Jørn Dalgaard Mikkelsen

Pigment and acyl lipid composition of photosystem I and II vesicles and of photosynthetic mutants in barley

The pigment and acyl lipid composition of photochemically active stroma lamellae and inside-out photosystem II vesicles have been investigated. Mechanical disruption followed by phase partitioning in an aqueous two phase system resulted in a clean separation of the photosystems. A qualitative and quantitative determination of the pigment content was achieved using reversed-phase high performance liquid chromatography. A single chromatographic separation provided adequate resolution of neoxanthin, violaxanthin, lutein, chlorophyllb, chlorophylla, and β-carotene and was completed in 22 min. For each molecule ofP700, the stroma lamellae contained 166 molecules of chlorophylla, 14 molecules of chlorophyllb, 0 molecules of neoxanthin, 3 molecules of violaxanthin, 7 molecules of lutein, and 13 molecules of β-carotene. For each molecule ofP680 the photosystem II vesicles contained 250 molecules of chlorophylla, 100 molecules of chlorophyllb, 7 molecules of neoxanthin, 7 molecules of violaxanthin, 22 molecules of lutein, and 4 molecules of β-carotene. The pigment content of three photosynthetic mutantschlorina-f2, viridis-zb63 andviridis-zd69 was determined. On a chlorophyll basis, these mutants generally showed a reduced carotenoid content, except thatchlorina-f2 had an increased level of β-carotene andviridis-zd69 an unaltered level of lutein.The acyl lipid composition of stroma lamellae, photosystem II vesicles and lamellar systems was also determined. Although the photochemical activities of the stroma lamellae and photosystem II vesicles were enriched compared to the parent lamellar system, more than 50% of the acyl lipids in the subchloroplastic membranes were degraded or severely modified resulting in a drastically reduced content of monogalactosyldiglyceride and digalactosyldiglyceride in the stroma lamellae and photosystem II vesicles. Due to the lipolytic degradation the level of saturation in the galactolipids was higher in stroma lamellae and photosystem II vesicles compared with the lamellar system. Stroma lamellae do not contain the light-harvesting chlorophylla/b-protein, but were enriched in phosphatidylglycerol and trans-3-hexadecenoic acid.

Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

An acyl carrier protein (ACP) dependent fatty acid synthetase (fas) from barley chloroplast stroma was purified five-fold by ammonium sulphate precipitation and gel filtration on Sephacryl S-300. The β-ketoacyl-ACP reductase, β-ketoacyl-ACP synthetase, acetyl-CoA:ACP transacylase and malonyl-CoA:ACP transacylase activities were resolved on the Sephacryl S-300 column with apparent molecular weights of respectively 125, 92, 82 and 41 kilodalton. The fas activity exhibited an apparent molecular weight of 87 kilodalton resulting from the overlapping portions of the component activities. A fifth component of the active fas, ACP, was separated completely from the other four individual enzyme activities by the ammonium sulphate precipitation. When the fas purified by gel filtration was applied to a Mātrex Gel Blue B column, the component activities were separated into two groups. A bound fraction contained all the malonyl-CoA:ACP transacylase whereas the β-ketoacyl synthetase activity was exclusively present in the non-bound fraction. Neither the bound nor the non-bound fraction showed any fas activity alone, but complete reconstitution of fas activity was obtained when both protein fractions were combined. The barley chloroplast fas is therefore not a multifunctional protein but consists of at least five separable components. Characterization with respect to cofactor requirements was also performed. Variation of certain cofactor concentrations markedly altered the pattern of fatty acid synthesis.

