Distribution and nature of surface wax in fungal spores and mechanisms of wax biosynthesis in Brassica oleracea



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Normal alkanes were common in surface waxes of all Basidiomycete chlamydospores and uredospores analyzed as well as the conidiospores from the Ascomycete studied. The spore samples were extracted with organic solvents, the extracts fractionated by silica gel chromatography and then analyzed by gas chromatography and by a gas chromatographic-mass spectrometric technique. Odd carbon-numbered alkanes predominated. Carbon numbers ranged from C[lowered 14] to C[lowered 37]. The major n-alkane components and total hydrocarbons present were C[lowered 27] for Ustilago maydis (40 ppm), C[lowered 29] for Puccinia graminis (105 ppm), C[lowered 29] and C[lowered 31] for Urocystis agropyri (126 ppm), C[lowered 27] and C[lowered 35] for Ustilago nuda (58 ppm), C[lowered 29] and C[lowered 31] for Ustilago avenae (53 ppm) and C[lowered 29] for Sphacelotheca reiliana (146 ppm). The conidiospores of Aspergillus niger had n-C[lowered 25] as the predominant alkane and a total of 5-10 ppm hydrocarbons present. For the most part the alkanes present in fungal spores were similar to the distribution known to occur in many higher plants; however, each spore type possessed a different distribution pattern. The alkanes present in chlamydospores of U. maydis had a distinctly different distribution than those present in any part of the host fruit whether from an uninfected or infected corn plant. These findings were interpreted as meaning that the alkanes observed apparently did not represent mass contamination by host waxes or that the parasite withdrew large quantities of preformed long-chain alkanes from the host to produce its own wax. In fact the infected cob material demonstrated a slightly different distribution of alkanes than the normal host suggesting an involvement between the host and parasite at some point in their lipid metabolism. Methyl esters of fatty acids comprised a large portion of the surface wax present on the chlamydospores of U. maydis. The methyl esters of C[lowered 18] mono- and dienoic fatty acids were present in abundance. In both the free acids and the natural methyl esters, the distribution of carbon skeletons was similar with the predominant compounds having even carbon-numbered chains. P. graminis spores had a rather complex homologous series of both polycyclic and polyunsaturated compounds present in the benzene eluate of the wax extracts. Mass spectra of the polycyclic compounds suggest that a steroid nucleus makes up the cyclic portion of the molecule. Due to the large number and structural diversity of the alkanes present in the fungal spores investigated, fungi were not seriously considered as a candidate for initial studies for wax biosynthesis. Cabbage (Brassica oleracea var. Flat Dutch) was selected as the organism for study because of the simple composition and abundance of leaf wax. The major components of the lipid fraction of young cabbage leaves as determined by column chromatography, gas chromatography, thin-layer chromatography and gas chromatography-mass spectrometry techniques were n-alkanes, ketones, esters and free fatty acids. The saturated hydrocarbons range from about C[lowered 20] through C[lowered 31] with C[lowered 27] (2-3%), C[lowered 28] (<1%), C[lowered 29] (90-92%), C[lowered 31] (<1%) and (5-20%) comprising the major alkanes present. The benzene eluate from the silica gel column contained one major component identified as 15-nonacosanone. Palmitic acid was the major saturated fatty acid which comprised 12% of the methanol eluate. The predominant components of the methanol fraction were the unsaturated fatty acids, linoleic (18%) and linolenic acid (22%). Acetate was incorporated equally well into both alkanes and ketones irrespective of the position of the label in the acetate. Some label also went into the long-chain esters. Imidazole did not substantially reduce the incorporation of labeled acetate. Labeled fatty acids were good precursors for the synthesis of various wax components. Both the paraffins and ketones were labeled at approximately the same rate. In all experiments with fatty acids, C[lowered 14]>C[lowered 16]>C[lowered 14] in terms their effectiveness as wax precursors. The acids were incorporated into the wax components irrespective of the position of the label within the substrate molecule. Terminal labeled acids were not as effective precursors as the uniformly labeled acids. Crude cell-free systems were capable of low levels of wax synthesis in cabbage leaf extracts from a stearic-1-[raised 14]C acid substrate. All purification attempts resulted in a loss of activity. The soluble protein fraction of cabbage leaves, however, was capable of synthesizing esters from a stearic-1-[raised 14]C acid substrate even when moderately purified. A hypothesis was proposed which supports the concept that palmitic acid is the major natural precursor for the long-chain wax components through a process of elongation by C[lowered 2] units to C[lowered 30] acid which is then decarboxylated and reduced to yield, in the case of cabbage leaves, nonacosane. The enzyme system, up to the point of decarboxylation and reduction, may resemble the fatty acid synthetase system known to occur in higher plants. Stearic and other longer chain acids may enter the system one or more C[lowered 2] units ahead of palmitic acid. Ketones may be formed in this system if the 18-carbon skeleton attached to the proposed synthetase carrier complex undergoes a [gamma]-dehydration instead of a [beta]-dehydration, thereby leading to the formation of a double bond between the [beta] and [gamma] carbons of the 18-carbon intermediate. Hydration of this 18-carbon intermediate would lead to a hydroxylated compound with the oxygen on the [gamma]-carbon. By allowing the oxygen to remain throughout the extension process as described for the paraffins, the product would then be a symetrical ketone with 29 carbons atoms. The final decarboxylation and reduction, if the proposed mechanism is followed, must be carried out in conjunction with a reduction of the terminal carbon as the long-chain compound is released from the carrier complex. Very long-chain free fatty acids may also be synthesized by this same complex if they are released as the elongation process proceeds. A mechanism for the formation of secondary alcohols and ketones, such as 10-hydroxynonacosane, 10-hydroxy-15-oxononacosane and 10-oxo-15-hydroxynonacosane, was also proposed which involved the use of common 9-10 monoenoic fatty acids. The acids must undergo hydration of the 9-10 double bond, yielding a 10-hydroxysteryl-CoA which can be extended to the desired length by the proposed alkane synthetase complex.