Vacuole-Localized Berberine Bridge Enzyme-Like Proteins Are Required for a Late Step of Nicotine Biosynthesis in Tobacco

Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan.
Plant physiology (Impact Factor: 6.84). 02/2011; 155(4):2010-22. DOI: 10.1104/pp.110.170878
Source: PubMed


Tobacco (Nicotiana tabacum) plants synthesize nicotine and related pyridine-type alkaloids, such as anatabine, in their roots and accumulate them in their aerial parts as chemical defenses against herbivores. Herbivory-induced jasmonate signaling activates structural genes for nicotine biosynthesis and transport by way of the NICOTINE (NIC) regulatory loci. The biosynthesis of tobacco alkaloids involves the condensation of an unidentified nicotinic acid-derived metabolite with the N-methylpyrrolinium cation or with itself, but the exact enzymatic reactions and enzymes involved remain unclear. Here, we report that jasmonate-inducible tobacco genes encoding flavin-containing oxidases of the berberine bridge enzyme family (BBLs) are expressed in the roots and regulated by the NIC loci. When expression of the BBL genes was suppressed in tobacco hairy roots or in tobacco plants, nicotine production was highly reduced, with a gradual accumulation of a novel nicotine metabolite, dihydromethanicotine. In the jasmonate-elicited cultured tobacco cells, suppression of BBL expression efficiently inhibited the formation of anatabine and other pyridine alkaloids. Subcellular fractionation and localization of green fluorescent protein-tagged BBLs showed that BBLs are localized in the vacuoles. These results indicate that BBLs are involved in a late oxidation step subsequent to the pyridine ring condensation reaction in the biosynthesis of tobacco alkaloids.

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Available from: Tsubasa Shoji, Mar 01, 2014
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    • "These results suggest that NtMATE1 and NtMATE2 transport nicotine into root vacuoles (Figure 2C) (Shoji et al. 2009). NtMATE-mediated transport is considered to contribute mainly to the vacuolar retention of nicotine, as nicotine is thought to be formed in the vacuolar lumen (Kajikawa et al. 2011). Tobacco NUP1 (nicotine uptake permease1) is a plasma membrane-localized nicotine transporter of the PUP family (Hildreth et al. 2011). "
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    ABSTRACT: Plants produce a multitude of secondary metabolites, including alkaloids with biological activities, and many alkaloids have been used for medicinal purposes. The biosynthetic enzymes and genes involved in alkaloid metabolic pathways exhibit divergent localizations, implying that alkaloid metabolites, including pathway products and intermediates, travel from organelle to organelle, cell to cell, and organ to organ. Biochemical studies have indicated that specific transporters move these metabolites. Indeed, molecular and cellular approaches have identified alkaloid transporters of the ATP-binding cassette (ABC) protein, multidrug and toxic compound extrusion (MATE), and purine permease (PUP) families. Interestingly, some of these transporters were found to be required for the efficient biosynthesis of alkaloids in plants. Here, we provide an updated inventory of alkaloid transporters and discuss the possibility of genetically manipulating the expression of these transporters to increase the accumulation of valuable alkaloid compounds.
    Full-text · Article · Dec 2014 · Plant Biotechnology
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    • "The PCR primers used are listed in Supplemental Table S2. The QPT primers amplified both QPT1 and QPT2 (Shoji and Hashimoto, 2011b), while all four BBL members, BBLa, BBLb, BBLc, and BBLd, were collectively detected by the BBL primers (Kajikawa et al., 2011). "
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    ABSTRACT: Previously, the down-regulation of NUP1, a Nicotiana tabacum (tobacco) plasma membrane-localized nicotine uptake permease, was reported to reduce total alkaloid levels in tobacco plants. However, it was unclear how this nicotine transporter affected the biosynthesis of the alkaloid nicotine. When NUP1 expression was suppressed in cultured tobacco cells treated with jasmonate, which induces nicotine biosynthesis, the NICOTINE2-locus transcription factor gene ERF189 and its target structural genes, which function in nicotine biosynthesis and transport, were strongly suppressed, resulting in decreased total alkaloid levels. Conversely, NUP1 overexpression had the opposite effect. In these experiments, the expression levels of the MYC2 transcription factor gene and its jasmonate-inducible target gene were not altered. Inhibiting tobacco alkaloid biosynthesis by suppressing the expression of genes encoding enzymes in the nicotine pathway did not affect the expression of ERF189 and other nicotine pathway genes, indicating that ERF189 is not regulated by cellular alkaloid levels. Suppressing the expression of jasmonate-signaling components in cultured tobacco cells showed that NUP1 acts downstream of the COI1 receptor and MYC2, but upstream of ERF189. These results suggest that while jasmonate-activated expression of MYC2 induces the expression of both NUP1 and ERF189, expression of ERF189 may actually be mediated by NUP1. Furthermore, NUP1 overexpression in tobacco plants inhibited the long-range transport of nicotine from the roots to the aerial parts. Thus, NUP1 not only mediates the uptake of tobacco alkaloids into root cells, but also positively controls the expression of ERF189, a key gene in the biosynthesis of these alkaloids.
    Full-text · Article · Oct 2014 · Plant physiology
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    • "In Nicotiana tabacum (tobacco), these alkaloids are exclusively synthesized in the root, translocated to the aerial parts via the xylem, and stored in the vacuoles of leaf cells. Since berberine bridge enzyme-like oxidoreductases, which catalyze the last or a late step of nicotine (1) biosynthesis, are localized in the vacuoles, nicotine (1) is thought to be formed in the vacuoles (Kajikawa et al., 2011). Nicotine (1) thus appears to travel from its site of synthesis in the root vacuole to its distant storage site in the leaf vacuole, via the root cytosol, root apoplast, xylem sap, leaf apoplast, and leaf cytosol. "
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    ABSTRACT: The tobacco purine permease-family transporter, nicotine uptake permease 1 (NtNUP1), is shown here to import not only tobacco alkaloids but also vitamin B6, demonstrating relaxed substrate preference to compounds containing a pyridine ring.
    Full-text · Article · Jan 2014 · Phytochemistry
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