Determination of tobacco alkaloids in single plant cells by capillary electrophoresis

Lochmann, H.; Bazzanella, A.; Kropsch, S.; Bächmann, K.

doi:10.1016/s0021-9673(01)00627-6

Cited by 32 publications

(26 citation statements)

References 19 publications

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“…In tobacco, nicotine is synthesized exclusively in the root and distributed throughout the plant via the xylem, concentrating in the young tissues of aerial parts (Hashimoto and Yamada, 1995;Baldwin, 2001). As much as 60 mM of nicotine accumulates in the vacuoles of the leaf epidermal cells at the tip (Lochmann et al, 2001). Putrescine N-methyltransferase (PMT) catalyzes the first committed step in the nicotine-specific pathway, and a PIP-family reductase, called A622, was also suggested to function in a late step in nicotine biosynthesis (Hibi et al, 1994;Shoji et al, 2000aShoji et al, , 2000bDeBoer et al, 2009;Kajikawa et al, 2009).…”

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confidence: 99%

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

et al. 2008

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Nicotine is a major alkaloid accumulating in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATE1 and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells.

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confidence: 99%

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

et al. 2008

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“…Once produced in the root, nicotine is translocated to the aerial parts via the xylem (Baldwin 1989), and stored at high concentrations in the vacuoles of leaf cells (ca. 60 mM) (Lochmann et al 2001). A classic grafting experiment initially revealed the root-to-shoot translocation of nicotine (Solt and Dawson 1958), but the molecular mechanism underlying this process has remained elusive.…”

Section: Nicotine Transport In Nicotiana Tabacummentioning

confidence: 99%

Alkaloid transporters in plants

Shitan

Kato

Shoji

2014

Plant Biotechnology

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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.

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“…Although the concentration of endogenous nicotine is 60 mM in the vacuoles of the leaf tip (22), which is a level much higher than that needed to drive the maximal rate of nicotine demethylation, the actual concentration of nicotine in the cytosol that is accessible for the demethylase enzymes is not known. Therefore, we assayed large range of nicotine concentrations in vitro.…”

Section: Enantioselectivity Of Cyp82e4 Cyp82e5v2 and Cyp82e10-mentioning

confidence: 99%

Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

Cai

Siminszky

Chappell

et al. 2012

Journal of Biological Chemistry

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Background: Nornicotine composition in tobacco leaf has been a puzzle for more than half a century. Results: Three recombinant nicotine demethylases selectively use (R)-nicotine in vitro and in planta. Conclusion: Nornicotine composition in tobacco leaf can be reasonably explained by the combination of three nicotine demethylases. Significance: This knowledge lays the base for the optimization of nornicotine composition in tobacco leaf in the future.

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Determination of tobacco alkaloids in single plant cells by capillary electrophoresis

Cited by 32 publications

References 19 publications

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

Multidrug and Toxic Compound Extrusion-Type Transporters Implicated in Vacuolar Sequestration of Nicotine in Tobacco Roots

Alkaloid transporters in plants

Enantioselective Demethylation of Nicotine as a Mechanism for Variable Nornicotine Composition in Tobacco Leaf

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