The allotetraploid plant Nicotiana tabacum (common tobacco) is a major crop species and a model organism, for which only very fragmented genomic sequences are currently available. Here we report high-quality draft genomes for three main tobacco varieties. These genomes show both the low divergence of tobacco from its ancestors and microsynteny with other Solanaceae species. We identify over 90,000 gene models and determine the ancestral origin of tobacco mosaic virus and potyvirus disease resistance in tobacco. We anticipate that the draft genomes will strengthen the use of N. tabacum as a versatile model organism for functional genomics and biotechnology applications.

The 4-and 5-hydroxylations of phenolic compounds in plants are catalyzed by cytochrome P450 enzymes. The 3-hydroxylation step leading to the formation of caffeic acid from p-coumaric acid remained elusive, however, alternatively described as a phenol oxidase, a dioxygenase, or a P450 enzyme, with no decisive evidence for the involvement of any in the reaction in planta. In this study, we show that the gene encoding CYP98A3, which was the best possible P450 candidate for a 3-hydroxylase in the Arabidopsis genome, is highly expressed in inflorescence stems and wounded tissues. Recombinant CYP98A3 expressed in yeast did not metabolize free pcoumaric acid or its glucose or CoA esters, p-coumaraldehyde, or p-coumaryl alcohol, but very actively converted the 5-O-shikimate and 5-O-D-quinate esters of trans-p-coumaric acid into the corresponding caffeic acid conjugates. The shikimate ester was converted four times faster than the quinate derivative. Antibodies directed against recombinant CYP98A3 specifically revealed differentiating vascular tissues in stem and root. Taken together, these data show that CYP98A3 catalyzes the synthesis of chlorogenic acid and very likely also the 3-hydroxylation of lignin monomers. This hydroxylation occurs on depsides, the function of which was so far not understood, revealing an additional and unexpected level of networking in lignin biosynthesis.Systematic genome sequencing is revealing a large number of orphan genes and their phylogenetic relatedness to genes with characterized function. EST 1 sequences, on the other hand, are providing preliminary information on levels, patterns of expression, and conservation of genes among species. Taken together, such information can be exploited as a clue to gene function and to track down missing steps in important pathways.The sequencing of the whole genome of Arabidopsis thaliana has revealed 273 cytochrome P450 genes distributed into 45 families and subfamilies (drnelson.utmem.edu/CytochromeP450. html, www.biobase.dk/P450/). P450 proteins thus form the largest superfamily of enzymes involved in plant metabolism, but the function of 80% of these enzymes is still unknown. Our attention was first drawn to the CYP98 family by its phylogeny and structure. An analysis of P450 phylogeny in A. thaliana (Fig. 1) shows that the CYP98 family is most closely related to CYP73A5, coding for the cinnamic-acid 4-hydroxylase, the second enzyme and first P450 in the phenylpropanoid pathway (1). CYP73A5 and the CYP98 family seem to have evolved from the same ancestor as CYP79 enzymes, some of which also, in common with CYP73A5, use aromatic substrates derived from the shikimate pathway (2, 3). It was thus tempting to speculate that the substrate of CYP98 enzymes was derived from aromatic amino acids as well. The Arabidopsis CYP98 family is formed by only three genes. CYP98A3 is present in single copy; CYP98A8 and CYP98A9 are 69% identical to one another and only 52% identical to CYP98A3. All P450 genes in the phenylpropanoid pathway (CYP73A5, CYP84A1, and CYP...

BackgroundNicotiana sylvestris and Nicotiana tomentosiformis are members of the Solanaceae family that includes tomato, potato, eggplant and pepper. These two Nicotiana species originate from South America and exhibit different alkaloid and diterpenoid production. N. sylvestris is cultivated largely as an ornamental plant and it has been used as a diploid model system for studies of terpenoid production, plastid engineering, and resistance to biotic and abiotic stress. N. sylvestris and N. tomentosiformis are considered to be modern descendants of the maternal and paternal donors that formed Nicotiana tabacum about 200,000 years ago through interspecific hybridization. Here we report the first genome-wide analysis of these two Nicotiana species.ResultsDraft genomes of N. sylvestris and N. tomentosiformis were assembled to 82.9% and 71.6% of their expected size respectively, with N50 sizes of about 80 kb. The repeat content was 72-75%, with a higher proportion of retrotransposons and copia-like long terminal repeats in N. tomentosiformis. The transcriptome assemblies showed that 44,000-53,000 transcripts were expressed in the roots, leaves or flowers. The key genes involved in terpenoid metabolism, alkaloid metabolism and heavy metal transport showed differential expression in the leaves, roots and flowers of N. sylvestris and N. tomentosiformis.ConclusionsThe reference genomes of N. sylvestris and N. tomentosiformis represent a significant contribution to the SOL100 initiative because, as members of the Nicotiana genus of Solanaceae, they strengthen the value of the already existing resources by providing additional comparative information, thereby helping to improve our understanding of plant metabolism and evolution.

