In Vivo NADH/NAD⁺ Biosensing Reveals the Dynamics of Cytosolic Redox Metabolism in Plants

Abstract

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 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

Photosynthetic activity triggers pH and NAD redox signatures across different plant cell compartments

“…To monitor reductant export into the cytosol, we focussed on the redox dynamics of the cytosolic NAD pool using the NADH/NAD + biosensor Peredox-mCherry (Hung et al, 2011; Steinbeck et al, 2020). When exposing discs of mature leaves of 4-5-week-old Arabidopsis thaliana overexpressing cytosolic Peredox-mCherry to a 15 min period of pseudo-continuous light (15-30 s illumination, 1.6 s scan cycles), we observed striking and characteristic dynamics in sensor fluorescence ratio, indicating NADH/NAD + redox dynamics.…”

“…Apart from the fast response in the initial phase of the light period, we were able to capture the NAD reduction dynamics with detailed kinetics during 15 min pseudo-continuous light, revealing a characteristic biphasic behaviour ( Figure 4B ). In previous work establishing Peredox-mCherry-based biosensing we missed the kinetics in the light, and cytosolic NAD reduction by photosynthetic activity could only be deduced from the recovery dynamics after illumination (Steinbeck et al, 2020). The temporal resolution of the NAD redox dynamics is likely to be limited by the kinetic properties of Peredox-mCherry (binding and dissociation rates of NAD + and NADH) (Hung et al, 2011; Steinbeck et al, 2020), meaning that NADH/NAD + changes may be even quicker and more pronounced in amplitude.…”

“…In previous work establishing Peredox-mCherry-based biosensing we missed the kinetics in the light, and cytosolic NAD reduction by photosynthetic activity could only be deduced from the recovery dynamics after illumination (Steinbeck et al, 2020). The temporal resolution of the NAD redox dynamics is likely to be limited by the kinetic properties of Peredox-mCherry (binding and dissociation rates of NAD + and NADH) (Hung et al, 2011; Steinbeck et al, 2020), meaning that NADH/NAD + changes may be even quicker and more pronounced in amplitude. Yet, the characteristic biphasic behaviour at onset of illumination is independent of such limitations and reveals different phases of reductant export from the chloroplast stroma.…”

“…However, the kinetics of the sensor response at the dark-to-light transition was similar in wild type, mmdh1 , mmdh2 and cpNADP-mdh both in kinetics as well as in the maximum that was reached ( Figure 5 ). While it is possible that the sensor response to illumination is limited by sensor saturation with NADH (Steinbeck et al, 2020), the responses clearly show that reductant export remains functional even when MDH capacity in the organelles is limited. Instead, reduced MDH activity in both the chloroplasts ( cpNADP-mdh ) or in the mitochondria ( mmdh1 , mmdh2) led to elevated cytosolic NADH/NAD + ratios in the dark ( Figure 5 ).…”

“…Also, changes in glutathione redox potential and H2O2 have been monitored in an illumination-dependent manner (Exposito-Rodriguez et al, 2017; Haber and Rosenwasser, 2020; Müller‐Schüssele et al, 2020; Ugalde et al, 2020). Recently, also live biosensing strategies for NAD + /NADH and NADPH were introduced (Lim et al, 2020; Steinbeck et al, 2020; Wagner et al, 2019) revealing light dependent changes in ATP and NAD(P) redox status in the chloroplast and the cytosol. Capturing light-dependent dynamics has hitherto been constrained, however, by the difficulty to monitor sensor fluorescence while simultaneously illuminating the plant.…”