Abstract
Secondary metabolites in plants play important roles in defence against biotic and abiotic stresses. Although the biosynthesis pathways of secondary metabolites have been extensively studied, the regulatory mechanism of gene expression involved in these pathways remains poorly understood. In this study, we develop a virus-induced gene silencing (VIGS) system that enables a rapid analysis of the regulatory mechanism of genes involved in the biosynthesis of isoprenoids, one of the largest groups in secondary metabolites, using hydroponically-grown Nicotiana benthamiana. Using VIGS, we successfully reduced the transcript levels of 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMGR1), cycloartenol synthase 1 (CAS1), sterol side chain reductase 2 (SSR2) and S-adenosyl-L-Met-dependent C-24 sterol methyltransferase 1 (SMT1) in leaf, stem and root tissues in approximately 2 weeks. We identified novel feedback and feed-forward regulation of isoprenoid biosynthesis genes when CAS1, which encodes a key enzyme involved in the biosynthesis of sterols and steroidal glycoalkaloids, was down-regulated. Furthermore, the regulation of these genes differed among different tissues. These results demonstrate that our system can rapidly analyse the regulatory mechanisms involved in the biosynthesis of secondary metabolites.
Highlights
Secondary metabolites in plants play important roles in defence against biotic stresses, such as herbivores and pathogens, and against abiotic stress, such as UV light[1]
Comparison of virus-induced gene silencing (VIGS) efficiency between soil and hydroponic culture. Since growth conditions, such as nutrient levels, affect the efficiency of RNA silencing[22,23], we compared the efficiency of VIGS between hydroponically- and soil-grown plants of N. benthamiana using the phytoene desaturase (PDS) gene; this gene is widely used for the evaluation of the VIGS efficiency, as its knockdown results in the bleaching of plant tissues, such as leaves, which is easy to recognize visually[24,25]
We showed that NbCAS1 knockdown up-regulated NbHMGR1 expression; knockdown of NbSMT1 or NbSSR2 did not increase NbHMGR1 expression (Fig. 4a)
Summary
Secondary metabolites in plants play important roles in defence against biotic stresses, such as herbivores and pathogens, and against abiotic stress, such as UV light[1]. The biosynthesis and accumulation of secondary metabolites are regulated in an organ-, tissue- and cell-specific manner[2,3,4]. We demonstrated the successful down-regulation of genes including 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMGR1), cycloartenol synthase 1 (CAS1), sterol side chain reductase 2 (SSR2) and S-adenosyl-L-Met-dependent C-24 sterol methyltransferase 1 (SMT1) in leaf, stem and root tissues. Using this experimental system, we identified novel feedback and feed-forward regulation of isoprenoid biosynthesis genes, and the differential regulation of these genes in different tissues
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