expression ofthe yeasta-9 fatty acid desaturase in nicotiana

Plant Physiol. (1992) 100, 894-9010032-0889/92/100/0894/08/$01.00/0

Received for publication April 1, 1992Accepted June 16, 1992

Expression of the Yeast A-9 Fatty Acid Desaturase inNicotiana tabacum'

James J. Polashock, Chee-Kok Chin, and Charles E. Martin*Department of Biological Sciences (J.J.P., C.E.M.), Department of Horticulture (C.-K.C), and Bureau of Biological

Research (C.E.M.), Rutgers University, Nelson Biological Laboratories, P.O. Box 1059,Piscataway, New Jersey 08855-1059

ABSTRACT

To examine the processes of plant cytoplasmic fatty acid desat-uration and glycerolipid biosynthesis, the protein coding sequenceof the endoplasmic reticulum cytochrome b5-dependent, A-9 fattyacid desaturase gene from Saccharomyces cerevisiae was intro-duced into Nicotiana tabacum via Agrobacterium transformation.All transformed plants expressing the yeast gene at the mRNA levelexhibited an approximately 10-fold increase in the levels of pal-mitoleic acid (16:1) in leaf tissue. This fatty acid species is foundin very low levels (less than 2%) in wild-type plants. These resultsindicate that the yeast desaturase can function in plants, presum-ably by using a leaf microsomal cytochrome b5-mediated electrontransport system. Lipid analysis demonstrated that the overpro-duced 16:1 is incorporated into most of the major polar lipidclasses, including the cytoplasmically produced "eukaryotic" frac-tion of the chloroplast galactolipids. 16:1 was not found, however,in phosphatidyl glycerol, which is considered to be produced almostexclusively in the chloroplast. Despite these changes in membranelipid composition, no obvious phenotypic differences were appar-ent in the transformed plants. Positional analysis shows that thecytoplasmically produced 16:1 is found primarily in the sn-2 posi-tion of phosphatidylcholine, phosphatidylethanolamine, monoga-lactosyldiacylglycerol, and digalactosyldiacylglycerol. The posi-tional data suggest that the sn-2 acyltransferases responsible forthe "eukaryotic" arrangement of 16- and 18- carbon fatty acids inglycerolipids are selective for unsaturated fatty acids rather thanchain length.

In plants, unsaturated fatty acids are formed and incorpo-rated into complex lipids in two distinct cellular compart-ments. The initial products of de novo fatty acid synthesis,which occurs almost exclusively in the plastids (12), are thesaturated species 16:0-ACP2 and 18:0-ACP. 18:1-ACP is

'This work was supported by National Science Foundation grantDMB84-17802, Biomedical Research Group grant RR-07058-21from the U.S. Public Health Service, and by a grant from the Bureauof Biological Research Charles and Johanna Busch Memorial Fund.

2 Abbreviations: ACP, acyl carrier protein; X:Y, fatty acyl groupscontaining X carbon atoms and Y cis double bonds; AN X:Y, as abovewith cis double bonds located at position N relative to the carboxylend of the chain; MGDG, monogalactosyldiacylglycerol; DGDG,digalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphati-dylethanolamine; DAG, diacylglycerol; PG, phosphatidylglycerol;CaMV, cauliflower mosaic virus.

rapidly formed from 18:0-ACP in the plastid by a soluble,ferredoxin-dependent, A-9 desaturase, which inserts a doublebond between carbons 9 and 10 of the fatty acyl chain (16,25). These fatty acids may then be shunted into one of twodistinct routes-a plastid-localized 'prokaryotic' pathway ora cytosolic/ER "eukaryotic" pathway (28)-for further modi-fication and acylation into glycerolipids. According to thismodel, fatty acids that are acylated into glycerolipids in thechloroplast are distinctive in that they tend to have 16-carbonspecies in the sn-2 position.

