DataSheet_1_First experimental evidence suggests use of glucobrassicin as source of auxin in drought-stressed Arabidopsis thaliana.pdf

The synthesis of indole-3-acetonitrile (IAN) from the indolic glucosinolate (iGSL) glucobrassicin (GB) is a unique trait of members of the Brassicales. To assess the contribution of this pathway to indole-3-acetic acid (IAA) synthesis under stress conditions, drought stress (DS) experiments with Arabidopsis thaliana were performed in vitro. Analysis of GSLs in DS plants revealed higher contents of GB in shoots and roots compared to control plants. Deuterium incorporation experiments showed the highest turnover of GB compared to all other GSLs during drought conditions. Evidence suggests the involvement of the thioglucosidase BGLU18 in the degradation of GB. The nitrile specifier proteins NSP1 and NSP5 are known to direct the GSL hydrolysis towards formation of IAN. Nitrilases like NIT2 are able to subsequently synthesize IAA from IAN. Expression of BGLU18, NSP1, NSP5 and NIT2 and contents of GB, IAN and IAA were significantly elevated in DS plants compared to control plants suggesting the increased use of GB as IAA source. Significantly higher contents of reactive oxygen species in DS bglu18 and epithionitrile specifier protein (esp) mutants compared to Col-0 indicate higher stress levels in these mutants highlighting the need for both proteins in DS plants. Furthermore, GB accumulation in leaves was higher in both mutants during DS when compared to Col-0 indicating enhanced synthesis of GB due to a lack of breakdown products. This work provides the first evidence for the breakdown of iGSLs to IAN which seems to be used for synthesis of IAA in DS A. thaliana plants.

从吲哚族芥子油苷（indolic glucosinolate, iGSL）芸苔葡糖硫苷（glucobrassicin, GB）合成吲哚-3-乙腈（indole-3-acetonitrile, IAN）是十字花目（Brassicales）类群成员的特有性状。为明确该通路在胁迫条件下对吲哚-3-乙酸（indole-3-acetic acid, IAA）合成的贡献，本研究以拟南芥（Arabidopsis thaliana）为材料开展了体外干旱胁迫（drought stress, DS）实验。对干旱胁迫植株的芥子油苷分析显示，其地上部与根部的GB含量均显著高于对照组植株。氘标记掺入实验表明，干旱胁迫条件下GB的周转速率高于所有其他芥子油苷。研究证据显示硫代葡糖苷酶BGLU18参与了GB的降解过程。已知腈类定向蛋白NSP1与NSP5可引导芥子油苷水解生成IAN，而诸如NIT2的腈水解酶可进一步由IAN合成IAA。与对照组相比，干旱胁迫植株中BGLU18、NSP1、NSP5及NIT2的表达量，以及GB、IAN与IAA的含量均显著升高，提示GB作为IAA合成前体的利用途径被激活。与哥伦比亚生态型Col-0相比，干旱胁迫下的bglu18突变体与环硫腈定向蛋白（epithionitrile specifier protein, ESP）突变体体内活性氧（reactive oxygen species, ROS）含量显著升高，表明这两类突变体的胁迫水平更高，凸显了这两种蛋白在干旱胁迫植株中的必要性。此外，干旱胁迫期间，上述两种突变体叶片中的GB积累量均高于Col-0，提示由于GB降解产物的缺失，GB的合成过程被增强。本研究首次提供证据表明，在干旱胁迫的拟南芥植株中，吲哚族芥子油苷可降解为IAN并用于IAA的合成。