The NADPH/NADP⁺ redox couple is central to metabolism and redox signalling. NADP redox state is differentially regulated by distinct enzymatic machineries at the subcellular compartment level. Nonetheless, a detailed understanding of subcellular NADP redox dynamics is limited by the availability of appropriate tools. Here, we introduce NAPstars, a family of genetically encoded, fluorescent protein-based NADP redox state biosensors. NAPstars offer real-time, specific measurements, across a broad-range of NADP redox states, with subcellular resolution. NAPstar measurements in yeast, plants, and mammalian cell models, reveal a conserved robustness of cytosolic NADP redox homoeostasis. NAPstars uncover cell cycle-linked NADP redox oscillations in yeast and illumination- and hypoxia-dependent NADP redox changes in plant leaves. By applying NAPstars in combination with selective impairment of the glutathione and thioredoxin antioxidative pathways under acute oxidative challenge, we find an unexpected and conserved role for the glutathione system as the primary mediator of antioxidative electron flux.

NADPH/NADP⁺ 氧化还原对是代谢和氧化还原信号传导的核心。NADP 氧化还原状态在亚细胞区室水平上受不同酶机制的差异调节。尽管如此，对亚细胞 NADP 氧化还原动力学的详细理解受到适当工具可用性的限制。在这里，我们介绍了 NAPstars，这是一个基因编码的、基于荧光蛋白的 NADP 氧化还原状态生物传感器家族。NAPstars 以亚细胞分辨率提供广泛的 NADP 氧化还原状态，提供实时、特异性的测量。酵母、植物和哺乳动物细胞模型中的 NAPstar 测量揭示了胞质 NADP 氧化还原稳态的保守稳健性。NAPstars 揭示了酵母中与细胞周期相关的 NADP 氧化还原振荡以及植物叶片中光照和缺氧依赖性 NADP 氧化还原变化。通过在急性氧化挑战下将 NAPstars 与谷胱甘肽和硫氧还蛋白抗氧化途径的选择性损伤相结合，我们发现谷胱甘肽系统作为抗氧化电子通量的主要介质具有意想不到的保守作用。