Plant Physiol. (1992) 100, 894-901 Received for publication April 1, 1992 0032-0889/92/100/0894/08/$01.00/0 Accepted June 16, 1992 Expression of the Yeast A-9 Fatty Acid Desaturase in Nicotiana 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 rapidly formed from 18:0-ACP in the plastid by a soluble, To examine the processes of plant cytoplasmic fatty acid desat- ferredoxin-dependent, A-9 desaturase, which inserts a double uration and glycerolipid biosynthesis, the protein coding sequence bond between carbons 9 and 10 of the fatty acyl chain (16, of the endoplasmic reticulum cytochrome b5-dependent, A-9 fatty 25). These fatty acids may then be shunted into one of two acid desaturase gene from Saccharomyces cerevisiae was intro- distinct routes-a plastid-localized 'prokaryotic' pathway or duced into Nicotiana tabacum via Agrobacterium transformation. a cytosolic/ER "eukaryotic" pathway (28)-for further modi- All transformed plants expressing the yeast gene at the mRNA level fication and acylation into glycerolipids. According to this exhibited an approximately 10-fold increase in the levels of pal- model, fatty acids that are acylated into glycerolipids in the mitoleic acid (16:1) in leaf tissue. This fatty acid species is found chloroplast are distinctive in that they tend to have 16-carbon in very low levels (less than 2%) in wild-type plants. These results species in the sn-2 position. indicate that the yeast desaturase can function in plants, presum- Other plastid enzymes that have desaturase functions have ably by using a leaf microsomal cytochrome b5-mediated electron been identified by of transport system. Lipid analysis demonstrated that the overpro- analysis isolated chloroplasts or Arabi- duced 16:1 is incorporated into most of the major polar lipid dopsis mutants (7, 30, 32). Most polyunsaturated 18-carbon classes, including the cytoplasmically produced "eukaryotic" frac- plant fatty acids, however, appear to be formed in the cytosol tion of the chloroplast galactolipids. 16:1 was not found, however, by ER-bound enzymes. Fatty acids that are transported out in phosphatidyl glycerol, which is considered to be produced almost of the chloroplast are thought to be converted to CoA esters exclusively in the chloroplast. Despite these changes in membrane and subsequently incorporated into glycerolipids by the eu- lipid composition, no obvious phenotypic differences were appar- karyotic route (2, 6, 18, 26). These lipids characteristically ent in the transformed plants. Positional analysis shows that the contain 18-carbon fatty acids in the sn-2 position. cytoplasmically produced 16:1 is found primarily in the sn-2 posi- Once incorporated into phospholipids, a cytoplasmic de- tion of phosphatidylcholine, phosphatidylethanolamine, monoga- saturase catalyzes the formation of the A-12 double bond in lactosyldiacylglycerol, and digalactosyldiacylglycerol. The posi- 18:1. This desaturase is tional data suggest that the sn-2 acyltransferases responsible for thought to be similar to the animal the "eukaryotic" arrangement of 16- and 18- carbon fatty acids in and fungal desaturases because it is membrane bound and glycerolipids are selective for unsaturated fatty acids rather than appears to require a Cyt b5-mediated electron transport chain chain length. (31). This enzyme, however, has not been purified from higher 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 eukaryotic arrangement of acids In plants, unsaturated fatty acids are formed and fatty (an unsaturated 18-carbon species incorpo- in the sn-2 position) are probably derived from intermediates rated into complex lipids in two distinct cellular compart- originating from ments. The initial of eukaryotic lipids such as PC. One possibility products de novo fatty acid synthesis, is the conversion of PC to DAG, which is transported into which occurs almost exclusively in the plastids (12), are the the where saturated chloroplast, it is used to synthesize galacto- and species 16:0-ACP2 and 18:0-ACP. 18:1-ACP is sulfo-lipids (10, 14, 28, 40). Thus, chloroplast lipids such as MGDG and DGDG are thought to be formed from two 'This work was supported by National Science Foundation grant distinct pools, with the prokaryotic type having 16-carbon DMB84-17802, Biomedical Research Group grant RR-07058-21 fatty acids in the sn-2 position and the eukaryotic type having from the U.S. Public Health Service, and by a grant from the Bureau 18-carbon species in the sn-2 position. of Biological Research Charles and Johanna Busch Memorial Fund. 2 Abbreviations: ACP, acyl carrier protein; X:Y, fatty acyl groups The metabolic flux of fatty acids between the two pathways containing X carbon atoms and Y cis double bonds; AN X:Y, as above appears to be highly regulated during growth and develop- with cis double bonds located at position N relative to the carboxyl ment. Many plants, for example, can contain significant end of the chain; MGDG, monogalactosyldiacylglycerol; DGDG, amounts of fatty acids, such as 16:3 in leaf tissue, that are digalactosyldiacylglycerol; PC, phosphatidylcholine; PE, phosphati- derived from the prokaryotic pathway, but these are usually dylethanolamine; DAG, diacylglycerol; PG, phosphatidylglycerol; missing from seed oils, which contain acyl species derived CaMV, cauliflower mosaic virus. almost exclusively from the cytosolic/ER route (36). 