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- Experimental procedures
About 15% of the epidermal wax on Hordeum vulgare cv. Bonus barley spikes is n-alkanes. Longer homologues are greatly reduced in the eceriferum mutants, cer-a6, cer-e8, cer-n26, cer-n53, cer-n985, cer-x60, cer-yc135 and cer-yl187. Simultaneously hydrocarbons accounting for only traces in the wild-type become prominent in the mutants, although their chain-length distributions remain unchanged. Accordingly several new hydrocarbon series were identified. The two major ones were C23–C35cis monoenoic alkenes (the major 9-ene isomer was part of a homologous series including 11, 13 and 15-enes), and the novel C27–C31 cyclopropanes (the ring carbons of major isomers were 9,10 and 11,12 with lesser amounts of 13,14). Three minor series included 2- and 3-methylalkanes plus C25–C33 internally branched alkanes (methyls on carbons 9, 11, 13, 15 or 17; shorter homologues dominated by the 9 isomer, longer homologues by 11, 13 or 15 isomers). Acyl chains destined for spike waxes are synthesized via acyl and polyketide elongase systems plus associated reductive and decarbonylative/decarboxylative enzyme systems. Both elongation systems are defective in synthesizing C32 acyl chains in all nine mutants. The similarities in the position of the chemical groups (primarily on carbon 9, secondarily on carbon 11) of the alkenes, cyclopropanes and internally branched methyl alkanes imply an origin from a common, hitherto unrecognized associated pathway in barley, designated the enoic pathway. The elongation system leading to the enoic derived hydrocarbons differs from the known elongation systems by inclusion of a mechanism for introducing a double bond.
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- Experimental procedures
Enabling the movement of the ancestors of plants from their aquatic environment onto land required the development of a lipid coating as protection against water loss. This lipid coat or cuticle on the aerial surface of plants frequently consists of an aliphatic, outer-most layer of epicuticular wax decorating a hydrophobic layer composed of the cutin polymer in which intracuticular waxes are embedded. The lipid classes composing the surface wax generally include very-long-chain n-alkanes, aldehydes, primary alcohols, esters and free fatty acids as in Zea mays (Bianchi et al., 1984), which are often supplemented by other lipids such as the secondary alcohols and ketones characterizing Arabidopsis thaliana (Kunst and Samuels, 2003). Early experiments suggested that C18 acyl chains synthesized by fatty acid synthase (FAS) served as precursors for further elongation by a membrane-localized enzyme complex, an elongase (ELS) consisting of a condensing enzyme that added a C2 unit, giving an acyl chain that was subjected to keto reduction, dehydration and enoyl reduction reactions yielding a saturated chain substrate for the next extension. Action of one or more ELS complexes gave elongated products that (i) served as precursors for associated enzyme complexes such as the acyl reduction pathway (giving even-chain-length aldehydes, primary alcohols and esters) and the acyl decarboxylation/decarbonylation pathways (forming odd-chain-length n-alkanes, secondary alcohols and ketones), or (ii) were exported to the plant surface giving free fatty acids (von Wettstein-Knowles, 1995). These early proposals are now being substantiated by the isolation and characterization of ELS genes, namely the Arabidopsis CER6 and KCS genes encoding condensing enzymes (Fiebig et al., 2000; Todd et al., 1999), the Zea mays gl8a and gl8b genes encoding keto reductases (Dietrich et al., 2005; Xu et al., 2002) and the Arabidopsis CER10 gene encoding an enoyl reductase (Zheng et al., 2005). Genes for reductive pathway components include Arabidopsis CER4 encoding an aldehyde reductase, closely related to the Simmondsia chinensis FAR gene that participates in primary alcohol synthesis in seeds (Metz et al., 2001). From the latter species, a gene encoding a wax synthase forming esters in seeds has also been isolated (Lardizabal et al., 2000). In addition, the Arabidopsis CER5 gene encodes a plasma membrane-localized ABC transporter that is involved in moving the wax lipids toward the cuticle surface (Pighin et al., 2004).