Other plastid enzymes that have desaturase functions havebeen identified by analysis of isolated chloroplasts or Arabi-dopsis mutants (7, 30, 32). Most polyunsaturated 18-carbonplant fatty acids, however, appear to be formed in the cytosolby ER-bound enzymes. Fatty acids that are transported outof the chloroplast are thought to be converted to CoA estersand subsequently incorporated into glycerolipids by the eu-karyotic route (2, 6, 18, 26). These lipids characteristicallycontain 18-carbon fatty acids in the sn-2 position.Once incorporated into phospholipids, a cytoplasmic de-

saturase catalyzes the formation of the A-12 double bond in18:1. This desaturase is thought to be similar to the animaland fungal desaturases because it is membrane bound andappears to require a Cyt b5-mediated electron transport chain(31). This enzyme, however, has not been purified fromhigher plants, and its structural gene has not been cloned.Movement of glycerolipids is also believed to occur in the

reverse direction, i.e. between the cytosol/ER and the plastids.Lipids localized in the chloroplast that contain the eukaryoticarrangement of fatty acids (an unsaturated 18-carbon speciesin the sn-2 position) are probably derived from intermediatesoriginating from eukaryotic lipids such as PC. One possibilityis the conversion of PC to DAG, which is transported intothe chloroplast, where it is used to synthesize galacto- andsulfo-lipids (10, 14, 28, 40). Thus, chloroplast lipids such asMGDG and DGDG are thought to be formed from twodistinct pools, with the prokaryotic type having 16-carbonfatty acids in the sn-2 position and the eukaryotic type having18-carbon species in the sn-2 position.The metabolic flux of fatty acids between the two pathways

appears to be highly regulated during growth and develop-ment. Many plants, for example, can contain significantamounts of fatty acids, such as 16:3 in leaf tissue, that arederived from the prokaryotic pathway, but these are usuallymissing from seed oils, which contain acyl species derivedalmost exclusively from the cytosolic/ER route (36).

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YEAST A-9 DESATURASE IN TOBACCO

The existence of two compartmentalized systems indicatesthat regulatory studies and efforts to modify plant lipids willrequire development of information not only about the func-tions of individual lipid metabolic enzymes, but also aboutthe coordinated movement of precursors and complex lipidsbetween the two compartments.We attempted to investigate the features of plant lipid

metabolism discussed above through the introduction of ayeast cytoplasmic lipid modifying enzyme into tobacco. TheSaccharomyces cerevisiae A-9 desaturase is a Cyt b5-depend-ent, ER enzyme that efficiently converts 16:0-CoA to 16:1 (4,34), a minor fatty acid species in plants. Other work in ourlaboratory has recently shown that there is a broad functionalhomology between Cyt b5-dependent desaturases by success-fully expressing the rat A-9 desaturase in yeast (35).

If significant amounts of 16:0-CoA are present in the plantcytosol, its desaturation to 16:1 by the yeast enzyme shouldprovide a useful reporter for the metabolic conversions ofcytosolic fatty acids into membrane lipids. The introductionof foreign lipid biosynthetic genes into specific subcellularlocations may also provide a useful tool for uncovering thereactions and components of key regulatory systems. Theincreased production of monounsaturated fats may also altermembrane fluid properties, allowing the examination of theeffects on membrane associated physiological functions. Totest these possibilities, the CaMV 35S promoter and polyad-enylation signal were fused to the coding sequence of theyeast gene. The chimeric gene was introduced into Nicotianatabacum via Agrobacterium transformation.

In this article, we demonstrate that the cytoplasmic yeastenzyme functions in plants and converts significant amountsof 16:0 to 16:1. The 16:1 product is found in high levelsprimarily in the sn-2 position of PC and other polar lipids,including the galactolipids of the chloroplast.

MATERIALS AND METHODS

Plants, Bacterial Strains, and Media

Nicotiana tabacum var W3 was used in these studies. Leafdisks used for transformations were taken from axenic plantsgrown in the light at 220C in Murashige and Skoog medium(24) with 2% sucrose. Escherichia coli strain HB101 was usedfor intermediate cloning and propagation of plasmids. Agro-bacterium strain LBA4404, which contains the helper plasmidpAL4404, was used for plant transformations. Agrobacteriumwas grown in LB medium at 250C with appropriate antibi-otics. All DNA manipulations were performed according tostandard techniques (3, 22).

