Multigenome analysis implicates miniature inverted-repeat transposable elements (MITEs) in metabolic diversification in eudicots.

Plants produce a plethora of natural products, including many drugs. It has recently emerged that the genes encoding different natural product pathways may be organized as biosynthetic gene clusters in plant genomes, with >30 examples reported so far. Despite superficial similarities with microbes, these clusters have not arisen by horizontal gene transfer, but rather by gene duplication, neofunctionalization, and relocation via unknown mechanisms. Previously we reported that two Arabidopsis thaliana biosynthetic gene clusters are located in regions of the genome that are significantly enriched in transposable elements (TEs). Other plant biosynthetic gene clusters also harbor abundant TEs. TEs can mediate genomic rearrangement by providing homologous sequences that enable illegitimate recombination and gene relocation. Thus, TE-mediated recombination may contribute to plant biosynthetic gene cluster formation. TEs may also facilitate establishment of regulons. However, a systematic analysis of the TEs associated with plant biosynthetic gene clusters has not been carried out. Here we investigate the TEs associated with clustered terpene biosynthetic genes in multiple plant genomes and find evidence to suggest a role for miniature inverted-repeat transposable elements in cluster formation in eudicots. Through investigation of the newly sequenced Amborella trichopoda, Aquilegia coerulea, and Kalanchoe fedtschenkoi genomes, we further show that the "block" mechanism of founding of biosynthetic gene clusters through duplication and diversification of pairs of terpene synthase and cytochrome P450 genes that is prevalent in the eudicots arose around 90-130 million years ago, after the appearance of the basal eudicots and before the emergence of the superrosid clade.

A metabolic gene cluster in Lotus japonicus discloses novel enzyme functions and products in triterpene biosynthesis.

Genes for triterpene biosynthetic pathways exist as metabolic gene clusters in oat and Arabidopsis thaliana plants. We characterized the presence of an analogous gene cluster in the model legume Lotus japonicus. In the genomic regions flanking the oxidosqualene cyclase AMY2 gene, genes for two different classes of cytochrome P450 and a gene predicted to encode a reductase were identified. Functional characterization of the cluster genes was pursued by heterologous expression in Nicotiana benthamiana. The gene expression pattern was studied under different developmental and environmental conditions. The physiological role of the gene cluster in nodulation and plant development was studied in knockdown experiments. A novel triterpene structure, dihydrolupeol, was produced by AMY2. A new plant cytochrome P450, CYP71D353, which catalyses the formation of 20-hydroxybetulinic acid in a sequential three-step oxidation of 20-hydroxylupeol was characterized. The genes within the cluster are highly co-expressed during root and nodule development, in hormone-treated plants and under various environmental stresses. A transcriptional gene silencing mechanism that appears to be involved in the regulation of the cluster genes was also revealed. A tightly co-regulated cluster of functionally related genes is involved in legume triterpene biosynthesis, with a possible role in plant development.

Formation of plant metabolic gene clusters within dynamic chromosomal regions.

In bacteria, genes with related functions often are grouped together in operons and are cotranscribed as a single polycistronic mRNA. In eukaryotes, functionally related genes generally are scattered across the genome. Notable exceptions include gene clusters for catabolic pathways in yeast, synthesis of secondary metabolites in filamentous fungi, and the major histocompatibility complex in animals. Until quite recently it was thought that gene clusters in plants were restricted to tandem duplicates (for example, arrays of leucine-rich repeat disease-resistance genes). However, operon-like clusters of coregulated nonhomologous genes are an emerging theme in plant biology, where they may be involved in the synthesis of certain defense compounds. These clusters are unlikely to have arisen by horizontal gene transfer, and the mechanisms behind their formation are poorly understood. Previously in thale cress (Arabidopsis thaliana) we identified an operon-like gene cluster that is required for the synthesis and modification of the triterpene thalianol. Here we characterize a second operon-like triterpene cluster (the marneral cluster) from A. thaliana, compare the features of these two clusters, and investigate the evolutionary events that have led to cluster formation. We conclude that common mechanisms are likely to underlie the assembly and control of operon-like gene clusters in plants.

Role of lupeol synthase in Lotus japonicus nodule formation.

