pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce auto-fluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intra-organelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0–8.5. We designated this sensor protein as “luminescent pH indicator protein” (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.

中文翻译：

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发光的 Nanoluc-GFP 融合蛋白能够读取光合生物体中的细胞 pH 值


pH 值是决定重要细胞活动（例如光合作用性能）的最关键生理参数之一。已经报道了能够测量动物细胞中原位 pH 值的荧光传感器蛋白。然而，这些蛋白质需要激发激光来测量 pH，这可能会影响光合作用性能并诱导叶绿素发出自发荧光。因此，不可能测量植物细胞内或细胞器内的 pH 变化。为了克服这个问题，我们通过将发光蛋白 Nanoluc 与独特设计的 pH 敏感 GFP 变体蛋白融合，开发了一种发光 pH 传感器。在该系统中，不需要激发激光，因为融合的 GFP 变体通过 Nanoluc 的生物发光共振能量转移来报告发光信号。传感器蛋白的两个发光峰的比率与 pH 在 7.0-8.5 范围内近似呈线性。我们将这种传感器蛋白命名为“发光 pH 指示蛋白”（Luphin）。我们将 Luphin 应用于光合生物在波动光照条件下的原位 pH 测量，使我们能够成功观察到蓝藻集胞藻 (Synechocystis sp) 中与光合电子转移相关的胞质 pH 变化。 PCC 6803。对该藻类细胞质中观察到的估计 pH 值变化机制的详细分析表明，光合电子转移受到光照条件下质体醌库减少的抑制。这些结果表明 Luphin 可以作为一种有用的工具，进一步阐明整个光合生物体中 pH 依赖性过程。