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

Abstract

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...

APETALA2/ETHYLENE RESPONSE FACTOR and basic helix–loop–helix tobacco transcription factors cooperatively mediate jasmonate‐elicited nicotine biosynthesis

“…Similar to the involvement of ORCA3 in TIA biosynthesis, NIC2-locus ERFs activate most of the known structural genes in the tobacco nicotine biosynthesis pathway and bind a GCC motif in the PMT promoter (Shoji et al, 2010). In agreement with these findings, we previously showed that two JA-inducible AP2/ERF TFs from tobacco, ORC1 and JAP1, positively regulate the PMT promoter in a protoplast-based transient expression assay (Goossens et al, 2003;De Sutter et al, 2005). ORC1 and JAP1 both belong, similar to all the NIC2 AP2/ERFs, to group IX of ERF genes, and are also known as ERF221 (one of the NIC2 AP2/ERFs in clade 2-1) and ERF10 (clade 1), respectively (Rushton et al, 2008;Shoji et al, 2010).…”

“…Statistical significance was determined by Student's t-test (**P < 0.01). previously observed in BY-2 cells (Goossens et al, 2003;De Sutter et al, 2005) and hairy roots (Shoji et al, 2010).…”

“…The expression of structural genes involved in nicotine biosynthesis is stimulated by methyl jasmonate (MeJA) in cultured cells or hairy roots of Nicotiana species (Imanishi et al, 1998;Goossens et al, 2003;Cane et al, 2005). These genes include those coding for ornithine decarboxylase (ODC), arginine decarboxylase (ADC) and putrescine N-methyltransferase (PMT), all three essential for the synthesis of the pyrroline ring, as well as aspartate oxidase (AO) and quinolinate phosphoribosyltransferase (QPRT), essential for the synthesis of the pyridine ring.…”

“…To test whether ORC1 controls the transcription of additional nicotine biosynthesis genes, we made new promoter-reporter constructs for the tobacco ADC, ODC and QPRT genes. Gene duplication was taken into consideration because differential MeJA induction has been reported within the ODC and QPRT families and promoter sequences of distinct relatives are now documented in public databases for some families (Imanishi et al, 1998;Goossens et al, 2003;J. D. Hamill, unpublished data).…”

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

“…7). Previous studies show a good correlation of concerted increases in the expression of multiple nicotine biosynthetic genes with increases in nicotine accumulation levels (8,39,40), indicating metabolic control mediated by the abundance of transcripts encoding nicotine biosynthetic enzymes. In contrast, the NUP1-reduced expression lines did not show a good correlation of five nicotine biosynthetic gene expression levels with reduced nicotine accumulation levels, suggesting that NUP1 may influence mechanisms of alkaloid metabolic control other than transcript abundance levels.…”