RESUMO
Plants that contain high concentrations of the defense compounds of the phenylpropene class (eugenol, chavicol, and their derivatives) have been recognized since antiquity as important spices for human consumption (e.g. cloves) and have high economic value. Our understanding of the biosynthetic pathway that produces these compounds in the plant, however, has remained incomplete. Several lines of basil (Ocimum basilicum) produce volatile oils that contain essentially only one or two specific phenylpropene compounds. Like other members of the Lamiaceae, basil leaves possess on their surface two types of glandular trichomes, termed peltate and capitate glands. We demonstrate here that the volatile oil constituents eugenol and methylchavicol accumulate, respectively, in the peltate glands of basil lines SW (which produces essentially only eugenol) and EMX-1 (which produces essentially only methylchavicol). Assays for putative enzymes in the biosynthetic pathway leading to these phenylpropenes localized many of the corresponding enzyme activities almost exclusively to the peltate glands in leaves actively producing volatile oil. An analysis of an expressed sequence tag database from leaf peltate glands revealed that known genes for the phenylpropanoid pathway are expressed at very high levels in these structures, accounting for 13% of the total expressed sequence tags. An additional 14% of cDNAs encoded enzymes for the biosynthesis of S-adenosyl-methionine, an important substrate in the synthesis of many phenylpropenes. Thus, the peltate glands of basil appear to be highly specialized structures for the synthesis and storage of phenylpropenes, and serve as an excellent model system to study phenylpropene biosynthesis.
Assuntos
Alcenos/metabolismo , Ocimum basilicum/metabolismo , Fenóis/metabolismo , Biotransformação , Ocimum basilicum/ultraestrutura , Folhas de Planta/metabolismoRESUMO
The evolution of new genes to make novel secondary compounds in plants is an ongoing process and might account for most of the differences in gene function among plant genomes. Although there are many substrates and products in plant secondary metabolism, there are only a few types of reactions. Repeated evolution is a special form of convergent evolution in which new enzymes with the same function evolve independently in separate plant lineages from a shared pool of related enzymes with similar but not identical functions. This appears to be common in secondary metabolism and might confound the assignment of gene function based on sequence information alone.
Assuntos
Evolução Molecular , Plantas/genética , Plantas/metabolismo , Duplicação Gênica , Expressão Gênica/genética , Genes de PlantasRESUMO
Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.
Assuntos
Furanos/metabolismo , Lignanas , Lignina/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/metabolismo , Árvores/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Complementar , Furanos/química , Lignina/química , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/genética , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Árvores/enzimologiaRESUMO
BACKGROUND: Although the lignins and lignans, both monolignol-derived coupling products, account for nearly 30% of the organic carbon circulating in the biosphere, the biosynthetic mechanism of their formation has been poorly understood. The prevailing view has been that lignins and lignans are produced by random free-radical polymerization and coupling, respectively. This view is challenged, mechanistically, by the recent discovery of dirigent proteins that precisely determine both the regiochemical and stereoselective outcome of monolignol radical coupling. RESULTS: To understand further the regulation and control of monolignol coupling, leading to both lignan and lignin formation, we sought to clone the first genes encoding dirigent proteins from several species. The encoding genes, described here, have no sequence homology with any other protein of known function. When expressed in a heterologous system, the recombinant protein was able to confer strict regiochemical and stereochemical control on monolignol free-radical coupling. The expression in plants of dirigent proteins and proposed dirigent protein arrays in developing xylem and in other lignified tissues indicates roles for these proteins in both lignan formation and lignification. CONCLUSIONS: The first understanding of regiochemical and stereochemical control of monolignol coupling in lignan biosynthesis has been established via the participation of a new class of dirigent proteins. Immunological studies have also implicated the involvement of potential corresponding arrays of dirigent protein sites in controlling lignin biopolymer assembly.