Acyl ELS complexes differ with respect to their specificity for the elongation substrate, as do FAS complexes (von Wettstein-Knowles, 1993). Radioactive tracer, inhibitor and genetic studies established that, in barley, a minimum of three acyl ELS complexes participate in the seven C2 additions extending 18 carbon chains to give 20, 22–26 and 28–32 carbon chains Mikkelsen, 1978). The resulting acylCoAs are substrates for aldehydes, primary alcohols, esters, n-alkanes, secondary alcohols and free fatty acids. Of these, the primary alcohols are the predominating lipid class on leaves and lower leaf sheaths. On the uppermost leaf sheaths and internodes plus the spikes (floral structure), synthesis of the predominating wax lipids requires a different type of ELS. These polyketide ELS complexes omit the three tailoring reactions following specific C2 extensions, resulting in a substrate for the next elongation containing a 3-oxo group (von Wettstein-Knowles, 1993). The polyketide ELS complexes exhibit specificity for the elongation substrate, and 3-oxo groups are retained in only one or two successive elongation steps. The resulting oxoacyl-CoAs serve as substrates for (i) the decarboxylative pathway in which associated enzymes produce alkan-2-ol esters and oxoalkan-2-ol esters, and (ii) the decarbonylative pathway in which associated enzymes give rise to β-diketones, hydroxy-β-diketones and oxo-β-diketones. On wild-type Bonus barley spikes, the β-diketone lipids account for at least 50% of the epicuticular wax, and the primary alcohols and hydrocarbons account for 3–4 and 15–16%, respectively (von Wettstein-Knowles and Søgaard, 1980). The major cuticular surface of the spike that is responsible for its overall wax phenotype is that of the lemmas enfolding the seeds, while paleas, glumes, rachis and awns make minor contributions. Structural and/or wax lipid class analyses reveal that β-diketone lipids are present on all surfaces except those of the awns (Simpson and von Wettstein-Knowles, 1980; von Wettstein-Knowles, unpublished results). Hydrocarbons are more prominent constituents of awn and glume wax than they are of the total spike wax (Simpson and von Wettstein-Knowles, 1980). Moreover, awn waxes are so far the only cuticle surface in barley that has been shown to bear secondary alcohols (von Wettstein-Knowles and Netting, 1976).
A total of 1580 eceriferum (cer) mutants distributed among 79 loci were isolated in barley based on a visual decrease in the glaucousness of the leaves, uppermost leaf sheaths and internodes and/or spikes (Lundqvist and Lundqvist, 1988). The wax phenotype on each of these three surfaces is designated by ‘−’ (absent), ‘+’ (less than wild-type) or ‘++’ (as wild-type). Thus, the wild-type wax phenotypic formula is ‘++ ++ ++’ with the cuticle surfaces in the order specified above. The 55 recessive and one dominant mutation at the cer-n locus, affecting spike and uppermost leaf sheath and internode wax, exhibit a range of phenotypes that are susceptible to environmental factors. The overall spike wax composition of ten cer-n mutants was therefore investigated (Lundqvist and von Wettstein-Knowles, 1983) using plants grown under controlled conditions in the Stockholm Phytotron (Dormling et al., 1969, 1972). In the mutant waxes, the β-diketone lipids varied from wild-type amounts to 8% thereof, and the dominating C31 alkane varied from approximately 71% as in wild-type to 8% of the hydrocarbons. The effect on the two lipid classes was not correlated, however. The primary alcohol spectrums dominated by the C26 homologue could resemble that of the wild-type or have increased amounts of C20, C22, C30 or C32 homologues. In the most extreme case, the C32 alcohol accounted for 38%, versus 19% for C26. The observed changes in the β-ketoacyl ELS derived lipids were not correlated with those of the acyl ELS derived lipids, nor were those of the two acyl ELS derived lipids, the n-alkanes and primary alcohols, correlated. Interestingly, the reduction in long-chain n-alkanes was accompanied by apparent increases in long-chain minor hydrocarbons in all the mutants. This implies that a different elongation system may be involved in their synthesis, where the term ‘elongation system’ includes not only the ELS complexes but also the origin of the substrates they use. As a first step to probe this hypothesis, the identity of the minor hydrocarbons has to be established. Material for this investigation included four mutants of the cer-n locus plus a mutant at each of five additional loci, namely cer-a, − − ++; cer-e, − ++ ++; cer-x, − − ++; cer-yc, − ++ ++, and cer-yl, − − ++, in which the long-chain n-alkanes are also reduced without apparently affecting the chain-length distributions of the minor hydrocarbons. The results identify five new hydrocarbon series in wild-type and eceriferum (cer) mutant barley spike epicuticular waxes. The prominent ones are monoalkenes (C23–C35) and cyclopropanes (chain lengths C27–C31), novel wax lipids. The minor series include internal methyl branched alkanes (chain lengths C25–C33), plus 2- and 3-methyl branched alkanes. The nature of the potential elongation systems participating in synthesis of the monoenoic alkenes, the cyclopropanes and the internally branched alkanes is considered.
