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Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier protein thioesterase

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We have examined production of mediumchain fatty acids by Brassica napus L. plants transformed with a California bay (Umbellularia californica) medium-chain acyl-acyl carrier protein (ACP) thioesterase (UcFatB1) cDNA under the control of the constitutive cauliflower mosaic virus 35S promoter. These plants were found to accumulate medium-chain fatty acids in seeds but not in leaves or roots. Assay of thioesterase activity in extracts of leaves indicated that lauroyl-ACP thioesterase activity is comparable to oleoyl-ACP thioesterase (EC activity in transformant leaves. Furthermore, leaf lauroyl-ACP thioesterase activity was in excess of that which produced a significant increase in the amount of laurate (12:0) in seed. Studies in which isolated chloroplasts were 14C-labelled were used to evaluate whether medium-chain fatty acids were produced in transformed leaves. Up to 34% of the fatty acids synthesized in vitro by isolated chloroplasts were 12:0. These results demonstrate that the normally seed-localized lauroyl-ACP thioesterase can be expressed in active form in leaves, imported into chloroplasts and can access acyl-ACP intermediates of leaf de-novo fatty acid synthesis. The most likely explanation for the lack of accumulation of 12:0 in transformed leaves is its rapid degradation by β-oxidation. In support of this hypothesis, isocitrate lyase (EC activity was found to be significantly increased in plants transformed with 35S-UcFatB1.

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acyl carrier protein


cauliflower mosaic virus


Brassica napus cultivar 212/86

event 8:


event 11:



fatty acid synthase


isocitrate lyase


β-keto-acyl ACP synthase


malate synthase


oleoyl-ACP thioesterase



UcFatB1 :

California bay medium-chain acyl-ACP thioesterase


  1. Arnon DI (1948) Copper enzymes in isolated chloroplasts. Plant Physiol 24: 1–15

  2. Bafor M, Stobart AK, Stymne S (1990) Properties of the glycerol acylating enzymes in microsomal preparations from the developing seeds of safflower (Carthamus tinctorius) and turnip rape (Brassica campestris) and their ability to assemble cocoa-butter type fats. J Am Oil Chem Soc 67: 217–225

  3. Banas A, Johansson I, Stymne S (1992) Plant microsomal phospholipases exhibit a preference for phosphatidylcholine with oxygenated acyl groups. Plant Sci 84: 137–144

  4. Battey JF, Ohlrogge JB (1989) A comparison of the metabolic fate of fatty acids of different chain lengths in developing oilseeds. Plant Physiol 90: 835–840

  5. Beevers H (1979) Microbodies in higher plants. Annu Rev Plant Physiol 30: 159–193

  6. Bligh EG, Dyer WJ (1959) A rapid method for total lipid extraction and purification. Can J Biochem Physiol 37: 911–917

  7. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal Biochem 72: 248–254

  8. Caboon EB, Shanklin J, Ohlrogge JB (1992) Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. Proc Natl Acad Sci USA 89: 11184–11188

  9. Chen Z-L, Pan N-S, Beachy RN (1988) A DNA sequence element that confers seed-specific enhancement to a constitutive promoter. EMBO J 7: 297–302

  10. Cooper G, Beevers H (1969) Mitochondria and glyoxysomes from castor bean endosperm. J Biol Chem 244: 3507–3513

  11. Davies HM (1993) Medium chain acyl-ACP hydrolysis activities of developing oilseeds. Phytochemistry 33: 1353–1356

  12. Davies HM, Anderson L, Fan C, Hawkins DJ (1991) Developmental induction, purification and further characterization of 12:0-ACP thioesterase from immature cotyledons of Umbellularia californica. Arch Biochem Biophys 290: 37–45

  13. DeBellis L, Picciarelli P, Pistelli L, Alpi A (1990) Localisation of glyoxylate-cycle marker enzymes in peroxisomes of senescent leaves and green cotyledons. Planta 180: 435–439

  14. Dormann P, Frentzen M, Ohlrogge JB (1994) Specificities of the acyl-acyl carrier protein (ACP) thioesterase and glycerol 3-phosphate acyltransferase for octadecenoyl-ACP isomers. Plant Physiol 104: 839–844

  15. Ettinger WF, Harada JJ (1990) Translational or post-translational processes affect differentially the accumulation of isocitrate lyase and malate synthase proteins and enzyme activities in embryos and seedlings of Brassica napus. Arch Biochem Biophys 281: 139–143

  16. Fuhrmann J, Heise K-P (1993) Factors controlling medium-chain fatty acid synthesis in plastids from maturing Cuphea embryos. Z Naturforsch 48c: 616–622

  17. Graham IA, Leaver CJ, Smith SM (1992) Induction of malate synthase gene expression in senescent and detached organs of cucumber. Plant Cell 4: 349–357

  18. Gut H, Matile P (1988) Apparent induction of key enzymes of the glyoxylic acid cycle in senescent barley leaves. Planta 176: 548–550