• Triterpenes are plant secondary metabolites, derived from the cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs). Here, we investigated the role of lupeol synthase, encoded by OSC3, and its product, lupeol, in developing roots and nodules of the model legume Lotus japonicus. • The expression patterns of OSC3 in different developmental stages of uninfected roots and in roots infected with Mesorhizobium loti were determined. The tissue specificity of OSC3 expression was analysed by in situ hybridization. Functional analysis, in which transgenic L. japonicus roots silenced for OSC3 were generated, was performed. The absence of lupeol in the silenced plant lines was determined by GC-MS. • The expression of ENOD40, a marker gene for nodule primordia initiation, was increased significantly in the OSC3-silenced plant lines, suggesting that lupeol influences nodule formation. Silenced plants also showed a more rapid nodulation phenotype, consistent with this. Exogenous application of lupeol to M. loti-infected wild-type plants provided further evidence for a negative regulatory effect of lupeol on the expression of ENOD40. • The synthesis of lupeol in L. japonicus roots and nodules can be solely attributed to OSC3. Taken together, our data suggest a role for lupeol biosynthesis in nodule formation through the regulation of ENOD40 gene expression.

High throughput screening of mutants of oat that are defective in triterpene synthesis.

The triterpenes are a large and diverse group of plant natural products that have important functions in plant protection and food quality, and a range of pharmaceutical and other applications. Like sterols, they are synthesised from mevalonate via the isoprenoid pathway, the two pathways diverging after 2,3-oxidosqualene. During triterpene synthesis 2,3-oxidosqualene is cyclised to one of a number of potential products, the most common of these being the pentacyclic triterpene beta-amyrin. Plants often produce complex mixtures of conjugated triterpene glycosides which may be derived from a single triterpene skeleton. The delineation, functional analysis and exploitation of triterpene pathways in plants therefore represent a substantial challenge. Here we have carried out high throughput screening to identify mutants of diploid oat (Avena strigosa) that are blocked in the early steps of triterpene synthesis. We also show that mutants that are affected in the first committed step in synthesis of beta-amyrin-derived triterpenes, and so are unable to cyclise 2,3-oxidosqualene to beta-amyrin (sad1 mutants), accumulate elevated levels of primary sterols. The major differences were in Delta-7-campesterol and Delta-7-avenasterol, which both increased several fold relative to wild-type levels. This is presumably due to accumulation of squalene and 2,3-oxidosqualene and consequent feedback into the sterol pathway, and is consistent with previous reports in which specific oxidosqualene cyclase inhibitors and elicitors of triterpene biosynthesis were shown to have inverse effects on the flux through the sterol and triterpene pathways.

Operons.

Operons (clusters of co-regulated genes with related functions) are common features of bacterial genomes. More recently, functional gene clustering has been reported in eukaryotes, from yeasts to filamentous fungi, plants, and animals. Gene clusters can consist of paralogous genes that have most likely arisen by gene duplication. However, there are now many examples of eukaryotic gene clusters that contain functionally related but non-homologous genes and that represent functional gene organizations with operon-like features (physical clustering and co-regulation). These include gene clusters for use of different carbon and nitrogen sources in yeasts, for production of antibiotics, toxins, and virulence determinants in filamentous fungi, for production of defense compounds in plants, and for innate and adaptive immunity in animals (the major histocompatibility locus). The aim of this article is to review features of functional gene clusters in prokaryotes and eukaryotes and the significance of clustering for effective function.

Metabolic diversification--independent assembly of operon-like gene clusters in different plants.

Operons are clusters of unrelated genes with related functions that are a feature of prokaryotic genomes. Here, we report on an operon-like gene cluster in the plant Arabidopsis thaliana that is required for triterpene synthesis (the thalianol pathway). The clustered genes are coexpressed, as in bacterial operons. However, despite the resemblance to a bacterial operon, this gene cluster has been assembled from plant genes by gene duplication, neofunctionalization, and genome reorganization, rather than by horizontal gene transfer from bacteria. Furthermore, recent assembly of operon-like gene clusters for triterpene synthesis has occurred independently in divergent plant lineages (Arabidopsis and oat). Thus, selection pressure may act during the formation of certain plant metabolic pathways to drive gene clustering.

Capillary-induced contact guidance.

Topographical features are known to impose capillary forces on liquid droplets, and this phenomenon is exploited in applications such as printing, coatings, textiles and microfluidics. Surface topographies also influence the behavior of biological cells (i.e., contact guidance), with implications ranging from medicine to agriculture. An accurate physical description of how cells detect and respond to surface topographies is necessary in order to move beyond a purely heuristic approach to optimizing the topographies of biomaterial interfaces. Here, we have used a combination of Langmuir-Blodgett lithography and nanoimprinting to generate a range of synthetic microstructured surfaces with grooves of subcellular dimensions in order to investigate the influence of capillary forces on the biological process of contact guidance. The physical-chemical properties of these surfaces were assessed by measuring the anisotropic spreading of sessile water droplets. Having established the physical properties of each surface, we then investigated the influence of capillary forces on the processes of cellular contact guidance in biological organisms, using mammalian osteoblasts and germinating fungal spores as tester organisms. Our results demonstrate that capillary effects are present in topographical contact guidance and should therefore be considered in any physical model that seeks to predict how cells will respond to a particular surface topography.

