AP2/ERF Transcription Factor, Ii049, Positively Regulates Lignan Biosynthesis in Isatis indigotica through Activating Salicylic Acid Signaling and Lignan/Lignin Pathway Genes

Abstract

Lignans, such as lariciresinol and its derivatives, have been identified as effective antiviral ingredients in <i>Isatis indigotica</i>. Evidence suggests that the APETALA2/ethylene response factor (AP2/ERF) family might be related to the biosynthesis of lignans in <i>I. indigotica</i>. However, the special role played by the AP2/ERF family in the metabolism and its underlying putative mechanism still need to be elucidated. One novel AP2/ERF gene, named <i>Ii049</i>, was isolated and characterized from <i>I. indigotica</i> in this study. The quantitative real-time PCR analysis revealed that <i>Ii049</i> was expressed highest in the root and responded to methyl jasmonate, salicylic acid (SA) and abscisic acid treatments to various degrees. Subcellular localization analysis indicated that <i>Ii049</i> protein was localized in the nucleus. Knocking-down the expression of <i>Ii049</i> caused a remarkable reduction of lignan/lignin contents and transcript levels of genes involved in the lignan/lignin biosynthetic pathway. <i>Ii049</i> bound to the coupled element 1, RAV1AAT and CRTAREHVCBF2 motifs of genes <i>IiPAL</i> and <i>IiCCR</i>, the key structural genes in the lignan/lignin pathway. Furthermore, <i>Ii049</i> was also essential for SA biosynthesis, and SA induced lignan accumulation in <i>I. indigotica</i>. Notably, the transgenic <i>I. indigotica</i> hairy roots overexpressing <i>Ii049</i> showed high expression levels of lignan/lignin biosynthetic genes and SA content, resulting in significant accumulation of lignan/lignin. The best-engineered line (OVX049-10) produced 425.60 μg·g^-1 lariciresinol, an 8.3-fold increase compared with the wild type production. This study revealed the function of <i>Ii049</i> in regulating lignan/lignin biosynthesis, which had the potential to increase the content of valuable lignan/lignin in economically significant medicinal plants.