Fig 1.
Subcellular compartmentalisation of nicotine / nornicotine biosynthetic enzymes—modified from [13].
A622: isoflavone reductase-like protein; ADC: arginine decarboxylase; BBL: berberine bridge enzyme-like; MPO: N-methylputrescine oxidase; NND: nicotine N-demethylase; ODC: ornithine decarboxylase; PMT: putrescine methyltransferase; QPT: quinolinate phosphoribosyltransferase. Genes that have been overexpressed in the present study are shown in green.
Fig 2.
Colocalisation of NtabMPO1-GFP and of PTS1-mCherry in BY-2 cells overexpressing NtabMPO1-GFP showed peroxisome localisation of NtabMPO1.
NtabMPO-GFP fluorescence (a) PTS1-mCherry fluorescence (b) and merged fluorescence of a and b (c) Orange colour indicates areas where the images overlap and where the two markers colocalise (Bars = 10 μm).
Fig 3.
Phenotyping and alkaloid accumulation patterns for BY-2 cells overexpressing NtabMPO1 compared to non-transformed BY-2 cells (WT).
(a) cell viability (mean of n = 1000), (b) mitotic index (mean of n = 1000), (c) cell elongation as ratio of cell length over cell width at days 4 and 7 (mean of n = 500), (d, e) from the time course of cell density a cell cycle duration of 27.96 h for the non-transformed BY-2 cells (d) and of 32.9 h for BY-2 cells overexpressing NtabMPO1 (e) can be inferred. All experimental data are derived from three independent experimental series; error bars = SE. (f, g) Alkaloid profiles measured after 3 days of culture in presence of 100 μM jasmonic acid either intracellularly (f) or secreted to the medium (g). The levels of nornicotine were below detection limit (indicated by N.D.). Note the difference in scales between (f) and (g). For the alkaloid measurement, mean and SE are shown from six independent experimental series. Significant differences to the non-transformed WT cells assessed by a Student’s t-test are indicated by * (P < 0.05) or ** (P < 0.01), respectively.
Fig 4.
Localisation and metabolic impact of overexpressed NtabCYP82E5v2-GFP.
(a) NtabCYP82E5v2-GFP signal, (b) rhodamine-conjugated ER-tracker, (c) merged signal of both channels showing the tight colocalisation of the nicotine demethylase NtabCYP82E5v2 GFP with the endoplasmic reticulum. (d, e) Alkaloid profiles measured after 3 days of culture in presence of 100 μM jasmonic acid either intracellularly (d) or secreted to the medium (e). The levels of nornicotine were below detection limit (indicated by N.D.). Note the difference in scales between (d) and (e) (the level of secreted alkaloids is in some case around tenfold lower). For the alkaloid measurement, mean and SE are shown from six independent experimental series. Significant differences to the non-transformed WT cells assessed by a Student’s t-test are indicated by * (P < 0.05) or ** (P < 0.01), respectively.
Fig 5.
Nicotine and nornicotine contents in senescent leaves of different Nicotiana species and metabolic impact of overexpressed NomCYP82E4-GFP.
(a) The low ratio of nornicotine to nicotine in N. tabacum and the high level of nornicotine compared to nicotine in N. tomentosiformis are evident. Error bars represent SE from three independent experiments. (b, c) Alkaloid profiles measured after 3 days of culture in presence of 100 μM jasmonic acid either intracellularly (b) or secreted to the medium (c). The levels of nornicotine below detection limit is indicated by non-detectable (N.D.). Note the difference in scales between (b) and (c) (the level of secreted alkaloids is in some case around tenfold lower). For the alkaloid measurement, mean and SE are shown from six independent experimental series. Significant differences to the non-transformed WT cells assessed by a Student’s t-test are indicated by * (P < 0.05) or ** (P < 0.01), respectively.
Fig 6.
Alkaloid accumulation in non-transformed BY-2 cells (WT) compared to cells overexpressing NtabCYP82E5v2 and NtomCYP82E4, respectively after feeding with 15 μg.ml-1 of pure nicotine (Nic).
The level of nornicotine below detection limit is indicated by non-detectable (N.D.). Error bars represent SE from 3 independent experimental series. Significant differences in nornicotine production to the WT treated with nicotine and also between two CYP overexpressing cell lines in a Student’s t-test are indicated by two (P < 0.01) asterisks.
Fig 7.
Profile of intracellular alkaloids accumulated in the NtomCYP82E4 overexpressor in response to conditioned medium.
Stimulation of intracellular alkaloid accumulation by conditioned medium collected from non-transformed wild type (M1) or cells overexpressing MPO1 (M2). Unconditioned medium (M0) was used as negative control. Alkaloid synthesis was elicited by 100 μM of jasmonic acid for three days. Error bars indicate SE, from three independent experimental series. Significant differences to the non-transformed WT cells assessed by a Student’s t-test are indicated by * (P < 0.05) or ** (P < 0.01), respectively.
Fig 8.
Nornicotine contents in BY-2 cells overexpressing NtabMPO1 (I) and cells overexpressing NtomCYP82E4 (II) compared to NtomCYP82E4 cells cultivated in a mixture composed of medium of a three days old NtabMPO1 cell line (M2) (III) and when co-cultivated together with NtabMPO1 cells (IV).
Error bars represent SE (n = 3). Significant differences to the NtomCYP82E4 in a Student’s t-test are indicated by two asterisks (P < 0.01).