NADH and NAD⁺ are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD⁺ in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis (Arabidopsis thaliana) and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism.

NADH 和 NAD ⁺是细胞中普遍存在的氧化还原对。尽管 NAD 在植物代谢中的核心作用及其调节作用已在生化水平上得到广泛研究，但分析活体植物组织中 NAD 的亚细胞氧化还原动力学一直具有挑战性。在这里，我们使用基因编码荧光生物传感器 Peredox-mCherry 建立了植物中 NADH/NAD ⁺的实时监测。我们建立了拟南芥 ( Arabidopsis thaliana ) 的 Peredox-mCherry 品系，并验证了传感器的生物物理和生化特性，这些特性对于植物测量至关重要，包括特异性、pH 稳定性和可逆性。我们生成了活拟南芥幼苗细胞质的 NAD 氧化还原图谱，揭示了不同器官和组织之间 NAD 氧化还原状态的显着差异。通过从暗到亮的转变、呼吸抑制、糖补充和激发子暴露来操纵代谢状态，揭示了胞质 NAD 氧化还原状态的显着程度的可塑性，并证明了叶片细胞区室之间的代谢氧化还原耦合。最后，我们利用蛋白质工程生成了一种传感器变体，扩大了可解析的 NAD 氧化还原范围。总之，我们建立了一种植物内 NAD 氧化还原监测技术，可以为植物细胞质氧化还原代谢的体内动态提供重要的见解。