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

Abstract

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.

Tobacco nicotine uptake permease (NUP1) affects alkaloid metabolism

“…4) and altered the root's capacity to retain nicotine (Figs. 4 and 6), whereas NtMATE1/2 reduced expression plants do not affect nicotine accumulation levels (26). NUP1-RNAi seedlings had roots that grew significantly faster regardless of treatment ( Fig.…”

“…NUP1 did not efficiently transport two tropane alkaloids (Fig. 1B); in contrast, NtJAT1 and NtMATE1/2 both efficiently transport tropane alkaloids (25,26). NUP1-RNAi lines showed decreased nicotine accumulation levels (Fig.…”

“…NUP1-GFP localized to the plasma membrane of BY2 protoplasts (Fig. 2), whereas NtJAT1 and NtMATE1/2 mostly localize to plant vacuole membranes (25,26). NUP1 did not efficiently transport two tropane alkaloids (Fig.…”

“…Induced de novo nicotine production confers a short-term reduction of N. sylvestris root growth (20). Exogenous nicotine treatment reduces both root and shoot biomass (37,38) and root elongation (26). Given that NUP1 was a plasma membrane-localized (Fig.…”

“…Based on nicotine tissue accumulation profiles over plant ontology, one can infer nicotine uptake and efflux transport activities, but only two transporters capable of nicotine uptake have been characterized. Nicotiana tabacum jasmonate-inducible alkaloid transporter 1 (NtJAT1) and multidrug and toxic compound extrusion (NtMATE1/2) transporters are vacuole-localized multisubstrate proton antiporters that mobilize nicotine into the vacuole (25,26).…”