Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells?
Why does anatabine, but not nicotine, accumulate in jasmonate-elicited cultured tobacco BY-2 cells?
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DOI：
10.1093/pcp/pcn096
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发表时间：
2008-08
影响因子：
4.9
通讯作者：
T. Shoji;T. Hashimoto
中科院分区：
文献类型：
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作者：
T. Shoji;T. Hashimoto
烟草悬浮培养细胞。明亮黄-2(BY-2)在高度均一的种群中生长迅速，仍然表现出植物细胞的一般行为，因此经常被用作植物分子和细胞生物学领域的模型系统，包括次生代谢。虽然亲本烟草品种合成尼古丁作为主要生物碱，但培养的烟草细胞在茉莉酸酯诱导下主要产生一种相关的生物碱Anatabine，而不是尼古丁。我们在这里报道，培养的BY-2细胞即使在茉莉酸诱导后也几乎不表达N-甲基腐胺氧化酶(MPO)基因。MPO是生物合成途径中的第二种酶，提供烟碱和去甲尼古丁的吡咯烷部分，但预计对于不含吡咯烷部分的anatabine、anabasine和anataline的生物合成是必不可少的。当MPO在烟草BY-2细胞中过表达时，尼古丁的合成显著增强，而Anatabine的形成有效地被抑制。作为补充，我们通过RNA干扰抑制了MPO在烟草毛状根中的表达，这些毛状根通常会积累尼古丁。在MPO抑制的根中，Anatabine、Anabasine和Anataline以及N-甲基腐胺和腐胺的含量显著增加，以补偿尼古丁和去甲烟碱的抑制形成。这些结果表明，MPO的转录调控是限制烟草BY-2细胞培养中尼古丁形成的关键限速步骤。
Suspension-cultured cells of Nicotiana tabacum cv. Bright Yellow-2 (BY-2) grow rapidly in a highly homogenous population and still exhibit the general behavior of plant cells, and thus are often used as model systems in several areas of plant molecular and cellular biology, including secondary metabolism. While the parental tobacco variety synthesizes nicotine as a major alkaloid, the cultured tobacco cells mainly produce a related alkaloid anatabine, instead of nicotine, when elicited with jasmonates. We report here that cultured BY-2 cells scarcely express N-methylputrescine oxidase (MPO) genes even after jasmonate elicitation. MPO is the second enzyme in the biosynthetic pathway that supplies the pyrrolidine moiety of nicotine and nornicotine, but is predicted to be dispensable for the biosynthesis of anatabine, anabasine and anatalline, which do not contain the pyrrolidine moiety. When MPO was overexpressed in tobacco BY-2 cells, nicotine synthesis was dramatically enhanced while anatabine formation was effectively suppressed. As a complementary approach, we suppressed MPO expression by RNA interference in tobacco hairy roots that normally accumulate nicotine. In the MPO-suppressed roots, the contents of anatabine, anabasine and anatalline, as well as N-methylputrescine and putrescine, markedly increased to compensate for suppressed formation of nicotine and nornicotine. These results identify the transcriptional regulation of MPO as a critical rate-limiting step that restricts nicotine formation in cultured tobacco BY-2 cells.