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- Experimental procedures
Hydrocarbons were isolated by preparative thin-layer chromatography (TLC) from the epicuticular waxes obtained from the spikes of the wild-type Bonus and nine cer mutants. The chain-length distributions of those from Bonus and the mutant cer-n985 are shown in Figure 1. That of the wild-type, which is analogous to previous determinations (see, for example, von Wettstein-Knowles and Netting, 1976), consists predominantly of odd-chain-length n-alkanes that are greatly reduced in cer-n985 and the other eight cer mutants. Most marked is the reduction of C31 from approximately 72% to 10%, which is compensated for by the increased relative abundance of the shorter n-alkanes, especially C23 and C25 (1–2 to 16–19%), and two other homologous series: the x series eluting just before the odd-chain-length n-alkanes and the y series eluting just before the even-chain-length n-alkanes. The objective of this study was to identify the x and y series of hydrocarbons whose syntheses were apparently not reduced in the spike waxes of cer mutants characterized by defects in the elongation system(s) leading to n-alkane and β-diketone formation. In the course of this work, several other hydrocarbon series were observed, and some of these were also identified.
Figure 1. Hydrocarbons from the epicuticular spike waxes of the wild-type Bonus barley (a) and its mutant cer-n985 (b) as resolved by GC using a 3% SP-2100 column. Chain lengths of the n-alkanes are specified. The letters x and y denote members of two homologous hydrocarbon series that are shown herein to be alkenes and cyclopropanes, respectively.
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Initially the hydrocarbons were subjected to AgNO3 preparative TLC which gave two bands, both of which were recovered and analyzed by gas chromatography (GC). The slower band contained the x series, suggesting they were unsaturated. Hydrogenation of the x series resulted in the formation of straight-chain, odd-numbered n-alkanes whose identities were confirmed by TLC, GC and mass spectrometry (MS). The chain-length distributions of the unsaturated homologues from Bonus and the nine cer mutants are given in Table 1. Examination of the data reveals that the distributions from all 10 genotypes are similar, i.e. odd homologues from C23–C37 characterize all genotypes, with C39 and C41 also being detected in some of the genotypes. The major homologue is always C29, amounting to 33% in Bonus and somewhat more in the mutants (42–54%). The mean distribution of the nine mutants that were grown at various times over a three-year period (with SEs) implies that the differences among them are not significant, i.e. the nine mutations have no effect on alkene synthesis. Combining results from the total hydrocarbon and alkene distributions (see Experimental procedures) reveals that the unsaturates represent approximately 0.3% in Bonus, and 10.9, 25.2, 18.9 and 15.7%, respectively, in cer-n26, −n53, −n625 and −n985, for example. To determine the configuration of the unsaturated bond, total spike wax of mutants was applied to the silica gel end of a hybrid TLC plate. Development with hexane separated the hydrocarbons from the other wax components. Upon encountering the AgNO3-containing gel, the mobility of the unsaturates was retarded. Co-chromatography with cis and trans 9-C23:1 standard alkenes, which had Rf values of 0.38 and 0.57, respectively, revealed that the barley unsaturates were cis alkenes. No band corresponding to alkenes was detected on the hybrid plates when wild-type awn wax having the same amount of hydrocarbons as the mutants was analyzed.
Table 1. Monoenoic alkenes present in the epicuticular spike waxes of wild-type Bonus and nine cer mutants (weight%)
|Carbon number||Bonus||a6||e8||n26||n53||n624||n985||x60||yc135||yl187||Mean mutant value ±SE|
|22||0.1|| || || || || || || || || || |
|23||3.0a|| 0.7|| 1.3|| 0.8||Trace|| 0.8|| 1.4|| 0.6|| 0.2|| 1.1||0.8 ± 0.16|
|24||1.2a||Trace|| 0.3|| 0.3||Trace||Trace|| 0.1||Trace||Trace|| 0.3||0.1 ± 0.05|
|25||14.7|| 7.0||12.3|| 9.2|| 6.4|| 7.2|| 9.9|| 7.8|| 6.5||10.3||8.5 ± 0.76|
|26||0.7||Trace|| 0.9||Trace||trace||Trace||Trace||Trace||Trace|| 0.5||0.2 ± 0.11|
|27||14.4||14.9||19.0||15.7||14.1||16.9||14.5||14.5||12.9||15.8||15.4 ± 0.59|