Plasmid Constructs

Plasmids for Agrobacterium transformation were con-structed as shown in Figure 1. The yeast A-9 desaturasecoding sequence was digested from YEp352/OLE4.8 withSfaNI (35). The resulting 1553-bp fragment was filled in byKlenow polymerization. Synthetic BamHI linkers (12-mer)were blunt-end ligated to the fragment to facilitate furthercloning steps.The chimeric GUS marker gene from the plasmid pBI101

was removed by digestion with EcoRI and HindlIl (17), and

I I

Gus Marker Gene

Removed to Allow Insertionof the Desatumase ConstructZ

Figure 1. Construction of the Agrobacterium vector for plant trans-formations. The GUS marker chimeric gene in the T-DNA regionof pBI101 was replaced with the yeast desaturase coding se-quences under the control of the CaMV 35S enhancer-promoter(Enh-Prom.).

replaced with the HindIII/EcoRI fragment of pFF19 contain-ing the CaMV 35S promoter with the enhancer duplicatedand the 35S polyadenylation signal (39). The yeast genefragment with the BamHI linkers was then inserted into theBamHI site in the multiple cloning region of the pFF19fragment. The resulting plasmid containing the yeast desat-urase coding region modified for plant expression and theselectable marker gene NPTII was introduced into the binaryAgrobacterium strain LBA4404 using the freeze-thaw method(1). Transformed control plants were formed using the con-struct described above (including the marker NPTII), butwithout the yeast desaturase coding sequence.

Plant transformations were performed by the leaf diskmethod as described by Horsch et al. (15) with the followingchanges. The Agrobacterium was centrifuged at 5OOg for 10min and resuspended in 50 mL of LB medium withoutantibiotics just prior to infection of the leaf disks. Aftercocultivation, the Agrobacterium was cleared from the leafdisks using augmentin (Beecham Chemicals) instead of car-benicillin. Kanamycin-resistant plantlets were transplantedto the greenhouse and grown under standard greenhouseconditions.

Analysis of Transformants

Southern blots were performed according to standard pro-cedures (33). Plant DNA was extracted by the method ofJunghans and Metzlaff (19). Typically, 10 ,ug of HindlIl-cutDNA per lane was run on an agarose gel. The DNA wastransferred to Gene Screen-Plus (DuPont) and hybridizedaccording to manufacturers' instructions using the yeast genefragment with BamHI linkers as a probe. The probe was 3p_labeled using the random primer method (5 Prime-3 Prime,Inc.). RNA was isolated for northem blots by the guanidiniumisothiocyanate method (8). RNA was denatured in SDS/formamide and run on formaldehyde gels. Transfer to GeneScreen-Plus and hybridizations were according to manufac-turers' directions.

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Plant Physiol. Vol. 100, 1992

1 2 3 4 5 6 7 8 9 10 11

I..

_- _VS.

Figure 2. Southern blot analysis of kanamycin-resistant plants.Transformed tobacco plants regenerated under kanamycin selec-tion were cleared of the Agrobacterium and tested for integrationof the yeast A-9 desaturase. All 26 plants selected for further analysiscontained one or more insertions of the yeast gene into genomicDNA. Lanes 1-9, Representatives of kanamycin-resistant plantstransformed with plasmid containing the yeast gene; lane 10, non-transformed wild type; lane 11, control plant transformed withplasmid lacking yeast gene sequences. Genomic DNA from isolateswas digested to completion with HindlIl (which cleaves one end ofthe inserted yeast sequence) and probed with a 1553-bp DNAfragment containing the yeast desaturase coding sequences.

