1989), the desA-mutant (Wada et al., 1990) and the Fad6/desA- mutant (Wada et al.,1992) were obtained as described in our previous reports. Cells were

propagated with aeration at 25, 30 and 35°C in light at 0.07 mmol/m2/s inBG1 1 medium supplemented with 20 mM HEPES (pH adjusted to 7.5with NaOH), as described previously (Wada and Murata, 1989). Thegrowth of cells was monitored in terms of turbidity at 750 nm with an

absorption spectrophotometer (UV300; Shimadzu, Kyoto, Japan).

Transformation with disrupted genes for desaturasesThe desA gene in pBluescript (Stratagene, La Jolla, CA) was disruptedby inserting kanamycin resistance (Kmr) and bleomycin resistance (Bler)gene cartridges into the HindIII site, as described previously by Wadaet al. (1990, 1992). The resultant plasmid was designated pdesA::(Kmr+Bler) (Figure IA). Wild-type cells of Synechocystis sp. PCC 6803were transformed with this plasmid to generate desA- cells, as describedpreviously (Wada et al., 1990).We amplified the coding region of the desD gene (Reddy et al., 1993)

by PCR using genomic DNA isolated from Synechocystis sp. PCC 6803as template and subcloned this region into the TA cloning site of plasmidpCRII (Invitrogen, San Diego, CA). For construction of the disrupteddesD gene, the chloramphenicol resistance (Cmr) gene cartridge was

excised with TthIII and BsaAI restriction endonucleases from pBR328(Boehringer Mannheim GmbH, Mannheim, Germany) and inserted intothe AccI site of the desD gene in pCRII with a DNA Blunting Kit(Takara, Shiga, Japan). The resultant plasmid was designated pdesD::Cmr(Figure 1B). The wild-type and desA- (Wada et al., 1990) cells ofSynechocystis sp. PCC 6803 were transformed with this plasmid to

generate desD- and desA-ldesD- cells by the method of Golden et al.(1987), with minor modifications. We placed 20 p1 of a solution ofchloramphenicol (20 mg/ml) at the edge of a plate of BG 1 agar(diameter 9 cm) to produce a concentration gradient of chloramphenicolon the plate. Transformed cells resistant to chloramphenicol appearedwithin a certain range of concentrations of the drug on the plate duringincubation for -3 weeks at 34°C with illumination at 0.07 mmol/m2/s.A plasmid with a disrupted desB gene was constructed by inserting

the Kmr cartridge, which had been excised with SmaI restrictionendonuclease from pUC4-KIXX (Pharmacia, Uppsala, Sweden), into theSacl site of the desB gene in pBluescript II KS(+) (Sakamoto et al.,1994a) with the DNA Blunting Kit (Takara). The resultant plasmid was

designated pdesB::Kmr (Figure IC). The wild-type and desD- cells ofSynechocystis sp. PCC 6803 were transformed with this plasmid to

produce desB7 and desB/desD- cells by the same method as that usedfor transformation with pdesD: :Cmr. Then desB7 and desB/desD- mutant

cells were selected by monitoring resistance to kanamycin by the same

method as that used for selection of desD- and desAldesD- mutant cellsthat were resistant to chloramphenicol.A plasmid with a disrupted desC gene was constructed by replacing

the region between the EcoRV and NcoI sites of the desC gene in

pBluescript II KS(+) (Sakamoto et al., 1994b) with a Kmr cartridge,which had been excised with EcoRI restriction endonuclease from pUC4-KISS (Pharmacia, Uppsala, Sweden), with the DNA Blunting Kit(Takara). The resultant plasmid was designated pdesC::Kmr (Figure ID).Wild-type cells of Synechocystis sp. PCC 6803 were transformed withthis plasmid by the same method as that used for transformation with

pdesdD::Cmr.Disruption with the antibiotic resistance cartridges of the desA, desB

and desD genes on all copies of the chromosome was examined byPCR. Mutated cells were lysed by incubation with 0.5% Triton-X100 at

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Y.Tasaka et al.

95°C for 10 min and each lysate was used directly for PCR. Thepositions of primers used for PCR are indicated in Figure 1.

Isolation and thermal analysis of thylakoid membranesThylakoid membranes were isolated from wild-type, desD-, desB-IdesD-and desA-/desD- mutant cells by centrifugation in a step-wise sucrosegradient as described by Murata and Omata (1988), but with minormodifications: the treatment with lysozyme was omitted and cells weredisrupted by shaking with glass beads as described by Nishiyamaet al. (1993).

The thermal analysis of isolated thylakoid membranes was performedby DSC in a microcalorimeter (DASM-4; Russian Academy of Science,Moscow, Russia) at a scanning rate of 1°C/min. Thylakoid membraneswere suspended in 50 mM HEPES-NaOH buffer, pH 7.5, at -1 mgchlorophyll/ml, a concentration that corresponded to -8 mg protein/ml.

Analysis of lipids, proteins and chlorophyllLipids were extracted from cells by the method of Bligh and Dyer(1959). The fatty acid composition and lipid class composition wereanalyzed as described by Wada and Murata (1989) and Sato and Murata(1988) respectively. Protein contents of thylakoid membranes weredetermined with a Protein Assay kit (Bio-Rad, Hercules, CA). SDS-PAGE of isolated thylakoid membranes was carried out on 13.5%polyacrylamide gels containing 6.0 M urea by the method of Laemmli(1970). The gel was stained with Coomassie brilliant blue R-250.Chlorophyll concentrations were determined by the method of Arnonet al. (1974).

Photosynthetic and respiratory activitiesThe photosynthetic activity of a suspension of cells was measured witha Clark-type oxygen electrode by monitoring light-dependent evolutionof oxygen, with CO2 as the terminal electron acceptor. Actinic light wasprovided by an incandescent lamp in combination with a red opticalfilter (R-62; Hoya Glass, Tokyo, Japan) and an infrared absorbing filter(HA-50; Hoya Glass) at an intensity of 3.5 mmol/m2/s. The activity ofphotosystem II was measured in a similar way but in the presence of1.0 mM 1,4-benzoquinone as electron acceptor. Respiratory activity wasmeasured by monitoring uptake of oxygen in darkness by cells that hadbeen incubated in darkness for 60 min.

AcknowledgementsThis work was supported in part by Grants-in-Aid for Scientific Researchon Priority Areas (Nos 04273102 and 04273103), for Specially PromotedResearch (No. 08102011) and for the International Scientific ResearchProgram (Joint Research, No. 06044233) from the Ministry of Education,Science and Culture of Japan to NM. It was also supported in part bythe NIBB Cooperative Research Program on the Stress Tolerance ofPlants (95-703) and by a grant from the Hungarian Science Foundation(OTKA, T 020293) to Z.G.

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Received on July 16, 1996; revised on August 16, 1996

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