CYP82E4-mediated nicotine to nornicotine conversion in tobacco is regulated by a senescence-specific signaling pathway

Abstract

Nicotine to nornicotine conversion in tobacco (Nicotiana tabacum L.) is regulated by an unstable converter locus which in its activated state gives rise to a high nornicotine, low nicotine phenotype in the senescing leaves. In plants that carry the high nornicotine trait, nicotine conversion is primarily catalyzed by a cytochrome P450 protein, designated CYP82E4 whose transcription is strongly upregulated during leaf senescence. To further investigate the regulation of CYP82E4 expression, we examined the spatiotemporal distribution and the stress- and signaling molecule-elicited expression patterns of CYP82E4 using alkaloid analysis and a fusion construct between the 2.2 kb upstream regulatory region of CYP82E4 and the beta-glucurodinase (GUS) gene. Histochemical and fluorometric analyses of GUS expression revealed that the CYP82E4 promoter confers high levels of expression in the senescing leaves and flowers, and in the green stems of young and mature plants, but only very low activity was detected in the roots. In the leaves, GUS activity was strongly correlated with the progression of senescence. Treatments of leaf tissue with various signaling molecules including abscisic acid, ethylene, jasmonic acid, salicylic acid and yeast extract; and stresses, such as drought, wounding and tobacco mosaic virus infection did not enhance nicotine conversion or GUS activity in the green leaves, but an increase in CYP82E4 expression was observed in response to ethylene- or tobacco mosaic virus-induced senescence. These results suggest that the expression of CYP82E4 is senescence-specific in the leaves and the use of the CYP82E4 promoter could provide a valuable tool for regulating gene expression in the senescing leaves.

Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

“…It has been reported that CYP82E4 is expressed at very low levels in green healthy leaves and is strongly induced during senescence (Siminszky et al, 2005;Gavilano et al, 2006;Chakrabarti et al, 2008). Therefore, we investigated whether the increased CYP82E4 expression could have been triggered by leaf senescence.…”

“…The synthesized nicotine is translocated through the xylem and transported to the leaves (Morita et al, 2009;Shoji et al, 2009;Hildreth et al, 2011), where it accumulates. In the leaf, nicotine can undergo N-demethylation to produce nornicotine (Griffith et al, 1955;Chakrabarti et al, 2008). Nornicotine is the second most abundant alkaloid in most tobacco cultivars (Saitoh et al, 1985), and its conversion is mainly controlled by the nicotine N-demethylase gene CYP82E4 (Siminszky et al, 2005;Xu et al, 2007).…”

Investigations into the pituri plant: nicotine content, nicotine conversion to nornicotine, nicotine release and cytotoxicity of Australian native Nicotiana spp.

“…Nornicotine is accumulated to high levels in some tobacco (Nicotiana tabacum) cultivars and many wild Nicotiana species (Chakrabarti et al, 2008, Chelvarajan et al, 1993, Pakdeechanuan et al, 2012, Siminszky et al, 2005.…”

“…Investigations conducted on conversion locus analysis in Nicotiana spp. are limited to the varieties of the important commercial tobacco, mainly N. tabacum (Cai et al, 2012a, Siminszky et al, 2005, Chakrabarti et al, 2008, Gavilano et al, 2006, Lewis et al, 2010) and its progenitor species N. tomentosiformis (Mann et al, 1964), and N. sylvestris (Chakrabarti et al, 2007). There is also a report of the loci and their functionality in a few South American wild species including N. langsdorffii and N. alata (Pakdeechanuan et al, 2012).…”

“…The conversion locus, mainly CYP82E4, has been found to be present in all Nicotiana investigated, but its transcription is strongly upregulated by curing and during leaf senescence only in high converters (Chakrabarti et al, 2008). Therefore, it was hypothesised that the nicotine to nornicotine conversion locus is present in all Australian Nicotiana species and is triggered by curing only in high converter species.…”