  19. Harwood JL (1988) Fatty acid metabolism. Annu Rev Plant Phys Plant Mol Biol 39: 101–138

  20. Hilditch TP, Williams PN (1964) In: Siegenthaler PA (ed) The chemical constitution of natural fats. Wiley, New York, pp 332–343

  21. Kates M (1972) Techniques of lipidology. In: Work TS, Work E (eds) Laboratory techniques in biochemistry and molecular biology, vol 3 part II. Elsevier/North Holland, Amsterdam, pp 527–529

  22. Kay R, Chan A, Daly M, McPherson J (1987) Duplication of CaMV 35S promoter sequences creates a strong enhancer for plant genes. Science 236: 1299–1302

  23. Kridl JC, McCarter DW, Rose RE, Scherer DE, Knutzon DS, Radke SE, Knauf VC (1991) Isolation and characterization of an expressed napin gene from Brassica rapa. Seed Science Res 1: 209–219

  24. Lynch DV, Thompson GA Jr., (1984) Retailored lipid molecular species: a tactical mechanism for modulating membrane properties. TIBS 9: 442–445

  25. McBride KE, Summerfelt KR (1990) Improved binary vectors for Agrobacterium-mediated plant transformation. Plant Mol Biol 14: 269–276

  26. McBride KE, Schaaf DJ, Daley M, Stalker DM (1994) Controlled expression of plastid transgenes in plants based on a nuclear DNA-encoded and plastid-targeted T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 91: 7301–7305

  27. Odell JT, Nagy F, Chua N-H (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812

  28. Ohlrogge JB, Kuhn DK, Stumpf PK (1979) Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad Sci USA 76: 1194–1198

  29. Ohlrogge JB, Shine WE, Stumpf PK (1978) Fat metabolism in higher plants: characterization of plant acyl-ACP and acyl-CoA hydrolases. Arch Biochem Biophys 189: 382–391

  30. Oo K-C, Huang AHC (1989) Lysophosphatidate acyltransferase activities in the microsomes from palm endosperm, maize scutellum and rapeseed cotyledons of maturing seeds. Plant Physiol 91: 1288–1295

  31. Pollard MR, Anderson L, Fan C, Hawkins DJ, Davies HM (1991) A specific acyl-ACP thioesterase implicated in medium chain fatty acid production in immature cotyledons of Umbellularia californica. Arch Biochem Biophys 284: 306–312

  32. Radke SE, Andrews BM, Moloney MM, Crouch ML, Kridl JC, Knauf VC (1992) Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene. Theor Appl Genet 75: 685–694

  33. Roughan G (1987) Long chain fatty acid synthesis and utilisation by isolated chloroplasts. Methods Enzymol 148: 327–337

  34. Slabas AR, Fawcett T (1992) The biochemistry and molecular biology of plant lipid biosynthesis. Plant Mol Biol 19: 169–191

  35. Somerville C, Browse J (1991) Plant lipids: metabolism, mutants and membranes. Science 252: 80–87

  36. Stumpf PK, Boardman NK (1970) Fat metabolism in higher plants XXXIX: effect of adenosine triphosphate and Triton X-100 on lipid synthesis by isolated spinach chloroplasts. J Biol Chem 245: 2579–2587

  37. Titus DE, Becker WM (1985) Investigation of the glyoxysome/peroxisome transition in germinating cucumber cotyledons using double-label immunoelectron microscopy. J Cell Biol 101: 1288–1299

  38. Van deLoo FJ, Fox BG, Somerville C (1993) Unusual fatty acids. In: Moore TS Jr (ed) Lipid metabolism in plants. CRC Press, Boca Raton, pp. 91–126

  39. Voelker TA, Davies HM (1994) Alteration of the specificity and regulation of fatty acid synthesis of E. coli by expression of a plant medium-chain acyl-acyl-carrier protein thioesterase. J Bacteriol 176: 7320–7327

  40. Voelker TA, Worrell AC, Anderson L, Bleibaum J, Fan C, Hawkins DJ, Radke SE, Davies HM (1992) Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants. Science 257: 72–74

  41. Weaire P, Kekwick R (1975) The synthesis of fatty acids in Avocado mesocarp and cauliflower bud tissue. Biochem J 146: 425–437

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Author information

Correspondence to John B. Ohlrogge.

Additional information

We are indebted to Calgene's Brossica-transformation, growth-chamber, greenhouse, and lipid-analysis personnel. Maelor Davies conducted the initial tranformant analysis. We thank Laura Olsen for IL and MS Western blot analysis and advice on IL and MS activity assays. This work was supported in part by a grant from the U.S. Department of Energy (No. DE-FG02-87ER12729). Acknowledgement is made to the Michigan Agricultural Experiment Station for its support of this research.

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Eccleston, V.S., Cranmer, A.M., Voelker, T.A. et al. Medium-chain fatty acid biosynthesis and utilization in Brassica napus plants expressing lauroyl-acyl carrier protein thioesterase. Planta 198, 46–53 (1996). https://doi.org/10.1007/BF00197585

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Key words

  • β-Oxidation
  • Brassica
  • Fatty acid synthesis
  • Leaf
  • Medium-chain acyl-ACP thioesterase
  • Umbellularia