In planta study of photosynthesis and photorespiration using NADPH and NADH/NAD+ fluorescent protein sensors

Abstract

The challenge of monitoring in planta dynamic changes of NADP(H) and NAD(H) redox states at the subcellular level is considered a major obstacle in plant bioenergetics studies. Here, we introduced two circularly permuted yellow fluorescent protein sensors, iNAP and SoNar, into Arabidopsis thaliana to monitor the dynamic changes in NADPH and the NADH/ NAD + ratio. In the light, photosynthesis and photorespiration are linked to the redox states of NAD(P)H and NAD(P) pools in several subcellular compartments connected by the malate-OAA shuttles. We show that the photosynthetic increases in stromal NADPH and NADH/ NAD + ratio, but not ATP, disappear when glycine decarboxylation is inhibited. These observations highlight the complex interplay between chloroplasts and mitochondria during photosynthesis and support the suggestions that, under normal conditions, photorespiration supplies a large amount of NADH to mitochondria, exceeding its NADH-dissipating capacity, and the surplus NADH is exported from the mitochondria to the cytosol through the malate-OAA shuttle.

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

“…The progression of cytosolic alkalisation during the light period was mirrored in the mitochondrial matrix, monitored with cpYFP ( Figure 2 ). Alkalisation of the mitochondrial matrix after a light period was observed recently albeit without any measurements in the cytosol, and therefore interpreted as increased mETC activity and proton pumping rate leading to a steepened proton gradient across the inner mitochondrial membrane (Lim et al, 2020; Voon et al, 2018). Here we refine those observations by (a) resolving the pH signature at high resolution and (b) by a side-by-side comparison with cytosolic measurements, that show that very similar signatures occur in both the mitochondrial matrix and the cytosol.…”

“…A similar effect was recently shown in Arabidopsis cotyledons expressing the NADH/NAD + -sensitive biosensor SoNar. When being exposed to a 3 min illumination period the cytosolic NAD pool showed reduction (Lim et al, 2020). However, despite the reported 10-fold larger dynamic range of SoNar compared to Peredox-mCherry (Zhao et al, 2015), which was employed here, the magnitude of cytosolic NAD reduction was comparably small.…”

“…However, despite the reported 10-fold larger dynamic range of SoNar compared to Peredox-mCherry (Zhao et al, 2015), which was employed here, the magnitude of cytosolic NAD reduction was comparably small. Interestingly, the increase in NAD reduction occurred after 1 min illumination, and at the same time, an increase in NADPH was recorded in the chloroplast stroma (Lim et al, 2020). The latter is surprising considering that photosynthetic electron transfer and the resulting NADPH production is initiated on a much faster time scale and the resulting NADPH increase is particularly expected in the initial phase, when NADPH consumption by the Calvin cycle is not yet at full capacity.…”

“…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.…”

“…Capturing light-dependent dynamics has hitherto been constrained, however, by the difficulty to monitor sensor fluorescence while simultaneously illuminating the plant. First insights into light-induced dynamic changes at subcellular level were achieved by switching between illumination and fluorescence monitoring, albeit with significant delays in the order of several seconds (Lim et al, 2020; Voon et al, 2018). That temporal resolution has made it difficult to resolve reliable signatures, limiting the power of the approach to extract mechanistic insight from live responses to the illumination status of photosynthetic tissues.…”