|28||0.9|| 0.7|| 0.7|| 0.7|| 0.5||Trace|| 0.5|| 0.8||Trace|| 0.8||0.5 ± 0.11|
|29||32.6||52.5||41.9||44.1||54.1||53.8||45.9||47.3||52.7||46.2||48.7 ± 1.53|
|30||1.6|| 1.6|| 1.4|| 1.3|| 1.4|| 0.4|| 1.5|| 2.7|| 1.4|| 1.3||1.4 ± 0.20|
|31||19.3||19.8||18.7||22.7||20.1||19.1||20.7||21.1||23.2||18.7||20.5 ± 0.55|
|32||0.7||Trace||Trace||Trace||Trace||Trace||Trace||Trace||Trace|| 0.4||Trace ± 0.04|
|33||4.6|| 1.5|| 2.0|| 2.6|| 1.7|| 1.3|| 3.2|| 2.4|| 1.9|| 2.3||2.1 ± 0.20|
|35||3.4|| 1.0|| 1.3|| 1.7|| 1.1|| 0.3|| 1.8|| 1.9|| 1.1|| 1.6||1.3 ± 0.17|
|37||1.8|| 0.4|| 0.2|| 0.7|| 0.4||Trace|| 0.5|| 0.8||Trace|| 0.7||0.4 ± 0.10|
|39||0.8|| || || 0.1||Trace|| || || || || 0.1||0.2 ± 0.01|
|41||0.3|| || || || || || || || || || |
|29b||0.1|| 3.4|| 5.5|| 5.5||12.9||10.0|| 7.8|| 4.1|| 3.5|| 0.5|| |
To determine the positions of the double bonds in the alkenes, total hydrocarbons were subjected to OsO4 oxidation converting the alkenes to diols, which were then analyzed by GC-MS of their trimethylsilyl (TMS) derivatives. GC traces of cer-e8 hydrocarbons before and after the oxidation are shown in Figure 2. The TMS derivatives elute 3.76 equivalent chain-length (ECL) units later than the respective alkene, for example the C29-TMS elutes before the C33n-alkane with an ECL of 32.43 (Figure 2b), while the alkene elutes before the C29n-alkane with an ECL of 28.67 (Figure 2a). That only one homologous series results from OsO4 oxidation implies that only one type of alkene is present. Upon GC-MS fragmentation between α-oxytrimethylsilyl (OTMS)-substituted carbons, strong intensity ions whose m/z in combination with that of the M-15 ion reveal the number of carbons on either side of the original double bond (Capella and Zorzut, 1968). When more than one isomer is present, the mass spectrum obtained depends on the time it was taken during elution of the GC peak. This is illustrated for the cer-a6 C29-TMS GC peak in Figure 3(a,b). Two major positional isomers with double bonds at 9 or 11 (Figure 3a) are revealed by the pairs of ion fragments at 215/369 and 243/341 plus an M-15 ion at 569. In addition, the ion fragment pairs at 271/313 and 285/299 intimate the presence of lesser amounts of the 13- and 14-ene isomers. The latter numbered from the other end of the chain is a 15-ene. Such differences are summarized in Table 2A by the chemical nomenclature designation/opposite end designation, in this case 14/15-ene. In the text, however, isomers are designated as members of a homologous series differing by C2-units. The spectrum in Figure 3(b) is dominated by the 215/369 ion fragment pair indicative of a double bond at position 9, with small amounts of the ion fragment pairs 243/341 and 187/397 plus an M-15 ion at 569, implying the presence of 7- and 11-ene isomers. Table 2A summarizes all alkene positional isomers occurring in cer-yl187 epicuticular spike wax detected during MS analyses at representative time points throughout the elution of each GC peak. For all homologues, the 9-ene was the major isomer, as at peak height, the diagnostic ion fragments of any other isomer never amounted to more than 20% of those of the C9 isomer, generally to less than 10%. While the 11-, 13- and 15-enes were generally present, whether or not 17- to 23-enes also occur is unknown at present as only one of the characteristic ion fragments from each pair was present in the spectra. Analogous positional isomers characterize the monoenoic cis alkenes from cer-a6 and cer-e8.
Figure 2. Hydrocarbons from the epicuticular spike wax of cer-e8 before (a) and after (b) formation of the diol-TMS derivatives of the alkenes by OsO4 oxidation as resolved by GC chromatography using a 3% SP-2100 column. Dotted lines are used for n-alkanes. Black and x denote the alkene homologues in (a), and their diol-TMS derivatives in (b). Chain lengths of the n-alkanes are specified.
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Figure 3. Mass spectra of the diol-TMS derivatives generated from the C29 alkene of cer-a6 epicuticular spike wax resolved by GC using a 5% OV-101 column and the VG 7070F instrument. (a) Leading side of the GC peak with two major isomers and two minor isomers. The origins of the major 11-ene 243/342 and minor 15-ene 285/299 fragments are shown. (b) Tailing side of the GC peak with one major isomer and two minor isomers. The origin of the major 9-ene ion fragment 215/369 is shown.