Assuntos
Lignanas/biossíntese , Lignina/biossíntese , Plantas/metabolismo , Sequência de Aminoácidos , Southern Blotting , Western Blotting , DNA de Plantas/biossíntese , DNA de Plantas/genética , Epitopos/metabolismo , Radicais Livres/química , Genoma , Lignanas/química , Lignina/química , Lignina/genética , Dados de Sequência Molecular , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Plantas/genética , Plantas/ultraestrutura , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Frações Subcelulares/metabolismoRESUMO
Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with beta-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.
Assuntos
Oxirredutases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Catálise , Cromatografia Líquida de Alta Pressão , Clonagem Molecular , Primers do DNA , DNA Complementar , Espectrometria de Massas , Dados de Sequência Molecular , Oxirredutases/química , Oxirredutases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , EstereoisomerismoRESUMO
Lignans are a widely distributed class of natural products, whose functions and distribution suggest that they are one of the earliest forms of defense to have evolved in vascular plants; some, such as podophyllotoxin and enterodiol, have important roles in cancer chemotherapy and prevention, respectively. Entry into lignan enzymology has been gained by the approximately 3000-fold purification of two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase, a pivotal branchpoint enzyme in lignan biosynthesis. Both have comparable ( approximately 34.9 kDa) molecular mass and kinetic (Vmax/Km) properties and catalyze sequential, NADPH-dependent, stereospecific, hydride transfers where the incoming hydride takes up the pro-R position. The gene encoding (+)-pinoresinol/(+)-lariciresinol reductase has been cloned and the recombinant protein heterologously expressed as a functional beta-galactosidase fusion protein. Its amino acid sequence reveals a strong homology to isoflavone reductase, a key branchpoint enzyme in isoflavonoid metabolism and primarily found in the Fabaceae (angiosperms). This is of great evolutionary significance since both lignans and isoflavonoids have comparable plant defense properties, as well as similar roles as phytoestrogens. Given that lignans are widespread from primitive plants onwards, whereas the isoflavone reductase-derived isoflavonoids are mainly restricted to the Fabaceae, it is tempting to speculate that this branch of the isoflavonoid pathway arose via evolutionary divergence from that giving the lignans.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/genética , Oxirredutases/isolamento & purificação , Plantas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Cromatografia de Afinidade , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Cinética , Dados de Sequência Molecular , Peso Molecular , Oxirredutases/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
We describe a method for isolating genomic DNA from teliospores of Tilletia caries (DC) Tul., T. controversa Kuhn and T. foetida (T. laevis) (Wallr.) Liro. for random-amplified polymorphic DNA (RAPD) analysis. DNA analysis of teliospores of covered smut or bunt has been difficult because of the thick wall and the high lipid content of the spores. This method overcomes these problems and yields sufficient quantities of DNA from the three species' teliospores for RAPDs. DNA quality appears to be good with very little degradation. RAPD amplifications of the extracted DNAs are reproducible and produce numerous large molecular weight bands from each individual. This procedure should permit the use of DNA analysis techniques to study species and races of Tilletia as well as fungi with similar spore structure.
Assuntos
Basidiomycota/genética , DNA Fúngico/isolamento & purificação , Reação em Cadeia da Polimerase/métodosRESUMO
Chrysothamnus nauseosus (rubber rabbitbrush) is used by browsing animals, especially mule deer (Odocoileus hemionus), as a forage in the winter months. It is used only slightly, if at all in the summer. This dietary difference may result from changes in the secondary chemical composition of the leaves. Solvent extracts from summer and winter rabbitbrush leaves were analyzed by gas chromatography and mass spectrometry, and the volatile compounds were quantified and identified. Hexane and chloroform extracts from winter leaves exhibit a marked concentration decrease in most chemicals when compared to summer extracts. The methanol extracts revealed the presence of several chemicals in the summer leaves that were absent in winter leaves. Rubber rabbitbrush has fewer secondary volatile chemicals in the winter than in the summer. These chemical differences may influence the seasonal dietary difference observed in mule deer and other browsing animals.