Philip Morris International R&D, Philip Morris Products S.A, Quai Jeanrenaud 5, CH-2000, Neuchâtel, Switzerland.
BMC Genomics. 2023 Sep 4;24(1):516. doi: 10.1186/s12864-023-09588-8.
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.
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.
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.
尽管安纳托宾是四大烟草生物碱之一，但在任何天然存在的茄属植物种中都从未大量积累过。因此，之前的研究集中在通过生成具有抑制腐胺甲基转移酶（PMT）活性的转基因系来合成安纳托宾，这导致了有希望的结果，但具有这种明显代谢的植物的全球基因表达尚未被分析。在本研究中，我们描述了这些植物如何对打顶做出反应以及对生物碱生物合成的下游影响。
PMT 转基因系在打顶处理后安纳托宾积累量的激增及其对基因表达变化的影响进行了分析。结果表明，异黄酮还原酶样（A622）和小檗碱桥连酶（BBLs）氧化还原酶基因的表达增加，先前已证明这些基因对于尼古丁生物合成的最后步骤至关重要。我们还观察到所有接受打顶处理的植物中 MPO 的表达显著增加。为了研究 MPO 抑制是否会产生与 PMT 相同的效果，我们生成了转基因植物。这些 MPO 表达受抑制的转基因植物叶片烟碱含量几乎完全下降，而叶片安纳托宾含量增加了约 1.6 倍。
我们的研究结果是第一个具体证据，证明 MPO 的抑制导致尼古丁减少，有利于烟草根系中的安纳托宾，并且这种安纳托宾成功地被运输到烟草叶片中。由于尼古丁在茄属植物和烟草品种之间的效率差异很大，因此植物中生物碱的运输仍需要更详细地研究。我们的研究为更好地理解吡咯烷环生物合成及其对基因表达和随后安纳托宾积累的影响增加了重要的一步。