The rice leaf blast pathogen undergoes developmental processes typical of root-infecting fungi.

Pathogens have evolved different strategies to overcome the various barriers that they encounter during infection of their hosts. The rice blast fungus Magnaporthe grisea causes one of the most damaging diseases of cultivated rice and has emerged as a paradigm system for investigation of foliar pathogenicity. This fungus undergoes a series of well-defined developmental steps during leaf infection, including the formation of elaborate penetration structures (appressoria). This process has been studied in great detail, and over thirty M. grisea genes that condition leaf infection have been identified. Here we show a new facet of the M. grisea life cycle: this fungus can undergo a different (and previously uncharacterized) set of programmed developmental events that are typical of root-infecting pathogens. We also show that root colonization can lead to systemic invasion and the development of classical disease symptoms on the aerial parts of the plant. Gene-for-gene type specific disease resistance that is effective against rice blast in leaves also operates in roots. These findings have significant implications for fungal development, epidemiology, plant breeding and disease control.

Purification, crystallization and preliminary X-ray diffraction analysis of a fungal saponin-detoxifying enzyme.

Tomatinase, an extracellular enzyme belonging to family 3 of the glycosyl hydrolases, is produced by the fungal tomato-leaf pathogen Septoria lycopersici and detoxifies the saponin alpha-tomatine. An efficient strategy for purification of the enzyme from fungal culture medium has been developed. Single crystals have been grown by vapour diffusion at 289 K from 17.5%(w/v) PEG 4K, 5%(v/v) 2-propanol and 0.1 M sodium acetate pH 4.5 as precipitant. When cryoprotected at 100 K, these crystals diffract to at least 3.0 A and belong to space group P2(1)2(1)2. Based on an estimated molecular weight of 110 kDa for the glycosylated protein and assuming two molecules in the asymmetric unit, the crystals contain approximately 46% solvent.

Molecular cloning and characterization of triterpene synthases from Medicago truncatula and Lotus japonicus.

Cloning of OSCs required for triterpene synthesis from legume species that are amenable to molecular genetics will provide tools to address the importance of triterpenes and their derivatives during normal plant growth and development and also in interactions with symbionts and pathogens. Here we report the cloning and characterization of a total of three triterpene synthases from the legume species Medicago truncatula and Lotus japonicus. These include a beta-amyrin synthase from M. truncatula (MtAMY1) and a mixed function triterpene synthase from Lotus japonicus (LjAMY2). A partial cDNA predicted to encode a beta-amyrin synthase (LjAMY1) was also isolated from L. japonicus. The expression patterns of MtAMY1, LjAMY1 and LjAMY2 and of additional triterpene synthases previously characterised from M. truncatula and pea differ in different plant tissues and during nodulation, suggesting that these enzymes may have distinct roles in plant physiology and development.

Saponins in cereals.

Saponins are a diverse family of secondary metabolites that are produced by many plant species, particularly dicots. These molecules commonly have potent antifungal activity and their natural role in plants is likely to be in protection against attack by pathogenic microbes. They also have a variety of commercial applications including use as drugs and medicines. The enzymes, genes and biochemical pathways involved in the synthesis of these complex molecules are largely uncharacterized for any plant species. Cereals and grasses appear to be generally deficient in saponins with the exception of oats, which produce both steroidal and triterpenoid saponins. The isolation of genes for saponin biosynthesis from oats is now providing tools for the analysis of the evolution and regulation of saponin biosynthesis in monocots. These genes may also have potential for the development of improved disease resistance in cultivated cereals.

Dissecting plant secondary metabolism - constitutive chemical defences in cereals.

Collectively plants synthesise a diverse array of secondary metabolites. Secondary metabolites are well known as agents that mediate pollination and seed dispersal. They may also act as chemical defenses that ward off pests and pathogens or suppress the growth of neighbouring plants. The ability to synthesise particular classes of secondary metabolite is commonly restricted to selected plant groups, and the evolution of different pathways in distinct plant lineages is likely to have been key for survival and for the generation of diversity at the organism level. An understanding of the evolution of secondary metabolism requires the characterisation of enzymes and genes for complete pathways in a broad range of plants in addition to the two model species, Arabidopsis thaliana and rice. Tracing the ancestry of the pathway components can then unravel the chain of events that led to the creation of individual pathways. This review summarises progress that has been made in the dissection of the pathways for constitutive chemical defences in cereals, namely saponins and benzoxazinoids.

