Dynamic imaging of glucose flux impedance using FRET sensors in wild-type Arabidopsis plants
Dynamic imaging of glucose flux impedance using FRET sensors in wild-type Arabidopsis plants
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DOI：
10.1093/jxb/erq444
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发表时间：
2011-04-01
影响因子：
6.9
通讯作者：
Frommer, Wolf B.
中科院分区：
文献类型：
--
作者：
Chaudhuri, Bhavna;Hoermann, Friederike;Frommer, Wolf B.
代谢物和信号分子的定量和动态分析受到在细胞和隔室水平获得时间分辨信息的技术挑战的限制。在基因编码的FRET（福斯特共振能量转移）纳米传感器的帮助下，可以获得亚细胞粒度的信号和代谢物水平的实时信息。FRET纳米传感器代表了基因发现和调控网络分析的强大工具，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，克服了这个缺点。然而，沉默突变体的使用需要产生RNA沉默缺陷的纯合系以及感兴趣的突变和纳米传感器的共表达。在此显示，在沉默对荧光强度具有主要影响之前，在发育的早期阶段（7-15 d），在野生型拟南芥植物中可以容易地定量胞质葡萄糖水平的动态变化。提供了每天筛选10-20株突变体幼苗的详细方案。这里提供的详细的成像协议是适合于分析糖通量在年轻的野生型植物，以及突变体影响糖信号，代谢，或运输使用广泛的FRET纳米传感器。
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 (Forster 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.