SUMMARYLeaves of tobacco (Nicotiana tabacum) are covered with glandular trichomes that produce sucrose esters and diterpenoids in varying quantities, depending on cultivar type. The bicyclic diterpene Z-abienol is the major labdanoid present in some oriental tobacco cultivars, where it constitutes a precursor of important flavours and aromas. We describe here the identification and characterization of two genes governing the biosynthesis of Z-abienol in N. tabacum. As for other angiosperm labdanoid diterpenes, the biosynthesis of Z-abienol proceeds in two steps. NtCPS2 encodes a class-II terpene synthase that synthesizes 8-hydroxy-copalyl diphosphate, and NtABS encodes a kaurene synthase-like (KSL) protein that uses 8-hydroxy-copalyl diphosphate to produce Z-abienol. Phylogenetic analysis indicates that NtABS belongs to a distinct clade of KSL proteins that comprises the recently identified tomato (Solanum habrochaites) santalene and bergamotene synthase. RT-PCR results show that both genes are preferentially expressed in trichomes. Moreover, microscopy of NtCPS2 promoter-GUS fusion transgenics demonstrated a high specificity of expression to trichome glandular cells. Ectopic expression of both genes, but not of either one alone, driven by a trichomespecific promoter in transgenic Nicotiana sylvestris conferred Z-abienol formation to this species, which does not normally produce it. Furthermore, sequence analysis of over 100 tobacco cultivars revealed polymorphisms in NtCPS2 that lead to a prematurely truncated protein in cultivars lacking Z-abienol, thus establishing NtCPS2 as a major gene controlling Z-abienol biosynthesis in tobacco. These results offer new perspectives for tobacco breeding and the metabolic engineering of labdanoid diterpenes, as well as for structure-function relationship studies of terpene synthases.

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Arabidopsis expressing the castor bean (Ricinus communis) oleate 12-hydroxylase or the Crepis palaestina linoleate 12-epoxygenase in developing seeds typically accumulate low levels of ricinoleic acid and vernolic acid, respectively. We have examined the presence of a futile cycle of fatty acid degradation in developing seeds using the synthesis of polyhydroxyalkanoate (PHA) from the intermediates of the peroxisomal ␤-oxidation cycle. Both the quantity and monomer composition of the PHA synthesized in transgenic plants expressing the 12-epoxygenase and 12-hydroxylase in developing seeds revealed the presence of a futile cycle of degradation of the corresponding unusual fatty acids, indicating a limitation in their stable integration into lipids. The expression profile of nearly 200 genes involved in fatty acid biosynthesis and degradation has been analyzed through microarray. No significant changes in gene expression have been detected as a consequence of the activity of the 12-epoxygenase or the 12-hydroxylase in developing siliques. Similar results have also been obtained for transgenic plants expressing the Cuphea lanceolata caproyl-acyl carrier protein thioesterase and accumulating high amounts of caproic acid. Only in developing siliques of the tag1 mutant, deficient in the accumulation of triacylglycerols and shown to have a substantial futile cycling of fatty acids toward ␤-oxidation, have some changes in gene expression been detected, notably the induction of the isocitrate lyase gene. These results indicate that analysis of peroxisomal PHA is a better indicator of the flux of fatty acid through ␤-oxidation than the expression profile of genes involved in lipid metabolism.Membranes of plant cells are composed primarily of five "common" fatty acids, namely stearic, palmitic, oleic, linoleic, and linolenic acids. In contrast, a very large diversity of fatty acids exists in the reserve triacylglycerols (TAG) of seeds. More than 300 naturally occurring fatty acids have been described in seeds to date (Badami and Patil, 1980; van de Loo et al., 1993). The structures of these fatty acids vary in a number of features, including the length of the acyl chains; the number, position, and nature of unsaturated bonds; and the presence of functional groups, such as hydroxy, epoxy, and acetylenic groups. These fatty acids are often referred as "unusual" fatty acids because their structure is different from the common fatty acids found in membranes.Synthesis of unusual fatty acids has attracted considerable interest, both in fundamental and applied areas of plant biology. Several genes have recently been identified that code for enzymes involved in the synthesis of fatty acids containing unusual groups, such as hydroxy, epoxy, acetylenic, or carbocyclic groups, as well as conjugated unsaturated bonds (for a recent review, see Jaworski and Cahoon, 2003). In the majority of cases, these enzymes were found to be variants of enzymes involved in the synthesis of common fatty acids, such as variants of the soluble stearoyl-a...

