Glandular trichomes play important roles in protecting plants from biotic attack by producing defensive compounds. We investigated the metabolic profiles and transcriptomes to characterize the differences between different glandular trichome types in several domesticated and wild Solanum species: Solanum lycopersicum (glandular trichome types 1, 6, and 7), Solanum habrochaites (types 1, 4, and 6), Solanum pennellii (types 4 and 6), Solanum arcanum (type 6), and Solanum pimpinellifolium (type 6). Substantial chemical differences in and between Solanum species and glandular trichome types are likely determined by the regulation of metabolism at several levels. Comparison of S. habrochaites type 1 and 4 glandular trichomes revealed few differences in chemical content or transcript abundance, leading to the conclusion that these two glandular trichome types are the same and differ perhaps only in stalk length. The observation that all of the other species examined here contain either type 1 or 4 trichomes (not both) supports the conclusion that these two trichome types are the same. Most differences in metabolites between type 1 and 4 glands on the one hand and type 6 glands on the other hand are quantitative but not qualitative. Several glandular trichome types express genes associated with photosynthesis and carbon fixation, indicating that some carbon destined for specialized metabolism is likely fixed within the trichome secretory cells. Finally, Solanum type 7 glandular trichomes do not appear to be involved in the biosynthesis and storage of specialized metabolites and thus likely serve another unknown function, perhaps as the site of the synthesis of protease inhibitors.Trichomes are epidermal structures widely conserved across the plant kingdom (Kim and Mahlberg, 1991;Wagner, 1991;Alonso et al., 1992;Yu et al., 1992;Kolb and Muller, 2003;Valkama et al., 2003;Giuliani and Bini, 2008). These structures perform important biological functions, such as discouraging herbivory, attracting pollinators, and maintaining a boundary layer (Nihoul, 1993;Van Dam and Hare, 1998;Kennedy, 2003;Moyano et al., 2003;Simmons and Gurr, 2005;Liu et al., 2006;Horgan et al., 2007;Gonzalez et al., 2008;Romero et al., 2008;Nonomura et al., 2009;Kang et al., 2010). Many of these functions are the result of the specialized nature of glandular trichomes (glands) as sites for the synthesis and storage of biologically active specialized metabolites (Alonso et al

SummaryThe glandular trichome is an excellent model system for investigating plant metabolic processes and their regulation within a single cell type. We utilized a proteomics-based approach with isolated trichomes of four different sweet basil (Ocimum basilicum L.) lines possessing very different metabolite profiles to clarify the regulation of metabolism in this single cell type. Significant differences in the distribution and accumulation of the 881 highly abundant and non-redundant protein entries demonstrated that although the proteomes of the glandular trichomes of the four basil lines shared many similarities they were also each quite distinct. Correspondence between proteomic, expressed sequence tag, and metabolic profiling data demonstrated that differential gene expression at major metabolic branch points appears to be responsible for controlling the overall production of phenylpropanoid versus terpenoid constituents in the glandular trichomes of the different basil lines. In contrast, post-transcriptional and post-translational regulation of some enzymes appears to contribute significantly to the chemical diversity observed within compound classes for the different basil lines. Differential phosphorylation of enzymes in the 2-C-methyl-D-erythritol 4-phosphate (MEP)/ terpenoid and shikimate/phenylpropanoid pathways appears to play an important role in regulating metabolism in this single cell type. Additionally, precursors for different classes of terpenoids, including mono-and sesquiterpenoids, appear to be almost exclusively supplied by the MEP pathway, and not the mevalonate pathway, in basil glandular trichomes.

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BackgroundThe template switching PCR (TS-PCR) method of cDNA synthesis represents one of the most straightforward approaches to generating full length cDNA for sequencing efforts. However, when applied to very small RNA samples, such as those obtained from tens or hundreds of cells, this approach leads to high background and low cDNA yield due to concatamerization of the TS oligo.ResultsIn this study, we describe the application of nucleotide isomers that form non-standard base pairs in the template switching oligo to prevent background cDNA synthesis. When such bases are added to the 5' end of the template switching (TS) oligo, they inhibit MMLV-RT from extending the cDNA beyond the TS oligo, thus increasing cDNA yield by reducing formation of concatamers of the TS oligo that are the source of significant background.ConclusionsOur results demonstrate that this novel approach for cDNA synthesis has valuable utility for application of ultra-high throughput technologies, such as whole transcriptome sequencing using 454 technology, to very small biological samples comprised of tens of cells as might be obtained via approaches like laser microdissection.

