Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

Abstract

An effective plant alkaloid chemical defense requires a variety of transport processes, but few alkaloid transporters have been characterized at the molecular level. Previously, a gene fragment encoding a putative plasma membrane proton symporter was isolated, because it was coordinately regulated with several nicotine biosynthetic genes. Here, we show that this gene fragment corresponds to a Nicotiana tabacum gene encoding a nicotine uptake permease (NUP1). NUP1 belongs to a plant-specific class of purine uptake permease-like transporters that originated after the bryophytes but before or within the lycophytes. NUP1 expressed in yeast cells preferentially transported nicotine relative to other pyridine alkaloids, tropane alkaloids, kinetin, and adenine. NUP1-GFP primarily localized to the plasma membrane of tobacco Bright Yellow-2 protoplasts. WT NUP1 transcripts accumulated to high levels in the roots, particularly in root tips. NUP1-RNAi hairy roots had reduced NUP1 mRNA accumulation levels, reduced total nicotine levels, and increased nicotine accumulation in the hairy root culture media. Regenerated NUP1-RNAi plants showed reduced foliar and root nicotine levels as well as increased seedling root elongation rates. Thus, NUP1 affected nicotine metabolism, localization, and root growth.

Tobacco Nicotine Uptake Permease Regulates the Expression of a Key Transcription Factor Gene in the Nicotine Biosynthesis Pathway

“…Although NUP1 preferentially transports pyridine alkaloids, it also transports pyridoxine and pyridoxamine (vitamin B 6 ; Kato et al, 2014). Unexpectedly, when NUP1 expression was suppressed in tobacco plants, total nicotine levels were reduced and seedling root growth was enhanced (Hildreth et al, 2011). Jasmonatetreated transgenic tobacco hairy root lines in which NUP1 expression was suppressed (Hildreth et al, 2011) did not show statistically significant differences in the expression levels of five nicotine biosynthesis genes tested, including PMT and QPT.…”

“…Unexpectedly, when NUP1 expression was suppressed in tobacco plants, total nicotine levels were reduced and seedling root growth was enhanced (Hildreth et al, 2011). Jasmonatetreated transgenic tobacco hairy root lines in which NUP1 expression was suppressed (Hildreth et al, 2011) did not show statistically significant differences in the expression levels of five nicotine biosynthesis genes tested, including PMT and QPT. However, in the absence of exogenous jasmonate, some of these root lines exhibited reduced expression levels of nicotine biosynthesis genes encoding Orn decarboxylase, PMT, and quinolinate synthase.…”

“…When expressed in yeast (Saccharomyces cerevisiae) cells or in cultured tobacco cells, NUP1 facilitates the import of tobacco alkaloids (nicotine and anatabine) from the culture medium into the cells (Hildreth et al, 2011;Kato et al, 2014). Although NUP1 preferentially transports pyridine alkaloids, it also transports pyridoxine and pyridoxamine (vitamin B 6 ; Kato et al, 2014).…”

“…NICOTINE UPTAKE PERMEASE1 (NUP1) is a plasma membrane-localized tobacco transporter belonging to the purine uptake permease family (Hildreth et al, 2011). When expressed in yeast (Saccharomyces cerevisiae) cells or in cultured tobacco cells, NUP1 facilitates the import of tobacco alkaloids (nicotine and anatabine) from the culture medium into the cells (Hildreth et al, 2011;Kato et al, 2014).…”

Nicotine biosynthesis is regulated by two more layers: Small and long non-protein-coding RNAs

“…These studies provided a wealth of inofmration at molecular level to understand observations made from classic genetic and biochemical studies regarding the regulation of nicotine biosynthesis by 2 distinct nicotine loci NIC-OTINE1 and NICOTINE2 (originally called the A and B loci) and wounding induced jasmonic acid elevation. In addition, it is not surprising to find that nicotine permeation, transportation and accumulation [8][9][10][11][12] could also affect the expression of the nicotine biosynthesis pathway genes via the cellular nicotine levels.…”