Functional characterization of plant volatile organic compound (VOC) biosynthetic genes and elucidation of the biological function of their products often involve the screening of large numbers of plants from either independent transformation events or mapping populations. The low time resolution of standard gas chromatographic methods, however, represents a major bottleneck for in planta genetic characterization of VOC biosynthetic genes. Here we present a fast and highly-sensitive method for the high-throughput characterization of VOC emission levels/patterns by coupling a Proton Transfer Reaction Time-of-Flight Mass Spectrometer to an autosampler for automation of sample measurement. With this system more than 700 samples per day can be screened, detecting for each sample hundreds of spectrometric peaks in the m/z 15-300 range. As a case study, we report the characterization of VOC emissions from 116 independent Arabidopsis thaliana lines transformed with a putative isoprene synthase gene, confirming its function also when fused to a C-terminal 3×FLAG tag. We demonstrate that the method is more reliable than conventional characterization of transgene expression for the identification of the most highly isoprene-emitting lines. The throughput of this VOC screening method exceeds that of existing alternatives, potentially allowing its application to reverse and forward genetic screenings of genes contributing to VOC emission, constituting a powerful tool for the functional characterization of VOC biosynthetic genes and elucidation of the biological functions of their products directly in planta.

中文翻译：

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通过PTR-ToF-MS对植物中VOC生物合成基因的特征进行高通量筛选。

植物挥发性有机化合物（VOC）生物合成基因的功能表征及其产品生物学功能的阐明通常涉及从独立转化事件或作图种群中筛选大量植物。然而，标准气相色谱方法的低时间分辨率代表了VOC生物合成基因在植物遗传学表征中的主要瓶颈。在这里，我们通过将质子转移反应飞行时间质谱仪与自动进样器耦合以实现样品测量的自动化，提出了一种用于高通量表征VOC排放水平/模式的快速而灵敏的方法。使用该系统，每天可以筛选700多个样品，为每个样品检测m / z 15-300范围内的数百个光谱峰。作为案例研究 我们报告了特征性的116个拟南芥合酶基因转化的拟南芥品系的VOC排放特征，证实了其与C末端3xFLAG标签融合时的功能。我们证明该方法比转基因表达的常规表征更可靠，可用于鉴定最高异戊二烯发射系。该VOC筛选方法的通量超过了现有替代方法的吞吐量，可能使其可以反向和转发对促发VOC排放的基因进行遗传筛选，从而构成了VOC生物合成基因功能表征和阐明其生物学功能的有力工具。产品直接在Planta中。