Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.
Live imaging of intra- and extracellular pH in plants using pHusion, a novel genetically encoded biosensor.
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DOI：
10.1093/jxb/ers040
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发表时间：
2012-05
影响因子：
6.9
通讯作者：
Fuglsang AT
中科院分区：
文献类型：
--
作者：
Gjetting KS;Ytting CK;Schulz A;Fuglsang AT
pH值的变化现在被广泛接受为细胞中的信号传导机制。在植物中，质膜和液泡膜中的质子泵在调节细胞内pH稳态和维持跨膜质子梯度中起关键作用。可以预期，响应于外部刺激的质子运输在空间和时间上被精细地调节。为了实时跟踪这种变化，一种新的基因编码传感器pHusion已经开发出来。pHusion专为质外体pH测量而设计。它在拟南芥中组成型表达，并在胞质溶胶或质外体（包括细胞内隔室）中靶向表达。pHusion由增强型绿色荧光蛋白（EGFP）和单体红色荧光蛋白（mRFP 1）的串联连接组成，并且用作比率式pH传感器。高空间和时间分辨率的活体显微镜高度依赖于用于显微镜检查的标本的适当固定。在这样的实验中经常使用的医用粘合剂破坏根部细胞的活力。在这里，提出了一种新的系统，用于固定拟南芥幼苗根灌注实验，不损害细胞活力。通过适当的固定，可以跟踪叶肉和根组织中质外体和胞质pH的变化。外部pH值变化后的快速pH值稳态反映了可忽略不计的胞质pH值波动，而质外体pH值变化剧烈。在整个组织的pH值调节分析的巨大潜力，生理背景下表现出立即碱化的外部吲哚-3-乙酸管理后的表皮下质外体。与根毛区和对照根相比，伸长区的这种变化非常显著。
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.
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影响因子：
3.5
作者：
Cormack, BP;Valdivia, RH;Falkow, S
通讯作者：
Falkow, S
影响因子：
2.9
作者：
Elsliger, MA;Wachter, RM;Remington, SJ
通讯作者：
Remington, SJ
DOI：
10.1073/pnas.96.5.2135
发表时间：
1999-03-02
影响因子：
11.1
作者：
Miyawaki, A;Griesbeck, O;Tsien, RY
通讯作者：
Tsien, RY
影响因子：
6.9
作者：
Chaudhuri, Bhavna;Hoermann, Friederike;Frommer, Wolf B.
通讯作者：
Frommer, Wolf B.
影响因子：
7.2
作者：
Clough, SJ;Bent, AF
通讯作者：
Bent, AF