Fatty Acid and Lipid Analysis

For screening, lipids were directly saponified and the fattyacids methylated for GC analysis from 100 mg of leaf tissue,as described by Browse et al. (6), or saponified in 4 N NaOH-methanol and extracted in hexane/ether (1:1) after acidifica-tion with HCl-methanol. If the latter method was used,methyl esters were prepared by the BF3-methanol procedure(23). Both methods gave comparable results, and were alsoused on young stems and roots, mature ovaries, and maturesingle seeds. Fatty acid methyl esters were suspended in asmall amount of hexane and analyzed on a Varian model3700 gas chromatograph fitted with a 0.75-mm i.d., 30-m,Supelcowax 10 capillary column (Supelco) and a HewlettPackard 3390A integrator. The injector and flame ionizationdetector were held at 2500C, and oven temperature was heldat 1900C. Total lipids were extracted from leaf tissue asdescribed by Kates (20). Polar lipid separations were on silicagel H TLC plates impregnated with 5% ammonium sulfate(Analtech) in a solvent system containing acetone:benzene:water(91:30:8) (21). Lipid classes were visualized by lightly stainingwith iodine and identified by comigration with authenticstandards (Sigma). Major lipid classes were scraped from theplates, eluted from the gel with CHCl3/methanol (1:1), andprepared for GC analysis as described above for total leaffatty acid analysis or used for lipase treatment.

Lipase Treatment

Fatty acid positional analyses using Rhizopus lipase (SigmaNo. L4384) for glycolipids and porcine pancreas (phospholi-pase A2, Sigma No. P6534) for phospholipids were carriedout as described in Fischer et al. (9) with the following

changes. Lipase reaction buffers contained 50 mm boric acidto prevent intramolecular acyl transfer in the lyso-productsduring the course of the reaction. The porcine pancreaticlipase was not pretreated because the enzyme preparationcontained no detectable lipid or fatty acid contaminants.These results were confirmed by a parallel analysis with Najanaja phospholipase A2. Deoxycholate was not used in thereaction mixture because Triton X-100 was sufficient to sol-ubilize the lipids for lipase treatment.

RESULTS

Over 100 independent kanamycin-resistant plantlets wereobtained from the Agrobacterium transformation. Of those,26 plants were chosen at random for further analysis.Southern blot analyses indicated that all of the putative

transformed plants contained one or more insertions of thechimeric yeast gene construct (Fig. 2). Northern blot analysisof total leaf RNA from all 26 plants indicated that althoughall of the plants contained at least one insertion of thechimeric yeast gene, only half of those tested expressed thegene at the mRNA level (Fig. 3).

Fatty acid analysis of total leaf lipids from expressing plants(as indicated by northern blot analysis) and nonexpressingplants indicated that all 'expressors' had altered fatty acidcompositions characterized by 7- to 13-fold increases in 16:1,as demonstrated in the representative chromatograms shownin Figure 4. The pooled data showing saturated and mono-unsaturated fatty acids from eight randomly chosen expres-sors and eight controls is given in Figure 5. In addition to thelarge increases in 16:1 levels, smaller, compensatory changeswere observed in other fatty acids, including decreases in16:0 and 18:0 levels of expressor plants. Although thereappear to be minor increases in 18:1 levels in the expressorplants, levels of the polyunsaturated species 18:2 (approxi-

4 06 ' I 1x i2 1& 14 P

Figure 3. Northern blot analysis of transformed plants. Total RNAfrom kanamycin-resistant plants was probed with a 1553-bp frag-ment containing the yeast desaturase gene coding sequences. Ap-proximately half of the transformed plants selected produced RNAcomplementary to the yeast gene. All of the hybridizing bands werethe correct size (approximately 2 kb) for a complete, polyadenylatedyeast desaturase mRNA. Lane 1, Wild type; lane 2, plant trans-formed with plasmid containing no yeast gene coding sequences;lane 3, transformed expressor plant N21; lanes 4-16, representativeplants transformed with plasmid containing yeast gene sequences.