894 YEAST A-9 DESATURASE IN TOBACCO 895 The existence of two compartmentalized systems indicates that regulatory studies and efforts to modify plant lipids will require development of information not only about the func- tions of individual lipid metabolic enzymes, but also about the coordinated movement of precursors and complex lipids between the two compartments. We attempted to investigate the features of plant lipid metabolism discussed above through the introduction of a yeast cytoplasmic lipid modifying enzyme into tobacco. The Saccharomyces cerevisiae A-9 desaturase is a Cyt b5-depend- ent, ER enzyme that efficiently converts 16:0-CoA to 16:1 (4, I I 34), a minor fatty acid species in plants. Other work in our laboratory has recently shown that there is a broad functional Gus Marker Gene homology between Cyt b5-dependent desaturases by success- Removed to Allow Insertion fully expressing the rat A-9 desaturase in yeast (35). of the Desatumase ConstructZ If significant amounts of 16:0-CoA are present in the plant cytosol, its desaturation to 16:1 by the yeast enzyme should Figure 1. Construction of the Agrobacterium vector for plant trans- formations. The GUS marker chimeric gene in the T-DNA region provide a useful reporter for the metabolic conversions of of pBI101 was replaced with the yeast desaturase coding se- cytosolic fatty acids into membrane lipids. The introduction quences under the control of the CaMV 35S enhancer-promoter of foreign lipid biosynthetic genes into specific subcellular (Enh-Prom.). locations may also provide a useful tool for uncovering the reactions and components of key regulatory systems. The increased production of monounsaturated fats may also alter replaced with the HindIII/EcoRI fragment of pFF19 contain- membrane fluid properties, allowing the examination of the ing the CaMV 35S promoter with the enhancer duplicated effects on membrane associated physiological functions. To and the 35S polyadenylation signal (39). The yeast gene test these possibilities, the CaMV 35S promoter and polyad- fragment with the BamHI linkers was then inserted into the enylation signal were fused to the coding sequence of the BamHI site in the multiple cloning region of the pFF19 yeast gene. The chimeric gene was introduced into Nicotiana fragment. The resulting plasmid containing the yeast desat- tabacum via Agrobacterium transformation. urase coding region modified for plant expression and the In this article, we demonstrate that the cytoplasmic yeast selectable marker gene NPTII was introduced into the binary enzyme functions in plants and converts significant amounts Agrobacterium strain LBA4404 using the freeze-thaw method of 16:0 to 16:1. The 16:1 product is found in high levels (1). Transformed control plants were formed using the con- primarily in the sn-2 position of PC and other polar lipids, struct described above (including the marker NPTII), but including the galactolipids of the chloroplast. without the yeast desaturase coding sequence. Plant transformations were performed by the leaf disk MATERIALS AND METHODS method as described by Horsch et al. (15) with the following changes. The Agrobacterium was centrifuged at 5OOg for 10 Plants, Bacterial Strains, and Media min and resuspended in 50 mL of LB medium without Nicotiana tabacum var W3 was used in these studies. Leaf antibiotics just prior to infection of the leaf disks. After disks used for transformations were taken from axenic plants cocultivation, the Agrobacterium was cleared from the leaf grown in the light at 220C in Murashige and Skoog medium disks using augmentin (Beecham Chemicals) instead of car- (24) with 2% sucrose. Escherichia coli strain HB101 was used benicillin. Kanamycin-resistant plantlets were transplanted for intermediate cloning and propagation of plasmids. Agro- to the greenhouse and grown under standard greenhouse bacterium strain LBA4404, which contains the helper plasmid conditions. pAL4404, was used for plant transformations. Agrobacterium was grown in LB medium at 250C with appropriate antibi- Analysis of Transformants otics. All DNA manipulations were performed according to standard techniques (3, 22). Southern blots were performed according to standard pro- cedures (33). Plant DNA was extracted by the method of Plasmid Constructs Junghans and Metzlaff (19). Typically, 10 ,ug of HindlIl-cut DNA per lane was run on an agarose gel.