Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba, Sendai, Miyagi 980-8577, Japan.
Plant Physiol Biochem. 2010 Jul;48(7):527-33. doi: 10.1016/j.plaphy.2010.01.013. Epub 2010 Jan 22.
Arabidopsis thaliana was thought to contain two spermine synthase genes, ACAULIS 5 (ACL5) and SPMS. Recent investigations, however, revealed that the ACL5 gene encodes thermospermine synthase. In this study, we have established a simple method to separate two isomers of tetraamine, spermine and thermospermine, in extracts from plant tissues of less than 500 mg. Polyamines (PAs) extracted from plant tissues were benzoylated, and the derivatives were completely resolved by high-performance liquid chromatography on a C18 reverse-phase column, by eluting with 42% (v/v) acetonitrile in water in an isocratic manner at 30 degrees C and monitoring at 254 nm. The relevance of the method was confirmed by co-chromatography with respective PAs and by the PA analysis of the single- and double-mutants of acl5 and spms, which could not synthesize thermospermine and/or spermine, respectively. Furthermore, with this method, we monitored the thermospermine contents in various tissues of A. thaliana and found that stems and flowers contain two- to three-fold more thermospermine compared to whole seedlings and mature leaves. The presence of thermospermine was confirmed in Oryza sativa and Lycopersicon pesculentum. Finally we addressed whether salinity stress changes the contents of PAs including thermospermine in Arabidopsis.
拟南芥被认为含有两个亚精胺合酶基因，ACAULIS 5 (ACL5)和 SPMS。然而，最近的研究表明，ACL5 基因编码热亚精胺合酶。在这项研究中，我们建立了一种从少于 500mg 的植物组织提取物中分离两种四胺异构体，亚精胺和热亚精胺的简单方法。从植物组织中提取的多胺（PA）被苯甲酰化，衍生物通过在 30°C 下以 42%（v/v）乙腈在水中的等度洗脱在 C18 反相柱上完全分离，并在 254nm 处监测。该方法的相关性通过与各自的 PA 进行共色谱分析以及通过 acl5 和 spms 的单突变体和双突变体的 PA 分析得到证实，这些突变体分别不能合成热亚精胺和/或亚精胺。此外，通过这种方法，我们监测了拟南芥各种组织中的热亚精胺含量，发现茎和花中热亚精胺的含量是整个幼苗和成熟叶的两倍到三倍。在水稻和番茄中也证实了热亚精胺的存在。最后，我们研究了盐胁迫是否会改变包括拟南芥中热亚精胺在内的多胺的含量。
