Epicuticular wax production was evaluated along the length of expanding leek (Allium porrum L.) leaves to gain insight into the regulation of wax production. Leaf segments from the bottom to the top were analyzed for (a) wax composition and load; (b) microsomal fatty acid elongase, plastidial fatty acid synthase, and acyl-acyl carrier protein (ACP) thioesterase activities; and (c) tissue and cellular morphological changes. The level of total wax, which was low at the bottom, increased 23-fold along the length of the leaf, whereas accumulation of the hentriacontan-16-one increased more than 1000-fold. The onset of wax accumulation was not linked to cell elongation but, rather, occurred several centimeters above the leaf base. Peak microsomal fatty acid elongation activity preceded the onset of wax accumulation, and the maximum fatty acid synthase activity was coincident with the onset. The C16:0-and C18:0-ACP-hydrolyzing activities changed relatively little along the leaf, whereas C18:1-ACP-hydrolyzing activity increased slightly prior to the peak elongase activity. Electron micrographic analyses revealed that wax crystal formation was asynchronous among cells in the initial stages of wax deposition, and morphological changes in the cuticle and cell wall preceded the appearance of wax crystals. These studies demonstrated that wax production and microsomal fatty acid elongation activities were induced within a defined and identifiable region of the expanding leek leaf and provide the foundation for future molecular studies.

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We have characterized the occurrence and expression of multiple acyl carrier protein (ACP) isoforms in Arabidopsis thaliana (L.) Heynh ecotype Columbia. Immunoblot analysis of ACPs from Arabidopsis tissues separated by native polyacrylamide gel electrophoresis and I molar urea polyacrylamide gel electrophoresis revealed a complex pattem of multiple ACP isoforms. All tissues examined (leaves, roots, and seeds) expressed at least three forms of ACP. The immunoblot identifications of ACP bands were confirmed by acylation of ACP extracts with Escherichia coli acyl-ACP synthetase. A full-length cDNA clone has been isolated that has 70% identity with a previously characterized Arabidopsis genomic ACP clone (ACP-1) (MA Post-Beittenmiller, A HlousekRadojcic, JB Ohirogge [1989] Nucleic Acids Res 17: 1777). Based on RNA blot analysis, the cDNA clone represents an ACP that is expressed in leaves, seeds, and roots. In order to identify the protein products of each known ACP gene, their mature coding sequences have been expressed in E. coli. Using polymerase chain reactions, exons 11 and IlIl of the genomic ACP-1 clone and the mature coding sequences of the ACP-2 cDNA clone were subcloned into E. coil expression vectors. Site-directed mutagenesis was used to convert the amino acid sequence of the ACP-2 cDNA clone to that of the A2 clone of Lamppa and Jacks ([1991] Plant Mol Biol 16: 469-474), ACP-3. The three E. coli-expressed proteins have different mobilities on polyacrylamide gel electrophoresis gels and each comigrates with a different Arabidopsis ACP isoform expressed in leaves, seeds, and roots. Thus, all of the three cloned ACPs appear to be constitutively expressed Arabidopsis ACPs. In addition to these three ACP isoforms, protein blots indicate that seed, leaf, and root each express one or more tissue-specific isoforms.

Summary The substrate specificity of fatty acid elongase was studied using an oil body fraction from developing seeds of Brassica napus. ATP was essential for high rates of elongase activity, but there was no apparent requirement for oleoyl‐CoA, oleic acid (18:1) or CoA. Furthermore, 14C from 18:1‐CoA was incorporated into eicosenoic (20:1) and erucic (22:1) acids at a much slower rate than 14C from malonyl‐CoA. Incubation of [14C]18:1‐CoA with the oil body fraction resulted in a rapid loss of [14C]18:1‐CoA into several lipid fractions whether in the absence or presence of ATP, but the loss of 18:1‐CoA had a comparatively small effect on the overall rate of elongation. Acyl‐CoAs were derivatized to their respective acylbutylamide and analyzed by gas chromatography‐mass spectrometry. This analysis of acyl‐CoAs demonstrated that there was no detectable 20:1‐CoA or 22:1‐CoA at 0 min incubation, while newly synthesized 20:1‐CoA and 22:1‐CoA were present at 10 min. Analysis of the %14C of the substrates and products of the elongation reaction revealed that the endogenous pool of 18:1‐CoA is quite small in elongase preparations. In addition, [14C]18:1‐CoA added to the incubation, although incorporated into lipids, was not significantly diluted by turnover or new synthesis. In contrast, the %14C of the 20:1‐CoA was two‐ to threefold less than that of the 18:1‐CoA. Taken together, these results indicate that the [14C]18:1 from the [14C]18:1‐CoA was diluted in an intermediate 18:1 pool and that the 18:1‐CoA was not the major donor of the acyl group to the elongase reaction.

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