Abstract：
Redox changes of pyridine nucleotides in cellular compartments are highly dynamic and their equilibria are under the influence of various reducing and oxidizing reactions. To obtain spatiotemporal data on pyridine nucleotides in living plant cells, typical biochemical approaches require cell destruction. To date, genetically encoded fluorescent biosensors are considered to be the best option to bridge the existing technology gap, as they provide a fast, accurate, and real-time readout. However, the existing pyridine nucleotides genetically encoded fluorescent biosensors are either sensitive to pH change or slow in dissociation rate. Herein, we employed the biosensors which generate readouts that are pH stable for in planta measurement of NADH/NAD+ ratio and NADPH level. We generated transgenic Arabidopsis lines that express these biosensors in plastid stroma and cytosol of whole plants and pollen tubes under the control of CaMV 35S and LAT52 promoters, respectively. These transgenic biosensor lines allow us to monitor real-time dynamic changes in NADH/NAD+ ratio and NADPH level in the plastids and cytosol of various plant tissues, including pollen tubes, root hairs, and mesophyll cells, using a variety of fluorescent instruments. We anticipate that these valuable transgenic lines may allow improvements in plant redox biology studies.
摘要：
细胞区室中吡啶核苷酸的氧化还原变化是高度动态的，它们的平衡受到各种还原和氧化反应的影响。为了获得活植物细胞中吡啶核苷酸的时空数据，典型的生化方法需要破坏细胞。迄今为止,基因编码的荧光生物传感器被认为是弥合现有技术差距的最佳选择，因为他们提供了一个快速的，准确,和实时读出。然而,现有的吡啶核苷酸基因编码的荧光生物传感器对pH变化敏感或解离速率慢。在这里，我们使用了生物传感器，这些传感器产生的读数对于NADH/NAD比率和NADPH水平的植物测量是pH稳定的。我们产生了转基因拟南芥系，这些系在CaMV35S和LAT52启动子的控制下，在整个植物的质体基质和胞质溶胶以及花粉管中表达这些生物传感器，分别。这些转基因生物传感器系使我们能够监测各种植物组织的质体和细胞质中NADH/NAD比率和NADPH水平的实时动态变化，包括花粉管,根毛,和叶肉细胞，使用各种荧光仪器。我们预计这些有价值的转基因品系可以改善植物氧化还原生物学研究。
