Biosynthesis of 1-alkenes in higher plants. A model study with the composite Carthamus tinctorius L

Uneven numbered 1-alkenes, such as 1-pentadecene, 1-heptadecene, 1,8-heptadecadiene, 1,8,11-heptadecatriene and 1,8,11,14-heptadecatetraene are the predominant hydrocarbons in germinating safflower (Carthamus tinctorius L.). According to their chain length and positions of double bonds they are derived from palmitic, stearic, oleic, linoleic or linolenic acid, respectively. This has been proved by administration of synthetic alpha-, beta-, or gamma-deuterated 12-phenyldodecanoic acids to germinating safflower. Mass spectrometric analysis of the resulting deuterium-labelled 11-phenyl-1-undecenes shows that only a single hydrogen from C-3 and carbon dioxide from C-1 of the precursor fatty acid is lost during vinyl group formation. A strong isotope effect (kH/kD greater than or equal to 10) during the conversion of racemic 12-phenyl[3-2h]dodecanoic acid to 11-phenyl-1-[2-2H]undecene is consistent with an initial enzymatic attack on a non-activated hydrogen at C-3 accompanied by simultaneous fragmentation into the respective 1-alkene and carbon dioxide. Mechanisms, based on ionic or radical abstraction of a hydrogen from C-3 of the fatty acid, followed by decarboxylation are discussed. The results are applicable to the formation of various vinylic substituents in natural products such as porphyrins, acetylenic hydrocarbons, pheromones from marine brown algae and terpenoids.     

Fat metabolism in higher plants. Properties of a soluble stearyl-acyl carrier protein desaturase from maturing Carthamus tinctorius

A soluble extract from maturing safflower seeds (Carthamus tinctorius) synthesized [¹⁴C]oleic acid from [¹⁴C]malonate, or [¹⁴C]stearyl-acyl carrier protein. Stearyl-acyl carrier protein was generated from [¹⁴C]malonate by the seed extract. The desaturase had only a trace of activity when stearyl-CoA was the substrate. The stearyl-acyl carrier protein desaturase had a specific requirement for ferredoxin which was only partially replaced by flavodoxin. While NADPH was an effective reductant, NADH was ineffective. However, the most effective reductant was a system composed of ferredoxin, grana lamellae, ascorbic acid, dichlorophenolindophenol, and light. No NADPH requirement was observed when this reducing system was employed. Stearylacyl carrier protein desaturase activity was enhanced by dithiothreitol and reduced glutathione, but was partially inhibited by β-mercaptoethanol. The desaturase activity was inhibited by 1 mm potassium cyanide but insensitive to carbon monoxide. No lipid micelle requirement could be demonstrated.     

Clastogenic effect of Carthamus lanatus L. (Asteraceae)

The clastogenic effect of total dichloromethane, methanol and water extracts, four bioactive fractions and three individual constituents from Carthamus lanatus aerial parts were evaluated in mice by bone marrow chromosome aberration assay with mitomycin C as positive control. Significant differences in the percentage of aberrant mitosis of the extracts were observed. The dichloromethane extract exhibited a considerable clastogenic effect and the water extract a negligible one. Different types of chromosome aberrations and time-dependant effects for the active fractions and individual compounds were found.     

Population genetic analysis of safflower (Carthamus tinctorius; Asteraceae) reveals a Near Eastern origin and five centers of diversity

Analyses of genetic variation in crop gene pools are a powerful tool for investigating the origin and early evolution of crop lineages. Such analyses also have the potential to identify unique genetic resources for continued crop improvement. The oilseed crop safflower (Carthamus tinctorius) is believed to have been domesticated in the Fertile Crescent region, but up to 10 geographic centers of similarity throughout the world have been proposed based on morphology. Nuclear microsatellite analysis of accessions from each of the 10 proposed centers of similarity, as well as individuals of the progenitor species, suggested the presence of five genetic clusters (1, Europe; 2, Turkey-Iran-Iraq-Afghanistan; 3, Israel-Jordan-Syria; 4, Egypt-Ethiopia; and 5, the Far East-India-Pakistan). North American accessions, products of a secondary introduction from the native range, suggest that a subset of the native accessions harbor unique genetic diversity that could be useful in future breeding efforts. Overall, a Near Eastern origin of safflower was confirmed based on the genetic similarity between the progenitor and the Near Eastern safflower accessions, as well as previous archaeological finds. Genetic differentiation between geographical clusters of accessions is evident, although not to the degree proposed based on morphology.     

