Suppression of pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine in leaves of tobacco (N. tabacum L.)

Abstract

Background: Anatabine, although being one of four major tobacco alkaloids, is never accumulated in high quantity in any of the naturally occurring species from the Nicotiana genus. Previous studies therefore focused on transgenic approaches to synthetize anatabine, most notably by generating transgenic lines with suppressed putrescine methyltransferase (PMT) activity. This led to promising results, but the global gene expression of plants with such distinct metabolism has not been analyzed. In the current study, we describe how these plants respond to topping and the downstream effects on alkaloid biosynthesis.

Results: The surge in anatabine accumulation in PMT transgenic lines after topping treatment and its effects on gene expression changes were analyzed. The results revealed increases in expression of isoflavone reductase-like (A622) and berberine bridge-like enzymes (BBLs) oxidoreductase genes, previously shown to be crucial for the final steps of nicotine biosynthesis. We also observed significantly higher methylputrescine oxidase (MPO) expression in all plants subjected to topping treatment. In order to investigate if MPO suppression would have the same effects as that of PMT, we generated transgenic plants. These plants with suppressed MPO expression showed an almost complete drop in leaf nicotine content, whereas leaf anatabine was observed to increase by a factor of ~ 1.6X.

Conclusion: Our results are the first concrete evidence that suppression of MPO leads to decreased nicotine in favor of anatabine in tobacco roots and that this anatabine is successfully transported to tobacco leaves. Alkaloid transport in plants remains to be investigated to higher detail due to high variation of its efficiency among Nicotiana species and varieties of tobacco. Our research adds important step to better understand pyrrolidine ring biosynthesis and its effects on gene expression and subsequent accumulation of anatabine.

Keywords: A622; Anatabine; BBLs; MPO; Nicotiana tabacum; Nicotine; PMT; Topping.

Fig. 1

Alkaloid biosynthesis pathway breakdown. Each biochemical conversion step is represented by an arrow, with defined steps accompanied by enzyme acronyms (full enzyme names with corresponding acronyms are provided in the box). Subcellular processes taking place in plastids and are root specific are enclosed in dark red space. Enzymes that were differentially expressed after topping are colored red, while the rest are colored green. Metabolites of alkaloid biosynthesis are indicated in black, and the final biosynthesis molecules are also presented with their structural formula

Fig. 2

Ultra-High-Performance Liquid Chromatography coupled with Mass Spectrometry (UHPLC-MS) alkaloid quantification box-and-whisker plots. Levels of nicotine, nornicotine, and anatabine are presented in µg/g for PMT and TN90 plants and treatment (control and topped), outliers are presented as circles. Leaf and root values are presented in panels A and B, respectively. Significantly different values are designated with letters and grouped into a, b or ab (paired t-tests, ***p < 0.001)

Fig. 3

NITAB041830 (MPO-S) and NITAB056529 (MPO-T) box-and-whisker plots of the expression in N. tabacum PMT-RNAi suppressed lines. The data units are in FPKM resulting from RNAseq analyses of roots from greenhouse topped plants. Significantly different values are designated with letters and grouped into a, ab, bc or c (paired t-tests, ***p < 0.001), outliers are presented as circles

Fig. 4

Box-and-whisker plots of the relative expression of MPO in control and RNAi lines. Lines T1-3, T1-15, and T1-4 were selected, based on the very low expression of MPO genes NITAB041830 (MPO-S) and NITAB056529 (MPO-T), outliers are presented as circles

Fig. 5

Box-and-whisker plots of the alkaloids measured by Ultra-High-Performance Liquid Chromatography coupled with Mass Spectrometry (UHPLC-MS, lower panels), from 8 WT plants, 3 independent MPO-RNAi lines, 10 plants each. Significantly different values are designated with letters and grouped into a, or b (paired t-tests, ***p < 0.001), outliers are presented as circles