The effects of inhibitors on the biosynthesis of the long chain lipids with even carbon numbers in barley spike epicuticular wax

The effects of inhibitors on the incorporation of label from [2-14C]-acetate into the epicuticular wax classes with even carbon numbers have been determined. They include the free fatty acids, the primary alcohols, the aldehydes, the ester acids and a newly identified wax class on barley spikes, the short chain esters. Dithiothreitol, which has no effect on β-diketone synthesis, inhibits decarboxylation of very long fatty acyl chains which are precursors of the alkanes. Mercaptoethanol, which greatly stimulates wax synthesis at low concentrations and has no effect on β-diketone synthesis, blocks further elongation of fatty acyl precursors having 20 carbons at higher concentrations. A relatively small inhibition of decarboxylation was also observed with mercaptoethanol. Arsenite, which inhibits β-diketone synthesis presumably by blocking the entrance of C16 precursors into the β-diketone elongation mechanism, was also found to prevent the C20–C22 elongation step in the sequence leading to the alkanes. Cyanide, which blocks β-diketone synthesis by an unknown mechanism, greatly stimulated synthesis of the other wax classes. At higher concentrations a block in the elongation sequence leading to the alkanes was also apparent between C26–C28. These results are incorporated into a composite figure illustrating the biosynthetic relationships among the wax classes. All identified chemical and genetic blocks have been included. The conclusion is reached that at least three elongation systems must be involved.The effects of inhibitors on the incorporation of label from [2-14C]-acetate into the epicuticular wax classes with even carbon numbers have been determined. They include the free fatty acids, the primary alcohols, the aldehydes, the ester acids and a newly identified wax class on barley spikes, the short chain esters. Dithiothreitol, which has no effect on β-diketone synthesis, inhibits decarboxylation of very long fatty acyl chains which are precursors of the alkanes. Mercaptoethanol, which greatly stimulates wax synthesis at low concentrations and has no effect on β-diketone synthesis, blocks further elongation of fatty acyl precursors having 20 carbons at higher concentrations. A relatively small inhibition of decarboxylation was also observed with mercaptoethanol. Arsenite, which inhibits β-diketone synthesis presumably by blocking the entrance of C16 precursors into the β-diketone elongation mechanism, was also found to prevent the C20–C22 elongation step in the sequence leading to the alkanes. Cyanide, which blocks β-diketone synthesis by an unknown mechanism, greatly stimulated synthesis of the other wax classes. At higher concentrations a block in the elongation sequence leading to the alkanes was also apparent between C26–C28. These results are incorporated into a composite figure illustrating the biosynthetic relationships among the wax classes. All identified chemical and genetic blocks have been included. The conclusion is reached that at least three elongation systems must be involved.

Identification of a 28,000 Dalton endochitinase in barley endosperm

One of the four major basic salt-soluble proteins in barley with a molecular weight of 28,000 Dalton was shown to be an endochitinase. A partial amino acid sequence has been determined, and strong homology to an endochitinase from bean (Phaseolus vulgaris) is demonstrated.

Structure and biosynthesis of β-diketones in barley spike epicuticular wax

The structure and composition of the β-diketones in barley spike and carnation leaf epicuticular waxes have been determined by gas liquid chromatography-mass spectrometry. Barley wax contained, in addition to the well known hentriacontan-14,16-dione, small amounts of tritriacontan-16,18-dione, nonacosan-12,14-dione and nonacosan-14,16-dione. Five β-diketones were identified in carnation wax: nonacosan-10,12-dione, nonacosan-12,14-dione, hentriacontan-12,14-dione, tritriacontan-12,14-dione and tritriacontan-14,16-dione. The biosynthesis of the β-diketones was studied by incorporating various precursors into tissue slices of barley spikes with awns removed. Incorporation of [1-14C]-pentadecanoic acid labelled a novel C30 β-diketone which upon cleavage contained the14C in the C16–30 end. This confirms that the carbon chain of the C31 β-diketone is formed in vivo by an elongation mechanism which proceeds from the C31 toward the C1 end of the molecule. A comparison of the position of label within the β-diketone chains synthesized from C12, C14, C16 and C18 labelled precursors revealed that the β-keto acyl elongase which is responsible for β-diketone carbon chain synthesis has the following specificities; C14 or C16 chains serve equally well as primers; C12 chains are first elongated to C14 or C16 before they can serve as primers; C18 chains can not serve as primers. After protection of the β-diketo groups by the β-keto acyl elongase either 7 or 8 additional C2 units are added before the presumed decarboxylation to yield the complete β-diketone molecule.