Taxonomic problems in Carthamus (Asteraceae): RAPD markers and sectional classification

Recent molecular studies have clarified the problem of the generic limits of Carthamus . However, taxonomic problems remain in the sectional classification: section Carthamus was confirmed as a natural group, but molecular phylogenies based on DNA sequences did not resolve the other sections, resulting in a confusing conglomerate. A survey based on RAPD markers has been carried out in this conflicting group of sections, with the aim of evaluating the usefulness of these markers at the section and species levels. On the basis of the results, correlated with morphological and karyological characters, some recommendations are made in relation to the taxonomy of Carthamus . The number of sections is reduced from five to two: section Thamnacanthus is currently classified as a different genus, Phonus ; section Carthamus s.s. is retained in its present delimitation, and section Atractylis is redefined, including current sections Atractylis , Lepidopappus and Odontagnathius . The commonly accepted treatment of some taxa as subspecies is not well founded, and C. alexandrinus , C. creticus , C. tenuis and C. turkestanicus should be considered as independent species. Our results confirm that methodological problems posed by RAPD markers can be avoided by careful laboratory procedures and appropriate data analyses; they also suggest that this kind of marker is useful at low taxonomic levels and is, furthermore, complementary to DNA sequence analysis.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 147 , 375–383.     

Novel repeated DNA sequences in safflower (Carthamus tinctorius L.) (Asteraceae): cloning, sequencing, and physical mapping by fluorescence in situ hybridization

Two novel repetitive DNA sequences, pCtKpnI-1 and pCtKpnI-2, were isolated from Carthamus tinctorius (2n = 2x = 24) and cloned. Both represent tandemly repeated sequences. The pCtKpnI-1 and pCtKpnI-2 clones constitute repeat units of 343-345 bp and 367 bp, respectively, with 63% sequence heterogeneity between the two. Fluorescence in situ hybridization (FISH) was employed on metaphase chromosomes of C. tinctorius using, simultaneously, pCtKpnI-1 and pCtKpnI-2 repeated sequences. The pCtKpnI-1 sequence was found to be exclusively localized at subtelomeric regions on most of the chromosomes. On the other hand, sequence of the pCtKpnI-2 clone was distributed on two nucleolar and one nonnucleolar chromosome pairs. The satellite, and the intervening chromosome segment between the primary and secondary constrictions, in the two nucleolar chromosome pairs were wholly constituted by pCtKpnI-2 repeated sequence. The pCtKpnI-2 repeated sequence, showing partial homology to intergenic spacer (IGS) of 18S-25S ribosomal RNA genes of an Asteraceae taxon (Centaurea stoebe), and the 18S-25S rRNA gene clusters were located at independent, but juxtaposed sites in the nucleolar chromosomes. Variability in the number, size, and location of the two repeated sequences provided identification of most of the chromosomes in the otherwise not too distinctive homologues within the complement. This article reports the start of a molecular cytogenetics program targeting the genome of safflower, a major world oil crop about whose genetics very little is known.     

Genome size variation in the genus Carthamus (Asteraceae, Cardueae): systematic implications and additive changes during allopolyploidization

BACKGROUND AND AIMS: Plant genome size is an important biological characteristic, with relationships to systematics, ecology and distribution. Currently, there is no information regarding nuclear DNA content for any Carthamus species. In addition to improving the knowledge base, this research focuses on interspecific variation and its implications for the infrageneric classification of this genus. Genome size variation in the process of allopolyploid formation is also addressed. METHODS: Nuclear DNA samples from 34 populations of 16 species of the genus Carthamus were assessed by flow cytometry using propidium iodide. KEY RESULTS: The 2C values ranged from 2.26 pg for C. leucocaulos to 7.46 pg for C. turkestanicus, and monoploid genome size (1Cx-value) ranged from 1.13 pg in C. leucocaulos to 1.53 pg in C. alexandrinus. Mean genome sizes differed significantly, based on sectional classification. Both allopolyploid species (C. creticus and C. turkestanicus) exhibited nuclear DNA contents in accordance with the sum of the putative parental C-values (in one case with a slight reduction, frequent in polyploids), supporting their hybrid origin. CONCLUSIONS: Genome size represents a useful tool in elucidating systematic relationships between closely related species. A considerable reduction in monoploid genome size, possibly due to the hybrid formation, is also reported within these taxa.     

