Institute of Chemical Technology, Prague, Department of Food Analysis and Nutrition, Prague, Czech Republic.
Anal Bioanal Chem. 2013 Mar;405(8):2671-83. doi: 10.1007/s00216-012-6692-1. Epub 2013 Jan 17.
The availability of rapid and reliable tools for monitoring of plants' cold tolerance is a prerequisite for research aimed at breeding of cold-tolerant crop plants. Therefore, we have tested the capacity of metabolomics-based methods employing ultra-high-performance liquid chromatography (UHPLC)-mass spectrometry and direct analysis in real time-mass spectrometry for high-throughput screening of cold tolerance in eight differentially cold-tolerant accessions of Arabidopsis thaliana. Metabolomic fingerprinting of leaf tissues was performed in methanolic extracts for (1) 6-week-old non-acclimated (NAC) plants grown at room temperature, (2) NAC plants cold-acclimated (ACC) at 4 °C for 2 weeks, and (3) cold-acclimated plants given sub-zero-temperature treatments by slow cooling at -4 °C for 8 h. The generated chromatograms and mass spectra were processed with the use of multivariate statistical analysis employing principal component analysis (PCA) and linear discriminant analysis. The PCA of metabolomic fingerprints classified the investigated A. thaliana accessions into three categories with low, intermediate, and high cold tolerance for both the cold-acclimated and the sub-zero-temperature-treated plants. This indicates the potential application of metabolomics-based fingerprinting for measuring cold tolerance in the cold-acclimated state, i.e., without treating plants at freezing temperatures that is required by currently available methods. Furthermore, we employed UHPLC coupled to the quadrupole-time-of-flight mass spectrometry to identify characteristic metabolites in ACC state and found the abundance of gluconapin and flavon-3-ol glycosides, respectively, in the cold-sensitive and the cold-tolerant accessions.
快速可靠的植物耐寒性监测工具的可用性是培育耐寒作物的研究的前提。因此，我们已经测试了基于代谢组学的方法的能力，该方法采用超高效液相色谱（UHPLC）-质谱法和实时直接分析质谱法，用于对 8 个拟南芥不同耐寒性品系进行高通量耐寒性筛选。对室温下生长的 6 周龄非驯化（NAC）植物的叶片组织进行了甲醇提取物的代谢组指纹图谱分析，包括（1）未经驯化（NAC）的植物，（2）在 4°C 下驯化 2 周的植物（ACC），以及（3）在 -4°C 下通过缓慢冷却 8 小时进行亚零温度处理的驯化植物。使用主成分分析（PCA）和线性判别分析的多元统计分析处理生成的色谱图和质谱图。代谢组指纹图谱的 PCA 将研究的拟南芥品系分为三类，具有低、中和高耐寒性，适用于驯化和亚零温度处理的植物。这表明基于代谢组学的指纹图谱在驯化状态下测量耐寒性的潜在应用，即在目前可用的方法中不需要将植物处理在冷冻温度下。此外，我们采用超高效液相色谱法与四极杆飞行时间质谱法相结合，鉴定了 ACC 状态下的特征代谢物，并在耐寒性敏感和耐寒性品系中分别发现了葡萄糖苷和类黄酮-3-醇糖苷的丰度。