Linoleate diol synthase of the rice blast fungus Magnaporthe grisea.

Mycelia of two strains of Magnaporthe grisea, Guy 11 and TH3, were incubated with linoleic acid, and the metabolites were isolated and identified by GC-MS and LC-MS. The two main metabolites were identified as 8-hydroxylinoleic and 7,8-dihydroxylinoleic acids, and the former was further oxidized by n-2 and by n-3 hydroxylation to 8,16- and 8,17-dihydroxylinoleic acids. Lipoxygenase metabolites of linoleic acid could not be detected. The sequence of the genome of M. grisea has been released from the Whitehead Institute; it contains a gene with close homology to the linoleate diol synthase gene of the take-all fungus Gaeumannomyces graminis. Both genes appear to have the same organization, with four exons and three short introns, and the intron-exon borders were determined by reverse-transcription PCR and sequencing. The linoleate diol synthase precursor of G. graminis consists of 978 amino acids, whereas the putative diol synthase precursor of M. grisea contains 987 amino acids. The diol synthases of G. graminis and M. grisea can be aligned with 65% identical and 78% positive amino acid residues, and catalytically important amino acid residues were conserved.

Cloning of the manganese lipoxygenase gene reveals homology with the lipoxygenase gene family.

Manganese lipoxygenase was isolated to homogeneity from the take-all fungus, Gaeumannomyces graminis. The C-terminal amino acids and several internal peptides were sequenced, and the information was used to obtain a cDNA probe by RT/PCR. Screening of a genomic library of G. graminis yielded a full-length clone of the Mn-Lipoxygenase gene. cDNA analysis showed that the gene spanned 2.6 kb and contained one intron (133 bp). Northern blot analyses indicated two transcripts (2.7 and 3.1 kb). The deduced amino-acid sequence of the Mn-Lipoxygenase precursor (618 amino acids, 67.7 kDa) could be aligned with mammalian and plant lipoxygenases with 23-28% identity over 350-400 amino-acid residues of the catalytic domains. Lipoxygenases have one water molecule and five amino acids as Fe ligands. These are two histidine residues in the highly conserved 30 amino-acid sequence WLLAK-X15-H-X4-H-X3-E of alpha helix 9, one histidine and usually an asparaine residue in the sequence H-X3-N-X-G of alpha helix 18, and the carboxyl oxygen of the C-terminal isoleucine (or valine) residue. The homologous sequence of alpha helix 9 of Mn-Lipoxygenase [WLLAK-X14-H(294)-X3-H(297)-X3-E] contained two single-amino-acid gaps, but otherwise His294 and His297 aligned with the two His residues, which coordinate iron. Mn-Lipoxygenase [H(478)-X3-N(482)-X-G] could be aligned with the two metal ligands of alpha helix 18, and the C-terminal residue was Val618. We conclude that Mn-Lipoxygenase belongs to the lipoxygenase gene family and that its unique biochemical properties might be related to structural differences in the metal centre and alpha helix 9 of lipoxygenases rather than to the metal ligands.

Biosynthesis of triterpenoid saponins in plants.

Many different plant species synthesise triterpenoid saponins as part of their normal programme of growth and development. Examples include plants that are exploited as sources of drugs, such as liquorice and ginseng, and also crop plants such as legumes and oats. Interest in these molecules stems from their medicinal properties, antimicrobial activity, and their likely role as determinants of plant disease resistance. Triterpenoid saponins are synthesised via the isoprenoid pathway by cyclization of 2,3-oxidosqualene to give primarily oleanane (beta-amyrin) or dammarane triterpenoid skeletons. The triterpenoid backbone then undergoes various modifications (oxidation, substitution and glycosylation), mediated by cytochrome P450-dependent monooxygenases, glycosyltransferases and other enzymes. In general very little is known about the enzymes and biochemical pathways involved in saponin biosynthesis. The genetic machinery required for the elaboration of this important family of plant secondary metabolites is as yet largely uncharacterised, despite the considerable commercial interest in this important group of natural products. This is likely to be due in part to the complexity of the molecules and the lack of pathway intermediates for biochemical studies. Considerable advances have recently been made, however, in the area of 2,3-oxidosqualene cyclisation, and a number of genes encoding the enzymes that give rise to the diverse array of plant triterpenoid skeletons have been cloned. Progress has also been made in the characterisation of saponin glucosyltransferases. This review outlines these developments, with particular emphasis on triterpenoid saponins.