Llić N, Normanly J, Cohen J D
Department of Plant Biology, University of Maryland, College Park 20742, USA.
Plant Physiol. 1996 Jul;111(3):781-8. doi: 10.1104/pp.111.3.781.
The genetic advantages to the use of Arabidopsis thaliana mutants for the study of auxin metabolism previously have been partially offset by the complexity of indolic metabolism in this plant and by the lack of proper methods. To address some of these problems, we developed isotopic labeling methods to determine amounts and examine the metabolism of indolic compounds in Arabidopsis. Isolation and indentification of endogenous indole-3-acetonitrile (IAN; a possible precursor of the auxin indole-3-acetic acid [IAA]) was carried out under mild conditions, thus proving its natural occurrence. We describe here the synthesis of 13C1-labeled IAN and its utility in the gas chromatography-mass spectrometry quantification of endogenous IAN levels. We also quantified the nonenzymatic conversion of IAN to IAA under conditions used to hydrolyze IAA conjugates. 13C1-Labeled IAN was used to assess the contribution of IAN to measured IAA following hydrolysis of IAA conjugates. We studied the stability and breakdown of the indolic glucosinolate glucobrassicin, which is known to be present in Arabidopsis. This is potentially an important concern when using Arabidopsis for studies of indolic biochemistry, since the levels of indolic auxins and auxin precursors are well below the levels of the indolic glucosinolates. We found that under conditions of extraction and base hydrolysis, formation of IAA from glucobrassicin was negligible.
此前，利用拟南芥突变体研究生长素代谢的遗传优势，在一定程度上被该植物吲哚代谢的复杂性以及缺乏合适的方法所抵消。为了解决其中一些问题，我们开发了同位素标记方法来测定拟南芥中吲哚类化合物的含量并研究其代谢。在温和条件下对内源吲哚 - 3 - 乙腈（IAN；生长素吲哚 - 3 - 乙酸 [IAA] 的一种可能前体）进行了分离和鉴定，从而证明了它的天然存在。我们在此描述了¹³C₁标记的IAN的合成及其在气相色谱 - 质谱法定量内源IAN水平中的应用。我们还在用于水解IAA共轭物的条件下，对内源IAN向IAA的非酶促转化进行了定量。¹³C₁标记的IAN用于评估在IAA共轭物水解后，IAN对测得的IAA的贡献。我们研究了吲哚硫代葡萄糖苷葡萄糖硫苷（已知存在于拟南芥中）的稳定性和分解情况。在利用拟南芥研究吲哚生物化学时，这可能是一个重要问题，因为吲哚类生长素和生长素前体的水平远低于吲哚硫代葡萄糖苷的水平。我们发现，在提取和碱水解条件下，葡萄糖硫苷生成IAA的量可忽略不计。
