MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, which comprises a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30-bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.

中文翻译：

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NtMYB305a 结合 NtPMT1a 启动子的茉莉酸反应性 GAG 区域来调节尼古丁生物合成


MYB 转录因子在调节植物次生代谢和茉莉酸 (JA) 信号传导中发挥重要作用。腐胺 N-甲基转移酶是尼古丁生物合成中 JA 调节的关键步骤，尼古丁是一种在烟草属中高度积累的生物碱化合物。在这里，我们报告了烟草（Nicotiana tabacum）中 NtMYB305a 的鉴定，该 NtMYB305a 是尼古丁生物合成的调节成分，并证明它与 JA 响应性 GAG 区域结合，该区域包含 G-box、富含 AT 的基序和 GCC- NtPMT1a 启动子中的盒状元件。酵母单杂交分析、电泳迁移率变动分析和染色质免疫沉淀分析表明，NtMYB305a 在体外和体内均与 GAG 区域结合。结合以不依赖于 G/C 碱基的方式特异性地发生在~30 bp 富含 AT 的基序上，从而将富含 AT 的基序定义为以前未知的 MYB 结合元件。 NtMYB305a 位于烟草细胞核中，能够以富含 AT 的基序依赖性方式激活 4×GAG 驱动的 GUS 报告基因的表达。 NtMYB305a 正向调节尼古丁生物合成以及 NtPMT 和其他尼古丁途径基因的表达。 NtMYB305a 与 NtMYC2a 协同作用来调节尼古丁生物合成，但没有检测到这两种蛋白质之间的相互作用。 NtMYB305a 的鉴定提供了对尼古丁生物合成调节的见解，并扩展了 MYB 转录因子在植物次生代谢中发挥的作用。