896 POLASHOCK ET AL.

v




SolventA. Peek 16:0

18:2

B Solvent* Peak 16:0

Figure 4. Representative gas chromatograms of expressing andcontrol plants. A, Fatty acid methyl esters of total leaf lipids ex-

tracted from wild type. B, Methyl esters from expressor plant N21transformed with the yeast desaturase gene.

mately 17%) and 18:3 (approximately 45%) were found tobe nearly identical to wild-type controls. Nonexpressors in-cluded two transformed controls (no yeast gene coding se-

quence), two wild-type plants, and four transformants thatcontained the yeast coding sequence, as indicated by South-ern blots, but were shown to be negative for yeast desaturasemRNA by northern blot analysis. None of the nonexpressors

showed differences in fatty acid composition compared towild-type controls.None of the expressing plants identified in these studies

displayed morphological abnormalities, and all grew nor-

mally under greenhouse conditions. Seed production andgermination rates also appeared to be similar to those of thecontrols (data not shown).

Organ Specificity of 16:0 Desaturation

Analysis of different plant tissues shows striking differ-ences in fatty acyl composition due to the expression of theyeast gene (Table I). The highest levels of 16:1 were observedin the stem tissues and root tissues, which contained 22.6and 13.7%, respectively, with corresponding decreases in16:0, 18:0, and 18:3 levels. Fatty acid analysis of seeds

1 Expressors

0

0

0

16:0 16:1 18:0 18:1

Fatty Acid Species

Figure 5. Saturated and monounsaturated fatty acid levels in ex-

pressing and control plants. Comparison of the distribution ofsaturated and monounsaturated fatty acid species in eight randomlychosen expressor and eight nonexpressor plants. Linoleic (18:2) andlinolenic (18:3) acids comprise approximately 60% of the total fattyacids in all plants. Those fatty acids showed no significant variationbetween expressor and nonexpressor plants and are not shown.Data points represent mean ± sample SD.

collected from three selfed, expressing plants chosen at ran-

dom indicated that about 50% exhibit a modest increase (2-to 3-fold) in the level of palmitoleic acid as compared to thecontrols (Table I). This agrees with the observation thatapproximately 50% of the seedlings from the same expressingplants exhibit the transformed phenotype, i.e. an increase inpalmitoleic acid in leaf tissue (data not shown).

Distribution of 16:1 in Membrane Lipids

Analysis of leaf polar lipids indicates that the overproducedpalmitoleic acid is present at high levels in most of the major

Table I. Distribution of Fatty Acidsa in Wild-Type and TransformedPlant Tissues

Fatty Acyl GroupPlant/Organ

16:0 16:1 18:0 18:1 18:2 18:3

WT leaves 19.6 1.2 2.9 4.3 17.9 47.8N leaves 15.9 9.8 1.7 5.1 17.6 43.2WT stem 22.7 1.9 5.2 4.4 36.5 29.3N stem 17.5 22.6 1.7 7.8 30.4 20.1WT root 22.6 1.9 4.9 2.5 52.7 15.4N root 9.5 13.7 1.6 17.4 51.5 6.3WT ovary 15.7 ndb 2.3 25.8 32.3 23.9N ovary 13.2 7.2 1.7 26.8 32.4 18.7WT seed 8.6 nd 2.6 10.7 78.3 ndN seed 8.0 2.0 2.1 9.1 78.2 0.6

a Values (mole %) are means obtained from three independentexperiments derived from wild-type (WT) or three independenttransformed expressor plants (N) with the exception of seeds, whichwere derived from expressor N21. b nd, Not detected (<0.5%).