Methylcinnamate, which is widely distributed throughout the plant kingdom, is a significant component of many floral scents and an important signaling molecule between plants and insects. Comparison of an EST database obtained from the glandular trichomes of a basil (Ocimum basilicum) variety that produces high levels of methylcinnamate (line MC) with other varieties producing little or no methylcinnamate identified several very closely related genes belonging to the SABATH family of carboxyl methyltransferases that are highly and almost exclusively expressed in line MC. Biochemical characterization of the corresponding recombinant proteins showed that cinnamate and p-coumarate are their best substrates for methylation, thus designating these enzymes as cinnamate/p-coumarate carboxyl methyltransferases (CCMTs). Gene expression, enzyme activity, protein profiling, and metabolite content analyses demonstrated that CCMTs are responsible for the formation of methylcinnamate in sweet basil. A phylogenetic analysis of the entire SABATH family placed these CCMTs into a clade that includes indole-3-acetic acid carboxyl methyltransferases and a large number of uncharacterized carboxyl methyltransferase-like proteins from monocots and lower plants. Structural modeling and ligand docking suggested active site residues that appear to contribute to the substrate preference of CCMTs relative to other members of the SABATH family. Site-directed mutagenesis of specific residues confirmed these findings.

BackgroundGinger (Zingiber officinale) and turmeric (Curcuma longa) accumulate important pharmacologically active metabolites at high levels in their rhizomes. Despite their importance, relatively little is known regarding gene expression in the rhizomes of ginger and turmeric.ResultsIn order to identify rhizome-enriched genes and genes encoding specialized metabolism enzymes and pathway regulators, we evaluated an assembled collection of expressed sequence tags (ESTs) from eight different ginger and turmeric tissues. Comparisons to publicly available sorghum rhizome ESTs revealed a total of 777 gene transcripts expressed in ginger/turmeric and sorghum rhizomes but apparently absent from other tissues. The list of rhizome-specific transcripts was enriched for genes associated with regulation of tissue growth, development, and transcription. In particular, transcripts for ethylene response factors and AUX/IAA proteins appeared to accumulate in patterns mirroring results from previous studies regarding rhizome growth responses to exogenous applications of auxin and ethylene. Thus, these genes may play important roles in defining rhizome growth and development. Additional associations were made for ginger and turmeric rhizome-enriched MADS box transcription factors, their putative rhizome-enriched homologs in sorghum, and rhizomatous QTLs in rice. Additionally, analysis of both primary and specialized metabolism genes indicates that ginger and turmeric rhizomes are primarily devoted to the utilization of leaf supplied sucrose for the production and/or storage of specialized metabolites associated with the phenylpropanoid pathway and putative type III polyketide synthase gene products. This finding reinforces earlier hypotheses predicting roles of this enzyme class in the production of curcuminoids and gingerols.ConclusionA significant set of genes were found to be exclusively or preferentially expressed in the rhizome of ginger and turmeric. Specific transcription factors and other regulatory genes were found that were common to the two species and that are excellent candidates for involvement in rhizome growth, differentiation and development. Large classes of enzymes involved in specialized metabolism were also found to have apparent tissue-specific expression, suggesting that gene expression itself may play an important role in regulating metabolite production in these plants.

An evaluation has been made of the potential of near-infrared (NIR) technologies in the assessment of essential oil components and in the identification of individual essential oils. The results showed that cross-validation models are able to predict accurately almost all of the components of essential oils. In different cinnamon (Cinnamomum zeylanicum) and clove (Syzygium aromaticum) essential oils, which showed a similar composition, 23 components (representing 97.8-99.9% of the oil) were accurately predicted, as well as 20 components (93.0-99.1%) in Cinnamomum camphora (ravintsara), 32 components (92.3-98.1%) in Ravensara aromatica (ravensara), and 26 components (96.6-98.4%) in Lippia multiflora. For almost all of the components, the modelled and reference values obtained by GC-FID were highly correlated (r2 > or = 0.985) and exhibited a low variance (less than 5%). The model was also able to discriminate between the ravintsara and ravensara essential oils. It was shown that two commercial oils labelled as R. aromatica were actually ravintsara (C. camphora), revealing the misidentification of these essential oils in the marketplace. The study demonstrates the application of NIR technology as a quality control tool for the rapid identification of individual essential oils, for product authentication, and for the detection of adulteration.

The genus Echinacea is comprised of nine species, which are perennial herbs indigenous to North America and which have been traditionally used as medicinal plants for centuries. Three Echinacea species, E. angustifolia, E. purpurea, and E. pallida, are currently being traded internationally in the natural products market. Echinacea products constitute a significant portion of this growing, multi-billion dollar industry. The increasing popularity of Echinacea products has led to the expansion of wildcrafting and commercial cultivation to meet the growing demand for plant material. Echinacea is considered of value as a nonspecific immune stimulant, and claims of its efficacy have been tentatively supported by both laboratory and clinical studies. This study used random amplified polymorphic DNA (RAPD) markers to determine the genetic relationships of the three Echinacea species of commercial interest, to evaluate the level of diversity present within germplasm of each of the three species, and to compare accessions of each species available from different sources. A total of 101 RAPD markers were generated for the 76 individuals of four species included in the analysis. NTSYS-pc was used to evaluate the genetic relationships of the three species and to determine the general level of overall diversity. Analysis of molecular variance (AMOVA) was performed using pruned marker sets corrected for the dominant nature of RAPD markers. AMOVA revealed that most of the variation occurred within accessions of the same species, though some accessions of both E. pallida and E. angustifolia were found to be significantly different from other accessions of the same species.