Chloroplast DNA study of the generaAmbrosia s.l. andHymenoclea (Asteraceae): Systematic implications

The relationship ofAmbrosia (ragweed) andFranseria has long been debated. Their treatment as separate genera has been repeatedly challenged. In this study, chloroplast DNA restriction site variation was examined for species from bothAmbrosia andFranseria as well as taxa from the closely related genusHymenoclea. The chloroplast genomes of members of these three genera were examined using 21 restriction endonucleases and the restriction mutations were used to construct phylogenetic trees. Wagner and Dollo parsimony as well as weighted parsimony were employed to compare the different phylogenies. The results support a close relationship betweenAmbrosia andFranseria, but indicate that the two groups are well separated. Compared toFranseria, Ambrosia is a much more strongly supported group, and the results indicate thatHymenoclea is closer toFranseria than toAmbrosia. The cpDNA phylogeny was used as a framework to examine evolutionary trends in morphology and secondary chemistry.     

The procaryotic nature of the fatty acid synthetase of developing Carthamus tinctorius L. (safflower) seeds

All component activities involved in the synthesis of fatty acid were detected in crude extracts of developing safflower seeds. The crude extracts were fractionated into three portions by polyethylene glycol (0–5, 5–15, and 15% supernatant). Acetyl-CoA:acyl carrier protein (ACP) transacylase was precipitated about 66% by 5% polyethylene glycol. β-Ketoacyl-ACP reductase and enoyl-ACP reductase I were completely recovered in the 5–15% fraction. β-Ketoacyl-ACP synthetase and enoyl-ACP reductase II were in the 15% supernatant fraction. Malonyl-CoA:ACP transacylase and β-hydroxyacyl-ACP dehydrase were distributed into both fractions of 5–15 and 15% supernatant. When the 5–15% fraction was gel-filtrated on Sephadex G-200 column, β-hydroxyacyl-ACP dehydrase and malonyl-CoA:ACP transacylase were clearly separated from other enzymes, but β-Ketoacyl-ACP reductase and enoyl-ACP reductase I overlapped. However, by hydroxyapatite chromatography, these two reductases were clearly separated. Properties of each enzyme were examined with the samples fractionated by polyethylene glycol. β-Ketoacyl-ACP reductase preferably utilized NADPH (K_m = 16 μM) as hydrogen donor. The K_m for acetoacetyl-ACP was 9 μm. β-Hydroxyacyl-ACP dehydrase had a K_m of 12 μm for crotonyl-ACP. Enoyl-ACP reductase had two forms, I and II, and these two reductases differed from each other as follows: (a) separation by polyethylene glycol (15%) fractionation; (b) the optimum pH; (c) the hydrogen donor specificity; (d) the substrate specificity. From these results, it is concluded that the FAS system of developing safflower seeds was nonassociated and similar to the procaryotic type of Escherichia coli.     

Evidence for an oleoyl phosphatidylcholine desaturase in microsomal preparations from cotyledons of safflower (Carthamus tinctorius) seed.

1. [14C]Oleoyl-CoA was metabolized rapidly and essentially completely by microsomal preparations from developing safflower (Carthamus tinctorius) cotyledons, and most of the [14C]oleate was incorporated into 3-sn-phosphatidylcholine. 2. In aerobic reaction mixtures containing NADH2 the [14C]oleate in 3-sn-phosphatidylcholine was converted into [14C]linoleate without any change in the specific radioactivity of the lipid. Over a 60 min incubation period the extent of conversion of [14C]oleoyl phosphatidylcholine into [14C]linoleoyl phosphatidylcholine was generally greater than 60%. The rate of desaturation of endogenous [14C]oleoyl phosphatidylcholine labelled from [14C]oleoyl-CoA was much greater that of exogenous [14C]dioleoyl phosphatidylcholine the specific radioactivity of the oleoyl moiety of the lipid remained constant, indicating that labelled and unlabelled oleate were desaturated at the same rate. On this assumption an initial rate of desaturation of about 15 nmol of oleate desaturated/min per mumol of 3-sn-phosphatidylcholine was estimated. 4. [14C]Oleate esterified at positions 1 and 2 of both endogenous and exogenous 3-sn-phosphatidylcholine was desaturated. 5. Attempts to demonstrate the presence of an oleoyl-CoA desaturase in safflower microsomal fractions by the appearance of linoleoyl-CoA in reaction mixtures were inconclusive.     

Fat Metabolism in Higher Plants XXVI. Biosynthesis of Fatty Acids in Tissues of Developing Seeds and Germinating Seedlings of Safflower (Carthamus tinctorius L.)

