Characterization of cDNAs differentially expressed in roots of tobacco (Nicotiana tabacum cv Burley 21) during the early stages of alkaloid biosynthesis

Wang, Jianmin; Sheehan, Moira; Brookman, Heather; Timko, Michael P.

doi:10.1016/s0168-9452(00)00293-4

Cited by 30 publications

(33 citation statements)

References 53 publications

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“…The most relevant previously reported ''transcriptome'' that can be compared with the MJM data set was the set of genes differentially expressed in tobacco roots during early stages of alkaloid biosynthesis (50). This set consists of 60 cDNAs, whose sequence has been released for 45 that were isolated by subtractive hybridization screening of a tobacco root library.…”

Section: Comparison Of the Jasmonate-modulated By-2 Transcriptome Withmentioning

confidence: 99%

A functional genomics approach toward the understanding of secondary metabolism in plant cells

Goossens

Häkkinen

Laakso

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

366

244

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Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonatemediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general. P lants are capable of synthesizing an overwhelming variety of low-molecular-weight organic compounds called secondary metabolites, usually with unique and complex structures. Presently, Ϸ100,000 such compounds have been isolated from higher plants (1). Numerous plant secondary metabolites possess interesting biological activities and find applications, such as pharmaceuticals, insecticides, dyes, flavors, and fragrances. Although secondary metabolism offers attractive targets for plant breeding, the enormous biosynthetic potential of plant cells is still not being exploited. In sharp contrast, metabolism of microorganisms has been successfully engineered for increased production of pharmaceuticals or novel compounds (2, 3). Despite a few decades of research, plant secondary metabolism remains poorly characterized (4). Genetic maps of biosynthetic pathways are still far from complete, whereas knowledge on the regulation of these pathways is practically nonexistent. However, such knowledge is of crucial importance to bypass the low product yield of various secondary metabolites in plants or plant cell cultures.Functional genomics approaches are powerful tools to accelerate comprehensive investigations of cellular metabolism in specialized tissues or whole organisms (5). Yet, related to plant secondary metabolism, only a few reports have been published on such studies, which include the use of comparative quantitative trait loci mapping (6), 2D gel electrophoresis-based proteomics (7), or transcript analysis tools, such as differential display (8, 9), EST databases (10-12), and microarrays (13,14). Nevertheless, still little is known about the genetics that control quantitatively and qualitatively natural variation in secondary metabolism.Because of the lack of extensive genomic data for the vast majority of medicinal plants, it is difficult to use the commonly used microarray-based approach for transcriptome analysis in these plant systems. Such an approach requires prior development of large EST or cDNA clone collections (13,14). As such, the cDNA-amplified fragment length polymorphism (AFLP) technology (15-17) offers an attractive alternative to identify genes involved i...

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Section: Comparison Of the Jasmonate-modulated By-2 Transcriptome Withmentioning

confidence: 99%

A functional genomics approach toward the understanding of secondary metabolism in plant cells

Goossens

Häkkinen

Laakso

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

366

244

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“…Xanthi leaf DNA (Clontech, Inc., Palo Alto, CA) was screened by plaque hybridization (Sambrook et al 1989) using a [ 32 P]-dCTP-labeled 1.6 kb EcoRI-XhoI fragment excised from PR46, a plasmid encoding the fulllength NtODC-2 cDNA (Wang et al 2000a) as probe. Hybridization was performed at 65°C for 16 h in a solution containing 0.25 M Na 2 HPO 4 (pH 7.2) and 7% (w/v) SDS.…”

Section: Screening Of Genomic Libraries and Phage Characterizationmentioning

confidence: 99%

“…The transferred RNA was UV crosslinked to the membrane using a UV Stratalinker (Stratagene, La Jolla, CA) and the membranes were prehybridized in Church and Gilbert solution (7% v/v SDS, 0.5 M Na 2 PO 4 , 1 mM EDTA, 1% w/v BSA, pH 7.2) for 2-4 h at 65°C. Hybridization was carried out for 16 h at 65°C in the same buffer in the presence of a [ 32 P]-dCTP-labeled 1.6 kb EcoRI-XhoI fragment excised from PR46, a plasmid encoding the fulllength NtODC-2 cDNA (Wang et al 2000a) as probe. The [ 32 P]-dCTP-labeled probes were prepared by random primed labeling using the Random Primed Labeling Kit (Boehringer Mannheim, Indianapolis, IN) as described in (Riechers and Timko 1999) and 25-50 ng of template DNA, purified by agarose gel electrophoresis and quantified by spectrophotometry.…”

Section: Rna Isolation and Gel Blot Analysismentioning

confidence: 99%

“…Ornithine decarboxylase (ODC) is encoded by a small nuclear gene family in tobacco (N. tabacum L.) consisting of four to five individual gene family members (Wang et al 2000a). The presence of multiple ODC genes in cultivated tobacco is consistent with the proposed evolutionary origin of N. tabacum by hybridization of different diploid progenitor species (i.e., N. sylvestris, N. tomentosiformis, and N. otophora) (Hashimoto et al 1998;Riechers and Timko 1999).…”

Section: Characterization Of Ntodc-1 and Ntodc-2 Genes In Tobaccomentioning

confidence: 99%

“…The nucleotide sequence of NtODC-2 is identical within the coding region and 5¢-and 3¢-UTRs to the cDNA PR46 (Genbank Accession No. AF127242; Wang et al 2000a), with the exception of four nucleotide changes (residues +2, +4, +6 and +8) in the 5¢-UTR region. These nucleotide differences likely reflect changes introduced during the cDNA synthesis reaction.…”

Section: Characterization Of Ntodc-1 and Ntodc-2 Genes In Tobaccomentioning

confidence: 99%

See 2 more Smart Citations

Differential induction of ornithine decarboxylase (ODC) gene family members in transgenic tobacco (Nicotiana tabacum L. cv. Bright Yellow 2) cell suspensions by methyl-jasmonate treatment

Sheehan

Timko

2004

Plant Growth Regul

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Impact of nicotine pathway downregulation on polyamine biosynthesis and leaf ripening in tobacco

et al. 2021

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Characterization of cDNAs differentially expressed in roots of tobacco (Nicotiana tabacum cv Burley 21) during the early stages of alkaloid biosynthesis

Cited by 30 publications

References 53 publications

A functional genomics approach toward the understanding of secondary metabolism in plant cells

A functional genomics approach toward the understanding of secondary metabolism in plant cells

Differential induction of ornithine decarboxylase (ODC) gene family members in transgenic tobacco (Nicotiana tabacum L. cv. Bright Yellow 2) cell suspensions by methyl-jasmonate treatment

Impact of nicotine pathway downregulation on polyamine biosynthesis and leaf ripening in tobacco

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