[Show abstract][Hide abstract]ABSTRACT: Background
Ginger (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.
In 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.
A 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.
Full-text available · Article · Feb 2013 · BMC Plant Biology
[Show abstract][Hide abstract]ABSTRACT: Distribution of EST sequence length. The average EST sequence length was 817 bp, with unitrans (unique transcripts, a.k.a. contigs) lengths ranging from 151 to 4021 bp, with the greatest number of unitrans having between 701 and 800 bp, and with 95% exceeding 300 bp. Figure S2. Gene Ontology (GO) annotations of ArREST unitrans. 87.6% of the ArREST unitrans could be annotated by GO classification. Eight GO categories had EST abundances greater than 5%, including protein modification (10.5%), transport (9.2%), metabolism (9.0%), transcription (8.6%), cellular process (8.0%), protein biosynthesis (6.9%), electron transport (5.6%), and biological process unknown (5.3%). In contrast, the GO category secondary metabolism contained relatively few ESTs (0.3%). Figure S3. Proposed metabolic map showing how the curcuminoids, gingerols and terpenoids are produced from sucrose in a large interconnected network. Names of enzymes identified in the ArREST database are colored blue; % values indicate fraction of unitrans in the database that are represented by genes encoding each protein. Figure S4. Phylogenetic tree of P450 monooxygenases. A neighbor joining tree was generated with 1136 P450s including 170 ginger and turmeric P450s from the ArREST database (indicated by black diamonds), 247 Arabidopsis P450s, 350 rice P450s and 369 P450s from other plants. P450 subfamily classifications are indicated. Additional file1: Table S6 contains a summary of these data for ginger and turmeric. As the tree is so large, it was impossible to display it at readable scale on a page size that is typical. Thus, details of tree can been seen by zooming in on this page of the PDF file. Figure S5. Overall gene expression in rhizomes is similar to but distinct from that observed for other plant tissues. Total ArREST ESTs, ESTs with shared expression in ginger or turmeric rhizomes and at least one other ginger or turmeric tissue, and ESTs found exclusively in ginger or turmeric rhizomes are represented as light grey, dark grey and black bars, respectively. Values used to generate this graph are presented in Additional file1 :Table S7.
[Show abstract][Hide abstract]ABSTRACT: cDNA library sources for sequences and unigene sets described in this study. Table S2. The most abundantly represented transcripts in the ArREST database (EST number ≥ 40). Table S3. The most abundantly represented transcripts in specific ginger and turmeric rhizome libraries (EST number ≥ 10 per library). Table S4. Normalized EST expression levels for selected enzymes in ginger and turmeric metabolic pathways. Table S5. Normalized EST expression levels for selected gene families. Table S6. Normalized EST expression levels for cytochrome P450 monooxygenases. Table S7. Normalized percentage of ArREST ESTs with GO associations. Table S8. Probable transcriptional regulator classes within ArREST associated with GO:0003677.
[Show abstract][Hide abstract]ABSTRACT: Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5μM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC-MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.
Full-text available · Article · Jul 2012 · Phytochemistry
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text available · Article · Jan 2011 · Plant physiology
[Show abstract][Hide abstract]ABSTRACT: The 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.
In 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.
Our 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.
Full-text available · Article · Jul 2010 · BMC Genomics
[Show abstract][Hide abstract]ABSTRACT: The 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.
Full-text available · Article · Jun 2008 · The Plant Journal
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text available · Article · Nov 2007 · The Plant Cell