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Table 2A. Monoenoic alkenes from cer-yl187 barley spike epicuticular wax
|Chain length||Double bond positions|
|25||9||11, 10/15, 12/13, (8/17)|
|27||9||11, 12/15, 10/17|
|29||9||11, 13, 14/15, (10/19), (8/21)|
|31||9||11, 13, 15, (10/21), (8/23)|
Even in the earliest studies of plant long-chain alkenes, double bond positions were identified. For example, in the wax from Papaver rhoeas anthers, cis 9-, 7- and 11-enes of C31 were identified, while C27 was the dominant long-chain n-alkane (Stránský and Streibl, 1969). In Rosa damascena flower waxes, the major alkene was C29 with cis 5 or 7 double bonds, and the major n-alkane was C31 (Wollrab, 1968). A later more detailed study on petal waxes of three Cistus species revealed that the C23–C33 alkenes were primarily cis 9-enes, but C23–C25 could also be 1-enes and C27–C33 could be 5- or 11-enes, while the major n-alkane was C25 (Gülz et al., 1979). Thus, in common with the barley monoenoic alkenes, those from other plant waxes are characterized by a series of positional isomers and homologue distributions that differ from those of the n-alkanes. Dienoic alkenes were also identified in early studies, for example in flower wax of R. damascene, but double bond positions were not characterized (Stoianova-Ivanova et al., 1971).
The chain-length distributions of the saturated hydrocarbons recovered from the faster running band on the AgNO3 TLC plates are given in Table 3. These compounds from the wild-type spikes are primarily odd-chain-length n-alkanes dominated by the C31 homologue, with trace amounts of even-chain-length homologues (von Wettstein-Knowles and Søgaard, 1980). In all nine mutant waxes, the importance of C31 is markedly decreased, while the relative amounts of the C23, C25 and C27 homologues are significantly increased. In addition, the z and y homologous series of hydrocarbons eluting with ECLs of 27.34, 29.34, 31.34 and 27.86, 29.86 and 31.86, respectively, become visible components of the saturated hydrocarbons (compare Figure 1a and b). As the SP-2100 column did not adequately resolve the z and y homologues from the adjacent n-alkane, the saturated hydrocarbon fraction was treated with a molecular sieve to reduce the relative amounts of the latter. This resulted in the simultaneous removal of the y series, revealing that they were not branched hydrocarbons.
Table 3. Saturated hydrocarbons from epicuticular waxes on spikes of wild-type Bonus and nine cer mutants after AgNO3 TLC removal of alkenes as determined by GC (weight%)
|20|| ||1.1|| || || || || || || ||0.1|
|21|| 0.4||5.5||2.1||2.7||5.5|| 6.5|| 6.5||4.9||1.7||7.5|
|22||Trace||0.9||0.3||0.3||0.8|| 0.7|| 0.7||0.7||0.1||0.6|
|24||Trace||1.0||0.7||0.6||3.4|| 0.8|| 1.1||0.9||0.3||1.0|
|26||Trace||0.5||0.4||0.3||1.0|| 0.3|| 0.3||0.4||0.1||0.8|
|26.64|| ||0.3||0.1|| || ||Trace||Trace||0.3|| ||0.2|
|27|| 3.2||11.9||8.8||8.7||10.3|| 8.3|| 6.7||9.6||5.7||11.3|
|27.17|| ||0.6||0.3|| || || 0.9|| 0.7||0.6|| ||0.6|
|27.86|| ||1.2||0.6||0.7||1.1|| 1.3|| 0.7||0.9||0.3||1.2|
|28|| 0.2||1.7||1.2||1.3||1.7|| 1.3|| 2.0||1.3||0.8||1.7|
|29 + 29.34||22.1||17.3||20.2||21.1||16.5||15.6||20.9||19.9||15.4||17.3|
|29.86|| 2.0||9.1||4.5||6.1||9.0|| 8.0|| 5.9||7.6||3.2||8.5|
|31 + 31.34||64.4||8.8||35.1||29.7||9.9|| 8.6||12.8||19.9||48.7||8.7|
|31.86|| ||4.2||2.5||3.2||3.7|| 3.7|| 3.1||3.6||2.2||4.1|
|33|| 1.9||1.7||2.2||2.6||1.2|| 1.6|| 1.6||1.9||7.5||0.8|
|35|| ||0.4|| || || || || || || || |
The molecular sieve-treated hydrocarbons were subjected to GC-MS to determine whether some of the minor branched components shown in Figure 4 could be identified. The molecular ion is generally not visible in electron-impact MS of long-chain methylalkanes which was used in the present study. Instead an M-15 ion and others of lesser mass are formed that can be used to deduce what the molecular ion was (Blomquist et al., 1987; Hamilton, 1995). A series with ECLs of 24.65, 26.65, 28.65 and 30.65 was characterized by M-CH3 and M-C3H7 ion fragments (337 + 309, 365 + 337, 393 + 365, 421 + 393), identifying them as iso or 2-methyl alkanes, namely 2-methyltetra-, hexa-, and octacoasane plus 2-methyltriacontane. In accordance with this, 13-methyltetra- and 15-methylhexadecanote from Supelco's bacterial fatty acid methyl ester mix have ECLs of 14.65 and 16.65 on the same HP-1 column. Two members of a second series with ECLs of 27.73 and 29.73 were characterized by M-CH3 and M-C2H5 (379 + 365, 407 + 393) ion fragments, identifying them as anteiso or 3-methylalkanes, namely 3-methylhepta- and nonacosane. In addition, members of two other homologous branched series having an ECL with either an odd or even carbon number + 0.34 are seen in Figure 4, labeled ‘ib’ (internal branch). The common ECL implies an analogous position of the methyl branch in both series. The MS data revealing the positions of the methyl branches are presented below. The additional unmarked peaks shown in Figure 4 were not investigated.