Mutants in the biosynthesis of isoleucine in a non-mating, non-sporulating brewing strain of Saccharomyces carlsbergensis

Mutants resistant to 2-amino-3-(methylthio)butanoic acid (thiaisoleucine) were selected from a non-mating, non-sporulating brewing strain of Saccharomyces carlsbergensis after treatment with N-methyl-N′-nitro-N-nitrosoguanidine. One closer studied mutant, C77-T70 had a threonine deaminase activity which was less sensitive tol-isoleucine than that of the parent strain. The inhibition with varying concentrations ofl-isoleucine showed that part of the activity was as sensitive as that of the parent strain, suggesting that the mutant is heterozygous for a dominant change in the structural gene for threonine deaminase. Of twelve mutants selected on 25mm-thiaisoleucine all are believed to have undergone a change similar to that of C77-T70, since their threonine deaminase activity was partly resistant to 1mm-l-isoleucine like that of C77-T70. Four such mutants were compared to the parent strain for the production of fusel alcohols during aerobic growth in minimal medium. The synthesis of 2-methyl-1-butanol (d-amyl alcohol) was 2–5 times higher in the mutants than in the parent strain, while the syntheses in the mutants of 3-methyl-1-butanol (isoamyl alcohol) and 2-methyl-1-propanol (isobutanol) were similar or slightly reduced compared to the level found in the parent strain.

Biosynthesis of esterified alkan-2-ols and β-diketones in barley spike epicuticular wax: Synthesis of radioactive intermediates

Thirteen different14C-and3H-labelled epicuticular wax precursors have been synthesized and their structure determined by gas chromatography-mass spectrometry analyses. The biosyntheses of β-diketones and alkan-2-ol containing esters were studied by incorporating these intermediates into tissue slices of barley spikes whose awns had been removed. A differential labelling pattern of the alkan-2-ol esters and the β-diketones was observed after feeding three selected mutants blocked in different steps catalyzed by a multifunctional enzyme encoded for by thecer-cqu gene. Incer-u69 tissue slices (9,10-3H)-3-oxopalmitoyl-CoA was incorporated into both the esterified alkan-2-ols and the β-diketones. Only the former wax component was synthesized by the mutantscer-c36, and-q42. When C14 and C16 fatty acyl chains were fed to the tissues slices, those ofcer-u69 and-c36 readily labelled the esterified alkan-2-ols, whereas those ofcer-q42 were totally inactive. In all three mutants (2-14C)-pentadecan-2-one, (10,11-3H)-heptadecan-2-one and (2-3H)-pentadecan-2-ol exclusively labelled the alkan-2-ol moieties of the specified esters. (9,10-3H)-l-3-hydroxypalmitoyl-CoA and (3-14C)-labelleddl-3-hydroxy fatty acids having 14, 16 and 18 carbon atoms were incorporated with a very low efficiency into the β-diketones and the esterified alkan-2-ols. (9,10-3H)-3-oxopalmitoyl-CoA is the primer for the enzyme system known as β-ketoacyl elongase which forms the C29 (nonacosan-14,16-dione), C31 (hentriacontan-14, 16-dione) and C33 (tritriacontan-16,18-dione) β-diketones. After protection of the β-dicarbonyl group, 7 or 8 C2 units are added before the presumed decarboxylation to yield the complete β-diketone carbon chain. The alkan-2-ol esters arise from the 3-oxoacyl-CoA derivative by an initial decarboxylation to form a methyl ketone, followed by a reduction to an alkan-2-ol. The latter is then esterified with a fatty acid to form the alkan-2-ol containing esters. The three steps involved in the alkan-2-ol ester synthesis are accomplished by the coordinated action of a decarboxylase, reductase and ester synthetase.