The capacity of both developing seeds and germinating seedlings of safflower for the incorporation of acetate-C¹⁴ into long-chain fatty acids is examined.

Intact tissue of the developing seed shows a low rate of acetate incorporation into fatty acid initially but between the tenth and twenty-fifth day after flowering the tissue has a high rate of synthesis, in particular with respect to the unsaturated fatty acids. Centrifuged fractionation of homogenates of this developmental tissue yielded several active fractions, the most active being the P_I fraction consisting mostly of plastids. Cofactor requirements and pH effects are examined.

Germinating tissue shows a more uniform capacity for synthesis of fatty acids since there is no marked change in synthetic capacity. The newly synthesized fatty acids are consistently palmitic, stearic, and oleic acid. No linoleic synthesis could be detected. The most active fraction of cell-free preparation of germinating tissue is the plastid fraction (P_I), similar to what was formed with developing tissue.

     

Stereochemical studies on algal pheromone biosynthesis. A model study with the flowering plant Senecio isatideus (Asteraceae)

Unsaturated C8 and C11 hydrocarbons act as chemical signals (chemotaxis) during sexual reproduction of many marine brown algae. One of these compounds, namely (+)-(6S)-6-(1Z-butenyl)cyclohepta-1,4-diene (= ectocarpene) is also formed as a major hydrocarbon by the flowering plant Senecio isatideus (Asteraceae). On administration of enantiospecifically labelled (8R)- or (8S)-[7,8-2H2]trideca-3,6,9-trienoic acid instead of the natural precursor dodeca-3,6,9-trienoic acid to this plant, the artificial C12 analogue of ectocarpene is formed. Mass spectroscopic analysis of the metabolites revealed this process to be enantiospecific for the C(8)-HRe atom of the precursor acid. The stereochemical course of the overall reaction is in agreement with a precise, U-shaped embedding of the trienoic acid into the active center of the enzyme(s) and Re-attack onto a hydrogen at the C(8) methylene group of this precursor. The reactive intermediate cyclizes by pi-orbital interaction between C(4) and C(6) of the acid and is accompanied by decarboxylation. The first product is a thermally unstable (1S,2R)-cis-1-(1E,3Z-hexadienyl)-2-vinylcyclopropane, which immediately rearranges to (+)-(6S)-ectocarpene (homo-Cope rearrangement). The present work provides first experimental evidence for a homo-Cope rearrangement as a naturally occurring electrocyclic reaction.     

A cladistic study of North AmericanCoreopsis (Asteraceae: Heliantheae)

A cladistic study of all 44 species of North AmericanCoreopsis was performed using 35 characters. The resulting cladogram indicated that all 11 sections are monophyletic. At the intersectional level, two lineages were revealed, one consisting of six sections occurring almost exclusively in Mexico and California, and another comprising five sections restricted largely to the eastern and southeastern United States. The cladogram is similar to phylogenies produced by less explicit methods but it differs in two major respects: the monotypic sect.Silphidium is placed with other sections from the southeastern United States rather than with Mexican sections, and sect.Anathysana from Mexico is more closely allied with the three California sections than with sect.Electra from Mexico.     

Pollen Studies in Subtribe Centaureinae (Asteraceae): The Carthamus Complex and the Genus Aegialophila Analyzed with Electron Microscopy

Abstract: Recent molecular and karyologic studies have deeply modified the delimitation of the genera of the Carthamus complex, and have led to a new classification of the group in four genera ( Carduncellus, Carthamus, Femeniasia and Phonus ). Molecular studies have confirmed the position of the group among the derived genera of the subtribe Centaureinae, and have suggested a faint relationship between the Carthamus complex and the small genus Aegialophila. However, despite the importance of pollen evolution in the subtribe, a comprehensive study of the pollen of the involved genera with electron microscopy was lacking. Therefore, we have carried out a survey of pollen morphology and ultrastructure in a wide sample of the genera, using both scanning and transmission electron microscopy. Our results suggest that pollen type of the Carthamus complex is homogeneous and should be classified as a new pollen type in the classification of Wagenitz. Pollen type of Aegialophila shows strong similarities with the Centaurea scabiosa pollen type of Wagenitz, but there are slight differences in shape and in ultrastructure of the exine. These differences are evaluated, together with morphological and molecular evidence; the authors conclude that similarities are the result of parallel evolution and that pollen of Aegialophila could be considered a new pollen type. Some considerations are made on the placement of the new types in the cadre of pollen and molecular evolution in the subtribe Centaureinae.