897




Table II. Fatty Acid Compositiona of Leaf Lipids from Wild-Type (WT) and N2 1 TransformantGlycerolipidb

MGDG DGDG PG PC PEAcyl Group'

WT N21 WT N21 WT N21 WT N21 WT N21

16:0 3.2 3.2 18.9 22.6 21.4 23.8 25.6 24.0 45.8 39.916:1 c 1.4 7.0 nd 9.1 nd nd nd 10.6 nd 3.616:1 t nd nd nd nd 31.2 32.5 nd nd nd nd16:3 13.0 11.5 nd nd nd nd nd nd nd nd18:0 0.6 0.5 3.3 3.6 3.0 2.9 5.2 3.6 5.6 4.318:1 1.7 2.7 2.5 4.5 9.1 9.0 7.5 9.8 2.1 4.518:2 5.3 5.7 5.3 4.9 15.2 13.1 44.4 38.0 33.5 31.618:3 74.8 69.5 70.0 55.2 20.2 18.8 17.0 14.0 13.0 13.0

a Values are means obtained from three independent experiments. b nd, Not detected(<0.5%). c 16:1c, 16:1 (A-9, cis); 16:1t, 16:1 (A-3, trans).

membrane lipid classes (Table II). The highest levels were

found in PC and the galactolipids. In the galactolipids, com-

pensatory changes were observed primarily in 18:3 levels.DGDG, for example, exhibited a reduction in 18:3 from 70to 55% of the total acyl fraction. In the PC fraction, reductionsin 18:2 and 18:3 were observed relative to the wild-typelevels. No change in the 16:1 (A-9 cis) level was observed inthe PG fraction, which normally contains high levels of thechloroplast-derived fatty acid A-3 trans 16:1

Specificity of 16:1 Incorporation in Polar Lipids

Positional analysis by phospholipase Al and A2 treatment(Tables III and IV) of two expressor plants indicates that themajority of the overproduced 16:1 is incorporated into thesn-2 position of PC, PE, MGDG, and DGDG. In the PC andPE fractions, 16:1 is exclusively located in the sn-2 position,whereas in the galactolipids, there appears to be a smallerbut significant amount in the sn-1 position. In this position,16:1 on the galactolipids may be representative of a fractionproduced by the prokaryotic pathway.

DISCUSSION

The introduction of yeast desaturase coding sequences

under the control of the CaMV 35S promoter and polyade-nylation signal resulted in the expression in leaf tissue ofunique mRNAs encoding the yeast enzyme. Fatty acid analy-sis of plants that expressed the gene also revealed an approx-

imate 10-fold increase in the levels of palmitoleic acid (A-916:1) in leaf lipids. Other plant organs had increased 16:1levels ranging from 2- to 20-fold (Table I). Although A-9 16:1is a minor species in plant lipids (<2%), it is the primaryproduct of the yeast enzyme that efficiently desaturates both16:0- and 18:0-CoA substrates. It is unlikely that a fractionof functional yeast enzyme would be localized in the chlo-roplast, since it would not be accessible to the appropriateelectron transport chains and it lacks the known characteris-tics of chloroplast targeting signals (i.e. the region comprisingthe first 35 amino acids of the desaturase is strongly acidicand does not contain the extremely high levels of serine or

threonine residues found in chloroplast transit peptides[13]). The yeast and rodent desaturases, in fact, contain no

N-terminal signal sequences, and, for the rat enzyme, mem-

Table l1l. Fatty Acid Compositiona of Major Chloroplast Lipids from Wild-Type (WT) and N2 1 Transformant Treated with Rhizopus Lipase (A1)Glycerolipidsb

MGDG DGDG PG

Acyl Group' WT N21 WT N21 WT N21

sn-1 sn-2 sn-1 sn-2 sn-1 sn-2 sn-1 sn-2 sn-1 sn-2 sn-1 sn-2

16:0 3.7 2.2 3.5 2.9 22.8 17.9 27.4 20.0 36.8 45.5 40.3 47.416:1 c nd 1.9 4.2 10.2 3.4 nd 4.5 11.3 nd nd 4.5 nd16:1t nd nd nd nd nd nd nd nd nd 41.2 nd 39.216:3 3.0 26.2 0.7 24.3 nd 0.9 nd 0.8 nd nd nd nd18:0 0.7 nd 0.6 nd 2.4 nd 4.1 0.9 12.1 9.6 15.0 6.418:1 2.0 0.9 3.0 1.4 2.4 2.0 4.1 2.7 12.7 11.3 14.3 12.818:2 5.4 5.3 6.0 5.2 5.3 4.8 5.0 4.4 14.7 15.7 7.6 12.518:3 84.9 63.4 82.0 56.0 66.1 74.1 54.8 59.8 23.8 17.9 18.3 21.0

