Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, CA 94305, USA.
J Exp Bot. 2011 Apr;62(7):2411-7. doi: 10.1093/jxb/erq444. Epub 2011 Jan 25.
Quantitative and dynamic analysis of metabolites and signalling molecules is limited by technical challenges in obtaining temporally resolved information at the cellular and compartmental level. Real-time information on signalling and metabolite levels with subcellular granularity can be obtained with the help of genetically encoded FRET (Förster resonance energy transfer) nanosensors. FRET nanosensors represent powerful tools for gene discovery, and analysis of regulatory networks, for example by screening mutants. However, RNA silencing has impaired our ability to express FRET nanosensors functionally in Arabidopsis plants. This drawback was overcome here by expressing the nanosensors in RNA silencing mutants. However, the use of silencing mutants requires the generation of homozygous lines deficient in RNA silencing as well as the mutation of interest and co-expression of the nanosensor. Here it is shown that dynamic changes in cytosolic glucose levels can readily be quantified in wild-type Arabidopsis plants at early stages of development (7-15 d) before silencing had a major effect on fluorescence intensity. A detailed protocol for screening 10-20 mutant seedlings per day is provided. The detailed imaging protocol provided here is suitable for analysing sugar flux in young wild-type plants as well as mutants affected in sugar signalling, metabolism, or transport using a wide spectrum of FRET nanosensors.
代谢物和信号分子的定量和动态分析受到在细胞和区室水平上获得时间分辨信息的技术挑战的限制。借助遗传编码的 FRET（Förster 共振能量转移）纳米传感器，可以获得具有亚细胞粒度的信号和代谢物水平的实时信息。FRET 纳米传感器是基因发现和调控网络分析的有力工具，例如通过筛选突变体。然而，RNA 沉默削弱了我们在拟南芥植物中功能性表达 FRET 纳米传感器的能力。通过在 RNA 沉默突变体中表达纳米传感器克服了这一缺点。然而，使用沉默突变体需要生成缺乏 RNA 沉默以及感兴趣的突变体的纯合系，并共同表达纳米传感器。这里表明，在早期发育阶段（7-15 天），在 RNA 沉默对荧光强度产生重大影响之前，野生型拟南芥植物中的细胞质葡萄糖水平的动态变化可以很容易地进行定量。提供了每天筛选 10-20 个突变体幼苗的详细方案。这里提供的详细成像方案适用于使用广泛的 FRET 纳米传感器分析年轻野生型植物以及影响糖信号转导、代谢或运输的突变体中的糖通量。