Figure 4. Segment of a GC trace of the saturated hydrocarbons from the epicuticular spike wax of cer-a6 present after treatment with a molecular sieve obtained using a methylsilicone HP column. Numbers denote the chain length of the hydrocarbon. 2-, 3- and ib- denote the presence of a methyl group on carbons 2 or 3 or internally (ib, internal branch), respectively.
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Cleavage of internally branched hydrocarbons upon chemical ionization occurs on either side of the methyl group, giving rise to two ion fragments. This is illustrated for the internally methyl branched C31z homologue (methylhentriacontane) from the epicuticular wax of cer-a6 spikes in Figure 5(a). A methyl group on carbon 15 of a C31 chain gives two characteristic ion fragment doublets, namely 225 + 224 and 253 + 252, where the doublets represent the primary and secondary carbonium ion fragments (Nelson et al., 1980). Hereafter, only the member of the doublet with the higher mass will be referred to. The ion fragment pairs 197/281, 169/309 and 141/337 would result from methyl groups on carbons 13, 11 or 9, respectively. Stronger spectra were subsequently obtained using the VG Trio-2 instrument, and the relevant portion for methyl branch determination of a MS from the analogous z homologue from cer-yl187 spikes is shown in Figure 5(b). As noted above, the observed isomer distribution depends on the time during homologue elution that the spectrum is taken. A summary of the methyl positions for all the cer-yl187z homologues is given in Table 2B. For the three shorter homologues, at peak height the major isomer had a methyl on carbon 9, whereas for the C31 and C33 homologues the methyls were prominent at two positions, namely 11 + 13 and 13 + 15, respectively. The diagnostic ion fragments of all other isomers never amounted to more than 10% of the specified isomer(s). Examination of Figure 5(b) also reveals fragment ions at 323, 295 and 267, which, although unaccompanied by clear doublets for the expected smaller ion fragment, imply that smaller amounts of isomers with the methyl group on carbons 10, 12 and 14 may also be present. A complete analysis of the cer-a6z homologues gave analogous results. Identification of the second series of internally branched methyl hydrocarbons eluting after the corresponding even-chain-length n-alkane (Figure 4) is based on weak spectra from a GC-MS analysis of the C30 homologue from the epicuticular waxes of cer-a6 and cer-yl187. Both spectra revealed the ion fragment pairs 155/281, 181/253 and 222/225, indicating methyl groups on carbons 10, 12 and 14 and an M-15 fragment ion at m/z 393. The tentative identification of the C28 homologue (Figure 4) is based on its having an analogous ECL (28.34) to that of the C30 homologue (30.34).
Figure 5. Mass spectra of internally branched methylhentriacontane, a series z homologue, from two mutants, and generated by acidic hydrogenation of the cyclopropane methylenehentriacontane, a series y homologue. (a) Complete spectrum of the homologue from cer-a6 epicuticular spike wax, resolved using a 5% OV-101 column with the VG 7070F instrument. (b) Portion of the mass spectrum of the homologue from cer-yl187 epicuticular spike wax, resolved using a methylsilicone HP column with the VG-Trio 2 instrument. (c) Portion of the mass spectrum of the compound generated by acidic hydrogenation of the cyclopropane homologue from cer-x60 epicuticular spike wax (see Figure 6c), resolved using a methylsilicone HP column with the VG-Trio 2 instrument. Only the m/z for the ion fragment of greater mass in each doublet representing the primary and secondary carbonium ions is specified in the spectra.