a Values are means obtained from three independent experiments. b nd, Not detected (<0.5%). C 16:1c, 16:1 (/-9, cis); 16:1t, 16:1(A-3, trans).





Table IV. Fatty Acida Composition of Phospholipids from Wild-Type (WT) and N2 1 TransformantTreated with Phospholipase A2

Phospholipidb

PC PE

Acyl Group WT N21 WT N21

sn-1 sn-2 sn-1 sn-2 sn-1 sn-2 sn-I sn-2

16:0 50.0 11.1 42.5 12.4 57.1 15.4 64.8 23.316:1 nd 2.8 nd 13.5 nd nd nd 3.118:0 10.8 5.1 14.9 3.3 9.3 5.2 8.5 5.618:1 5.2 10.0 8.8 12.7 2.3 3.9 3.8 4.318:2 27.9 50.2 30.0 43.5 14.3 55.9 15.7 47.218:3 6.9 19.8 5.9 14.6 17.0 19.5 13.1 16.6

Values are means obtained from three independent experiments. b nd, Not detected (<0.5%).

brane insertion appears to occur post-translationally (37, 38).Thus, it appears that a functional yeast enzyme is producedin the transformants, that it is able to interact with the plantmicrosomal redox systems, and that the increased 16:1 levelsare a sensitive indicator of the yeast enzyme activity. Con-sequently, the majority of the A-9 16:1 found in those plantsrepresents a fatty acyl species that is derived from the cyto-plasmic/ER eukaryotic pathway.

Incorporation of the 16:1 into lipids was expected to occurprimarily at the sn-1 position of the glycerol backbone. Thiswas based on current models that suggest that acyl-transfer-ases that form eukaryotic lipids exclude 16-carbon fatty acidsfrom the sn-2 position (27, 28). Our data suggest that theseacytransferases tend to exclude saturated fatty acids andselect for unsaturated species regardless of chain length. Theoccurrence of 16:1 in the sn-2 position in wild-type controlplants would not be obvious because 16:1 is normally presentat low levels. It is significant to note that of the majormembrane lipid species, only PG showed no change in A-916:1 levels. PG is a major chloroplast-derived lipid thatappears to contain fatty acids formed exclusively by theprokaryotic pathway. It has a unique A-3 trans 16:1 speciesthat is exclusively acylated to the sn-2 position.The ability of the transformants to promote the activity of

a Cyt b5-dependent acyl-CoA desaturase offers supportingevidence for several proposed mechanisms of plant fatty acidmetabolism. Fatty acids are known to be synthesized inchloroplasts and other plastids in the form of ACP derivatives(12). The cytosol serves, however, as a major site of furtherdesaturation of fatty acids and their assimilation into complexmembrane lipids. This requires transport of fatty acids acrossthe chloroplast envelope and their subsequent conversion toacyl-CoA derivatives that are required for acyltransferasereactions. The ability of the yeast enzyme to catalyze desat-uration of 16:0-CoA indicates that there is a substan-tial cytosolic pool of that species prior to its acylation intophospholipids.Although Cyt b5-dependent mechanisms have been pro-

posed for plant ER A-12 desaturation, early studies on themicrosomal electron transport components of safflower cot-yledons (11) failed to show evidence of Cyt bs involvement.The presence of yeast Cyt b5-dependent desaturase activity

