Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Copenhagen and Danish Basic Research Foundation Centre of excellence: Centre for Membrane Pumps in Cells and Disease-PUMPKIN, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark.
J Exp Bot. 2012 May;63(8):3207-18. doi: 10.1093/jxb/ers040. Epub 2012 Mar 9.
Changes in pH are now widely accepted as a signalling mechanism in cells. In plants, proton pumps in the plasma membrane and tonoplast play a key role in regulation of intracellular pH homeostasis and maintenance of transmembrane proton gradients. Proton transport in response to external stimuli can be expected to be finely regulated spatially and temporally. With the ambition to follow such changes live, a new genetically encoded sensor, pHusion, has been developed. pHusion is especially designed for apoplastic pH measurements. It was constitutively expressed in Arabidopsis and targeted for expression in either the cytosol or the apoplast including intracellular compartments. pHusion consists of the tandem concatenation of enhanced green fluorescent protein (EGFP) and monomeric red fluorescent protein (mRFP1), and works as a ratiometric pH sensor. Live microscopy at high spatial and temporal resolution is highly dependent on appropriate immobilization of the specimen for microscopy. Medical adhesive often used in such experiments destroys cell viability in roots. Here a novel system for immobilizing Arabidopsis seedling roots for perfusion experiments is presented which does not impair cell viability. With appropriate immobilization, it was possible to follow changes of the apoplastic and cytosolic pH in mesophyll and root tissue. Rapid pH homeostasis upon external pH changes was reflected by negligible cytosolic pH fluctuations, while the apoplastic pH changed drastically. The great potential for analysing pH regulation in a whole-tissue, physiological context is demonstrated by the immediate alkalinization of the subepidermal apoplast upon external indole-3-acetic acid administration. This change is highly significant in the elongation zone compared with the root hair zone and control roots.
pH 值的变化现在被广泛认为是细胞中的一种信号机制。在植物中，质膜和液泡膜中的质子泵在调节细胞内 pH 值稳态和维持跨膜质子梯度方面发挥着关键作用。对外界刺激的质子运输预计会在空间和时间上得到精细调节。为了实时跟踪这些变化，开发了一种新的遗传编码传感器 pHusion。pHusion 专门用于质外体 pH 值测量。它在拟南芥中组成型表达，并靶向表达于细胞质或质外体，包括细胞内区室。pHusion 由增强型绿色荧光蛋白 (EGFP) 和单体红色荧光蛋白 (mRFP1) 的串联串联组成，作为一种比率型 pH 传感器。高时空分辨率的活细胞显微镜高度依赖于对显微镜标本的适当固定。在这些实验中经常使用的医用粘合剂会破坏根中的细胞活力。这里提出了一种用于灌流实验固定拟南芥幼苗根的新系统，该系统不会损害细胞活力。通过适当的固定，可以观察到质外体和细胞质 pH 在叶肉和根组织中的变化。外部 pH 值变化引起的快速 pH 值稳态反映在细胞质 pH 波动可忽略不计，而质外体 pH 值变化剧烈。通过外部吲哚-3-乙酸处理后，表皮下质外体立即碱化，证明了在整个组织、生理环境中分析 pH 值调节的巨大潜力。与根毛区和对照根相比，伸长区的这种变化具有高度显著性。