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Table 2B. Internally branched hydrocarbons from cer-yl187 barley spike epicuticular wax
|Chain length||Carbon number||Methyl group positions|
|29||30||9||11, 13, 15|
|31||32||11, 13||9, 15|
|33||34||13, 15||9, 11, 17|
Internal methyl branched hydrocarbons in waxes from walnut leaves were first reported many years ago (Stránskýet al., 1970). The homologues ranging from C21–C33, with C27 and C29 being the most important, had methyl groups on carbons 9, 11, 13 or 15. Associated with the C28 GC peak were mass ions characteristic for methyl groups on carbons 12 and 14, indicating that the methylalkanes were not resolved from octacosane by the column used. These results are rather similar to those reported here for barley spikes, and for extracts of dried chamomile flowers where other typical wax lipids were also present (Stránskýet al., 1981).
Initial MS of the y series of homologues of the cer-e8 saturated hydrocarbons (not shown) unexpectedly implied that they might be alkenes with an even number of carbons. More interestingly, the ECLs of the y series of hydrocarbons relative to the other saturated components depended upon whether they were subjected to GC using a non-polar column (for example, SP-2100 in Figure 1) or a polar column. To illustrate, the y homologues from cer-x60 in Figure 6(a) resolved by GC on the polar HP-17 column have ECLs of 28.15, 30.15 and 32.15 versus 27.86, 29.86 and 31.86 on the SP-2100 column (Table 3) and 27.90, 29.90 and 31.90 on the HP-1 column. This effect of column polarity on retention is characteristic of cyclopropanes (see Experimental procedures). Moreover, if the y series were cyclopropanes, then the similarity of the initial MS to that of alkenes would be explained (Christie and Holman, 1966). To confirm this identification, the saturated hydrocarbons of cer-x60 epicuticular spike wax were brominated, resulting in the expected disappearance (Brian and Gardner, 1968) of the deduced cyclopropanes from the mixture as illustrated in Figure 6(b). Another aliquot of the cer-x60 saturated hydrocarbons was subjected to acidic hydrogenation, which opens a cyclopropane ring giving rise to a pair of internally branched hydrocarbons or a straight-chain n-alkane depending on which of the three carbon–carbon bonds in the ring is cleaved (McCloskey and Law, 1967). This is shown in Figure 6(c) in which the peaks representing the cyclopropane components are absent and two others are increased. For example, acidic hydrogenation of the cyclopropane y homologue eluting between the C29 and C31n-alkanes (Figure 6a) resulted in marked increases in the C30n-alkane peak and the internal methyl branched C29 peak (Figure 6c). These results are in accordance with the hypothesis that the y series of hydrocarbons comprises cyclopropanes with chain lengths of 27, 29 and 31 carbons, i.e. methyleneheptacosane, methylenenonacoasane and methylenehentricontane.
Figure 6. Saturated hydrocarbons from the epicuticular spike wax of cer-x60 (a) as recovered from AgNO3 TLC plates, (b) after bromination, and (c) after acidic hydrogenation as resolved by GC using an HP-17 column. cp, cyclopropanes of series y; n, normal; ib, internal methyl branch in (a) and (b) = series z.
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To confirm the identity of the branched compounds resulting from acidic hydrogenation as well as to determine the position of the ring, they were analyzed by GC-MS as described above for the internally branched methyl alkanes. Results for the C31 homologue from the epicuticular spike wax of cer-x60 are shown in Figure 5(c). The following ion fragment pairs resulted: 141/337, 155/323, 169/309, 183/295, 197/281 and 211/267. For the fragment ions at m/z 183, 197 and 211, only one member of the expected doublet can be seen. The relatively equal amounts of the 267 + 281, 295 + 309 and 323 + 337 ion fragments presumably reflects the randomness of the chemical acidic hydrogenation that breaks the carbon–carbon bonds in the cyclopropane ring. By comparison, the mass spectrum of the internally branched C31z homologue analyzed in Figure 5(b) from the epicuticular wax of cer-yl187 spikes reveals quite different amounts of the 281 and 309 versus the 267 and 295 ion fragments, presumably implying specificity of the enzyme participating in their synthesis. Analogous results were obtained for the cer-x60 C27 and C29z homologues as well as for the three cer-a6 homologues.