A gene, named AtECH2, has been identified in Arabidopsis thaliana to encode a monofunctional peroxisomal enoyl-CoA hydratase 2. Homologues of AtECH2 are present in several angiosperms belonging to the Monocotyledon and Dicotyledon classes, as well as in a gymnosperm. In vitro enzyme assays demonstrated that AtECH2 catalyzed the reversible conversion of 2E-enoyl-CoA to 3R-hydroxyacyl-CoA. AtECH2 was also demonstrated to have enoyl-CoA hydratase 2 activity in an in vivo assay relying on the synthesis of polyhydroxyalkanoate from the polymerization of 3R-hydroxyacyl-CoA in the peroxisomes of Saccharomyces cerevisiae. AtECH2 contained a peroxisome targeting signal at the C-terminal end, was addressed to the peroxisome in S. cerevisiae, and a fusion protein between AtECH2 and a fluorescent protein was targeted to peroxisomes in onion cells. AtECH2 gene expression was strongest in tissues with high ␤-oxidation activity, such as germinating seedlings and senescing leaves. The contribution of AtECH2 to the degradation of unsaturated fatty acids was assessed by analyzing the carbon flux through the ␤-oxidation cycle in plants that synthesize peroxisomal polyhydroxyalkanoate and that were over-or underexpressing the AtECH2 gene. These studies revealed that AtECH2 participates in vivo to the conversion of the intermediate 3R-hydroxyacyl-CoA, generated by the metabolism of fatty acids with a cis (Z)-unsaturated bond on an even-numbered carbon, to the 2E-enoyl-CoA for further degradation through the core ␤-oxidation cycle.The peroxisome is the site of numerous important biochemical reactions in plants, including photorespiration, the ␤-oxidation cycle, and the glyoxylate cycle. Although several enzymes involved in these pathways have been identified, analysis of the plant proteome for proteins possessing putative peroxisome targeting sequences have identified numerous candidate peroxisomal proteins for which no functions have been assigned (1). Furthermore, prediction of peroxisomal proteins can be made difficult by the absence of recognizable signal peptide, particularly for peroxisomal membrane protein. Thus, our present knowledge of the complexity of the biochemical pathways present in the peroxisome is fragmented.The peroxisomal ␤-oxidation cycle is of primary importance during seedling establishment following germination, because it is responsible for the breakdown of fatty acids into acetylCoA, which is subsequently converted to glucose via the glyoxylate cycle and gluconeogenesis (2). Although fatty acid ␤-oxidation is very active during germination in oleaginous seed and during senescence, this cycle is also present in mature photosynthetic tissues, such as leaves, as well as in developing seeds (3).Degradation of saturated fatty acids in the peroxisome occurs via four enzyme activities located on three proteins that form the core ␤-oxidation pathway. The first step is mediated by an acyl-CoA oxidase, converting acyl-CoA to 2E-enoyl-CoA. This is followed by the hydration of the 2E-enoyl-CoA to 3-hydroxyacyl-CoA by an...

Degradation of unsaturated fatty acids through the peroxisomal b-oxidation pathway requires the participation of auxiliary enzymes in addition to the enzymes of the core b-oxidation cycle. The auxiliary enzyme D 3,5 ,D 2,4 -dienoyl-coenzyme A (CoA) isomerase has been well studied in yeast (Saccharomyces cerevisiae) and mammals, but no plant homolog had been identified and characterized at the biochemical or molecular level. A candidate gene (At5g43280) was identified in Arabidopsis (Arabidopsis thaliana) encoding a protein showing homology to the rat (Rattus norvegicus) D Catabolism of fatty acids occurs primarily through the b-oxidation cycle. In plants, b-oxidation is located in peroxisomes, while in animal cells, it occurs in both peroxisomes and mitochondria (for review, see Gerhardt, 1992;Graham and Eastmond, 2002;Hooks, 2002). b-Oxidation is of primary importance for seedling establishment following germination since it allows the breakdown of fatty acids stored in triacylglycerides into acetyl-CoA, which is subsequently converted to Glc via the glyoxylate cycle and gluconeogenesis (Hayashi et al., 1998;Germain et al., 2001). Although b-oxidation is very active during germination, this cycle is also present in mature photosynthetic tissues, as well as in developing seeds Graham and Eastmond, 2002).The peroxisomal core b-oxidation cycle in plants is composed of four enzymatic activities located on three proteins. The first enzyme is acyl-CoA oxidase, converting acyl-CoA into 2-trans-enoyl-CoA. The second is a multifunctional enzyme that harbors two activities in a single polypeptide, namely, an enoyl-CoA hydratase and a 3-hydroxyacyl-CoA dehydrogenase, catalyzing the successive conversion of 2-trans-enoyl-CoA into 3-hydroxyacyl-CoA and 3-ketoacyl-CoA, respectively. The final enzyme is the 3-ketothiolase and is responsible for cleavage of 3-ketoacyl-CoA to form acetyl-CoA and an acyl-CoA that is two carbons shorter and that can reenter the b-oxidation spiral. In Arabidopsis (Arabidopsis thaliana), at least four genes have been characterized to encode for acyl-CoA oxidases with different chain-length specificities, two genes encode for multifunctional enzymes, and four genes encode 3-ketothiolases (Graham and Eastmond, 2002).In contrast to the degradation of saturated fatty acids, which can be mediated completely by the four enzymatic activities of the core b-oxidation cycle, the degradation of several types of unsaturated fatty acids has been shown to require the presence of auxiliary enzymes Hiltunen et al., 2003;van Roermund et al., 2003). This is because the core b-oxidation functions through a 2-trans-enoyl-CoA intermediate while many fatty acids have unsaturated bonds either on an odd-numbered carbon or in the Article, publication date, and citation information can be found at www.plantphysiol.org/cgi