Department of Plant Sciences (IPV), Faculty of Biosciences, Norwegian University of Life Sciences, Norway Campus Ås, Universitetstunet 3, 1430, Ås, Norway.
Department of Horticultural Sciences, Microbial and Plant Genomics Institute, University of Minnesota, 305 Alderman Hall, 1970 Folwell Avenue, Saint Paul, MN, 55108, USA.
BMC Plant Biol. 2018 Jun 25;18(1):133. doi: 10.1186/s12870-018-1352-z.
Quantitative measurement of actual auxin levels in plant tissue is complimentary to molecular methods measuring the expression of auxin related genes. Current analytical methods to quantify auxin have pushed the limit of detection to where auxin can be routinely quantified at the pictogram (pg) level, reducing the amount of tissue needed to perform these kinds of studies to amounts never imagined a few years ago. In parallel, the development of technologies like laser microdissection microscopy (LMD) has allowed specific cells to be harvested from discrete tissues without including adjacent cells. This method has gained popularity in recent years, especially for enabling a higher degree of spatial resolution in transcriptome profiling. As with other quantitative measurements, including hormone quantifications, sampling using traditional LMD is still challenging because sample preparation clearly compromises the preservation of analytes. Thus, we have developed and validated a sample preparation protocol combining cryosectioning, freeze-drying, and capturing with a laser microdissection microscope to provide high-quality and well-preserved plant materials suitable for ultrasensitive, spatially-resolved auxin quantification.
We developed a new method to provide discrete plant tissues for indole-3-acetic acid (IAA) quantification while preserving the plant tissue in the best possible condition to prevent auxin degradation. The method combines the use of cryosectioning, freeze-drying and LMD. The protocol may also be used for other applications that require small molecule analysis with high tissue-specificity where degradation of biological compounds may be an issue. It was possible to collect the equivalent to 15 mg of very specific tissue in approximately 4 h using LMD.
We have shown, by proof of concept, that freeze dried cryosections of plant tissue were suitable for LMD harvest and quantification of the phytohormone auxin using GC-MS/MS. We expect that the ability to resolve auxin levels with both spatial- and temporal resolution with high accuracy will enable experiments on complex processes, which will increase our knowledge of the many roles of auxins (and, in time, other phytohormones) in plant development.
定量测量植物组织中的实际生长素水平是对生长素相关基因表达进行分子方法测量的补充。目前用于定量测定生长素的分析方法已经将检测极限推到了可以常规地以皮克（pg）水平定量测定生长素的程度，从而减少了进行这些研究所需的组织量，而这在几年前是无法想象的。与此同时，像激光显微切割显微镜（LMD）这样的技术的发展使得可以从离散组织中收获特定的细胞，而不会包括相邻的细胞。近年来，这种方法越来越受欢迎，尤其是在转录组谱分析中实现更高的空间分辨率。与包括激素定量测定在内的其他定量测量一样，使用传统的 LMD 进行采样仍然具有挑战性，因为样品制备显然会影响分析物的保留。因此，我们开发并验证了一种结合冷冻切片、冷冻干燥和激光微切割显微镜捕获的样品制备方案，以提供高质量和保存完好的植物材料，这些材料非常适合超灵敏、空间分辨的生长素定量。
我们开发了一种新方法，用于提供用于吲哚-3-乙酸（IAA）定量的离散植物组织，同时以最佳可能的条件保存植物组织，以防止生长素降解。该方法结合了使用冷冻切片、冷冻干燥和 LMD。该方案也可用于其他需要具有高组织特异性的小分子分析的应用，其中生物化合物的降解可能是一个问题。使用 LMD 可以在大约 4 小时内收集相当于 15 毫克的非常特定组织。
我们通过概念验证表明，植物组织的冷冻干燥切片适合使用 GC-MS/MS 通过 LMD 进行收获和生长素的定量。我们预计，以高精度具有空间和时间分辨率解析生长素水平的能力将能够进行复杂过程的实验，这将增加我们对生长素（以及，随着时间的推移，其他植物激素）在植物发育中的许多作用的了解。
