Shimizu Takafumi, Miyakawa Shinya, Esaki Tsuyoshi, Mizuno Hajime, Masujima Tsutomu, Koshiba Tomokazu, Seo Mitsunori
RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, 230-0045 Japan.
Department of Biological Sciences, Tokyo Metropolitan University, Hachioji-shi, Tokyo, 192-0397 Japan.
Plant Cell Physiol. 2015 Jul;56(7):1287-96. doi: 10.1093/pcp/pcv042. Epub 2015 Mar 9.
Studies have indicated that endogenous concentrations of plant hormones are regulated very locally within plants. To understand the mechanisms underlying hormone-mediated physiological processes, it is indispensable to know the exact hormone concentrations at cellular levels. In the present study, we established a system to determine levels of ABA and jasmonoyl-isoleucine (JA-Ile) from single cells. Samples taken from a cell of Vicia faba leaves using nano-electrospray ionization (ESI) tips under a microscope were directly introduced into mass spectrometers by infusion and subjected to tandem mass spectrometry (MS/MS) analysis. Stable isotope-labeled [D(6)]ABA or [(13)C(6)]JA-Ile was used as an internal standard to compensate ionization efficiencies, which determine the amount of ions introduced into mass spectrometers. We detected ABA and JA-Ile from single cells of water- and wound-stressed leaves, whereas they were almost undetectable in non-stressed single cells. The levels of ABA and JA-Ile found in the single-cell analysis were compared with levels found by analysis of purified extracts with liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that stress-induced accumulation of ABA and JA-Ile could be monitored from living single cells.
研究表明，植物激素的内源性浓度在植物体内受到非常局部的调节。为了理解激素介导的生理过程背后的机制，了解细胞水平上确切的激素浓度是必不可少的。在本研究中，我们建立了一个系统来测定单个细胞中脱落酸（ABA）和茉莉酸异亮氨酸（JA-Ile）的水平。在显微镜下使用纳米电喷雾电离（ESI）尖端从蚕豆叶片的单个细胞中采集的样品，通过进样直接引入质谱仪，并进行串联质谱（MS/MS）分析。使用稳定同位素标记的[D(6)]ABA或[(13)C(6)]JA-Ile作为内标来补偿电离效率，电离效率决定了引入质谱仪的离子量。我们从水分胁迫和创伤胁迫叶片的单个细胞中检测到了ABA和JA-Ile，而在未受胁迫的单个细胞中几乎检测不到它们。将单细胞分析中发现的ABA和JA-Ile水平与通过液相色谱-串联质谱（LC-MS/MS）分析纯化提取物所发现的水平进行了比较。这些结果表明，可以从活的单个细胞中监测到胁迫诱导的ABA和JA-Ile的积累。