A summary of the positions of the cyclopropane rings in cer-x60 is given in Table 2C. Characteristic for all three y homologues is the approximately equal frequency of the ring on carbons 9,10 versus 11,12. Moreover, at peak height, the diagnostic ion fragments for the specified isomers amounted to at least 70% of all possible isomer fragments. The distribution of the three homologues is similar in all nine mutants with mean values ±SEs of 7.7 ± 0.4 for C27, 61.2 ± 0.8 for C29 and 31.0 ± 1.1 for C31, implying that synthesis of the cyclopropanes is not affected by these mutations. As only the major C29y homologue is detected in the wild-type saturated hydrocarbons, the increases in abundance of the cyclopropanes would appear to be significant in all the mutant hydrocarbons, with the smallest increase occurring in cer-yc135 and the largest in cer-a6, cer-n53, cer-n624 and cer-yl187. In the spike wax of cer-x60, the cyclopropanes account for 9.3% of the total hydrocarbons (weight percentage), which is similar to 8.7% for the alkenes. At best, the internally branched, odd–chain-length hydrocarbons (C25–C33) amount to 1.8%, which is more than any of the other methyl branched series identified (Figure 4).
Table 2C. Cyclopropanes from cer-x60 barley spike epicuticular wax
|Chain length||Carbon number||Cyclopropane ring carbons|
|27||28||9,10 + 11,12||13,14|
|29||30||9,10 + 11,12||13,14 + (14,15/15,16)|
|31||32||9,10 + 11,12||(13,14)|
Carbocyclic fatty acids occur in the Malvales and plants allied with this order, plus some gymnosperms. Generally they have a chain length of 18 or 17 carbons, with the ring including carbons 9 and 10 joined by either a single or double bond (Christie, 1970), and are known as cyclopropane and cyclopropene acids, respectively. While especially prevalent in seed oils, they occur also in vegetative tissues, among which roots have the highest concentration (Christie, 1970; Schmid and Patterson, 1988; Shenstone et al., 1965). In seed oils, they are primarily sequestered in triacylglycerols, but are also esterified to phospho- and glycolipids (Christie, 1970). In vegetative tissues, their concentration is always higher in neutral lipids than in the phospho- and glycolipids (Schmid and Patterson, 1988), and they may be localized in intracellular droplets (Yatsu and Kircher, 1987). Thus, the only similarity between the presently identified wax cyclopropanes and the previously known carbocyclic fatty acids in plants is the position of the ring. This generalization includes the cyclopropane fatty acids with chain lengths of 20, 21, 22 and 24, ring carbons unknown, in either sulfolipids or phosphatidyl choline of a number of early spring plants as noted by Kuiper and Stuiver (1972).
The other lipid classes in cer-a6, cer-e8, cer-x60, cer-yc135 and cer-yl187
The other lipid classes were characterized as detailed previously (Lundqvist and von Wettstein-Knowles, 1983). Results pertinent to the present study follow. The mutant waxes from cer-a6, cer-e8, cer-x60, cer-yc135 and cer-yl187 have approximately 8, 75, 16, 73 and 24%, respectively, of the β-diketone lipids compared with the wild-type per spike. That all five mutants have a ‘−’ spike wax phenotype implies that, at least in cer-e8 and cer-yc135, the total amount of wax per spike is greatly reduced. The chain-length distributions in the mutants are not affected; for example, in cer-yl187, C31 comprises 94% of the β-diketone lipids. Too little sample was available to analyze the chain-length distributions in cer-a6, cer-x60 and cer-n53. The C32 aldehyde homologue, accounting for 55% of this lipid class in wild-type, amounted to approximately 5, 37, 26 and 50% for cer-a6, cer-e8, cer-x60 and cer-yc135, respectively (there was too little material to analyze for cer-yl187). The other acyl ELS derived lipid classes are characterized as in the wild-type by prominent amounts of the shorter homologues, namely C22–C26 in the free and ester primary alcohols and C20 in the free fatty acids. The C32 homologues of the free primary alcohols amount to only 2–10%, and are almost non-existent in the other two lipid classes. The free fatty acids exhibit a rather uniform distribution from C20–C32, accounting for only 6% in the wild-type and 1–4% in the mutant waxes.
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I am indebted to J. Øgaard Madsen (Organic Chemistry Department, Technical University of Denmark) for obtaining and interpreting the mass spectra, to M. Petersen and M. Mortensen for outstanding technical assistance, and to G.J. Blomquist (University of Nevada, Reno, USA), B. Stoianova-Ivanova (University of Sofia, Bulgaria), K. Stránský (Academy of Sciences, Prague, Czech Republic) and E. Casagrande (AgriSense-BCS Ltd, Pontypridd, UK) for the standards. Support from the Danish Natural Sciences Research Council (grant number 272–05–0401) is gratefully acknowledged.