in these tissues provides evidence for the existence of a relatedplant ER electron transport chain that can support desatura-tion in leaf tissue. It also supports recent evidence of Cyt bsinvolvement in A-12 desaturation in developing safflowercotyledons (31). We have previously shown (35) that the ratdesaturase can be driven efficiently by the yeast cytoplasmicredox system. The ability of the yeast enzyme to catalyzedesaturation using plant ER cytochromes further extends thefunctional homology of Cyt b5-dependent desaturases be-tween modem eukaryotes, and suggests that the mechanismsinvolved in electron transfer to desaturases occurred at arelatively early stage in the evolution of eukaryotes.Most of the enzymological studies of plant microsomal

desaturases have been done on developing oilseed tissues,since there is a large induction of lipid biosynthetic enzymeactivities over a short developmental window (31, 36). Giventhe lower levels of A-9 16:1 found in the transformant seedoils, it appears that the CaMV promoter construct used inthese studies may not be as active during that developmentalstage as it is in leaf tissue. We would expect a similar fusionof the yeast gene to a seed-specific promoter in an appropriateoil seed plant to yield much higher levels of activity in thatorgan.Although the yeast desaturase has been shown in vitro to

desaturate both 16:0- and 18:0-CoA with equal efficiency (5),appreciable increases in the levels of 18:1 (oleic acid) werenot detected. This was not expected, since stearoyl-ACP(18:0-ACP) is efficiently converted to oleoyl-ACP (18:1-ACP)in the plastids by the native plant A-9 desaturase, resultingin relatively small amounts of 18:0-CoA available in thecytosol compared to those of 16:0-CoA. The rapid incorpo-ration in the cytosol of any available 18:0-CoA into glycero-lipids might further limit substrate availability to the yeastenzyme. Furthermore, since the bulk of 18-carbon fatty acidsin plants are polyunsaturated (about 90% of the total intobacco), the formation of any 18:1 by the yeast desaturasecould be obscured by further desaturation. It is interesting tonote that although the plants expressing the yeast desaturaseare independent transformants with presumably differentlevels of expression, all of those tested exhibit a narrowerthan expected range in 16:1 levels. This suggests that there

899




is some limiting factor other than enzyme concentration thatdetermines the extent of overproduction of this fatty acid.The expressors contain high levels of A-9 cis 16:1 in

chloroplast lipids such as MGDG and DGDG. This indicatesthat a significant fraction of the fatty acids modified by thecytoplasmic yeast desaturase show retrograde movement tothe chloroplast compartment. Prokaryotic lipids in wild-typeplants are conventionally identified by the presence of 16-carbon fatty acids (16:3 and 16:0), which are synthesized andacylated into the sn-2 position of MGDG within the chloro-plast. The contrast between the almost identical levels ob-served with 16:3 and 16:0 in the sn-2 position of MGDG inthe expressors and the controls as opposed to the increased16:1 (and the correspondingly reduced 18:3) in that sameposition suggests that the latter are derived from eukaryoticlipid precursors (Table III). This supports the prevailing ideathat there are two distinct pools of MGDG lipids, one formedin the chloroplast that contains 16:3 in the sn-2 position andone (containing 16:1- and 18-carbon unsaturates in the sn-2position) that is derived from DAGs formed by the cyto-plasmic eukaryotic pathway.

In conclusion, we have demonstrated that a foreign desat-urase can function in plants and alter fatty acyl and mem-brane lipid compositions. Unlike the plants described inrecently published reports of the effects of a yeast invertasegene that produced significant morphological and develop-mental changes when introduced into tobacco (29), the trans-formed plants in our study tolerated changes produced bythe yeast desaturase gene with little observable alteration inphenotype. These results suggest that it may be feasible toproduce broad changes in plant lipid composition throughthe introduction of synthetic or heterologous lipid biosyn-thetic genes. These may be useful in developing a range ofplant strains for a variety of applications. Furthermore, stud-ies to determine the limiting factor(s) involved in these fattyacid and lipid changes may lend insight into some of thecoordinate regulatory mechanisms involved in plant fattyacid and lipid biosynthesis.

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expression ofthe yeasta-9 fatty acid desaturase in nicotiana

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