Nonan-2-ol esters in sorghum leaf epicuticular wax and their collection by preparative gas chromatography

Epicuticular wax from sorghum seedling leaves was isolated and analyzed by TLC and GC. Its composition was remarkably similar to that of waxes on many other Gramineae leaves with the exception that three alkan-2-ol esters (nonan-2-ol docosanoate, nonan-2-ol tetracosanoate, and nonan-2-ol hexacosanoate) were present. This was unexpected as such esters have hitherto only been reported in waxes of plants with the capability of synthesizing β-diketone lipids. The C31−C35 alkan-2-ol esters were separated from the C38−C56 alkan-1-ol esters by preparative GC. The column effluent was passed through a small Dexsil-300 trap maintained at 150°C, and the esters eluted therefrom with chloroform. This technique gave better than 90% recovery of a [1-14C]−C32 ester.

Synthesis of epicuticular primary alcohols and intracellular fatty acids by tissue slices fromcer-j 59 barley leaves

The relative amounts of [1-14C]-acetate incorporated by tissue slices from primary leaves of Bonus barley into intracellular and epicuticular lipids were determined. Dark grown tissue incorporates about 2.5 times more label into intracellular fatty acids than into epicuticular lipids. Light grown tissue synthesizes the same amount of epicuticular lipids but ten times more intracellular fatty acids. The same total and relative amounts of incorporation were found when tissue slices fromcer-j59 were studied. Furthermore, no difference in the distributions of label among saturated, monoenoic, dienoic and trienoic fatty acids from the two genotypes were detected. This supports the contention that the block incer-j59 is independent of intracellular fatty acid synthesis.The locuscer-j59 blocks the production and use of C26 fatty acyl chains in the reductive pathway of epicuticular wax synthesis while increasing the relative importance of C22 chains. Wax on leaves of dark grown Bonus seedlings is a reasonable phenocopy of that present oncer-j59 light grown ones. The sites of control of wax synthesis by the locuscer-j and light as deduced from compositional analysis are confirmed by the present tissue slice incorporation experiments with [1-14C]-C2, −C16 and −C18 precursors. Although the distribution of label among the epicuticular lipid classes was not always a facsimile of the appropriate mass distribution, the distribution of label among the primary alcohols mimicked that of the corresponding mass distribution with respect to the relative importance of the C22 and C26 chain lengths. In wax synthesized by dark grown tissue the C22 alcohol was as well or better labelled than that with 24 carbons, whereas the latter was always more heavily labelled than the former in the wax from light grown tissue.

Effect of inhibitors on synthesis of fatty acyl chains present in waxes on developing maize leaves

Free fatty acids were identified as the fifth lipid class in wax from green leaf segments. They were not isolatable from the wax on yellow leaf segments, which also lacked aldehydes and appeared to have more esters and alkanes than primary alcohols. The chain length spectra of the alkanes and both ester moieties from yellow tissue are distinguishable from those of green tissue by the prominent amounts of shorter homologues, but the homologue distributions of the aldehydes and primary alcohols from the two types of tissue were similar. Tissue slices from yellow leaf segments incorporated 8.7% of the14C into wax lipids, whereas those from green segments were only half as efficient. In all wax fractions shorter homologues contained relatively more14C if they came from wax synthesized by yellow rather than green tissue. In wax from green tissue slices, 75% of the14C was in 32 carbon chains compared to 29% in that from yellow tissue slices. Pre-treatment of green tissue slices with 2.5 umol of Cl3AcONa inhibited total wax synthesis by 49%, primarily via blocking synthesis of C32 chains. While pre-treatment of yellow tissue slices with 2.5 μmol of triallate inhibited total wax synthesis by only 10%, C32 chain synthesis was reduced by 2,600 pmol (41%). In contrast to the Cl3AcONa inhibition, this reduction was to a large extent (1,700 pmol) compensated for by increases in C28 and C30 chain synthesis.