Simultaneous recording of biosensors and chlorophyll fluorescence reveals a tight correlation between ETR and oxidative signals

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The conversion of light into chemical energy through the light and carbon reactions of photosynthesis is a highly complex process that requires continuous coordination between dozens of enzymatic reactions to ensure adaptation to varying light intensities experienced by plants and avoid the production of harmful reactive oxygen species (ROS). The channeling of reductive and oxidative equivalents from the photosynthetic electron transport to regulated thiol proteins plays a substantial role in linking the reaction rates of photosynthetic enzymes to instantaneous changes in daytime photon fluxes (Buchanan and Balmer 2005; Foyer and Noctor 2009; Scheibe and Dietz 2012). Reductive signals, catalyzing the reduction of disulfide bonds, are transferred from ferredoxin to Thioredoxins (Trxs) and subsequently redox-regulated target chloroplast proteins via Ferredoxin-Thioredoxin reductase, or via ferredoxin-NADP(+) reductase, and NADPH thioredoxin reductase C (Serrato et al. 2004; Buchanan and Balmer 2005; Foyer and Noctor 2009; Scheibe and Dietz 2012). Counterbalanced oxidative signals, catalyzing disulfide bond formation, are generated by photosynthetically derived hydrogen peroxide (H₂O₂) and transmitted through 2-Cys-Prxs, highly efficient thiol peroxidases, and high midpoint potential atypical Trxs (Dangoor et al. 2012; Eliyahu et al. 2015; Ojeda et al. 2018; Vaseghi et al. 2018; Yoshida et al. 2018). Accordingly, the redox state of a wide range of photosynthetic enzymes is constantly adjusted through the simultaneous generation of opposing signals, which subsequently dictates the efficiency of light-capturing and carbon-assimilation processes.

Despite the tight link between photosynthetic activity and the redox regulatory network, the relationship between the electron transport rate and the activation of reductive/oxidative signals remains unexplored, mainly due to critical methodological differences in assessing photosynthetic activity versus thiol redox state. The development of genetically encoded redox fluorescent biosensors capable of probing the redox state of key redox components opened possibilities for studying the relationship between photosynthetic activity and redox signals (Meyer and Dick 2010; Molinari et al. 2023; Müller-Schüssele 2024). However, drawing a direct correlation between photosynthetic parameters and biosensor redox state is currently constrained by the standard tools used to analyze biosensor signals, which do not allow recording biosensor and chlorophyll fluorescence while illuminating leaves with actinic light over various light intensities.

We aimed to provide insights into the relationship between electron transport rates and the generation of reductive and oxidative signals in a crop plant model, by exploring a set of redox biosensors, allowing discrimination between reductive and oxidative signals and chlorophyll fluorescence-derived photosynthetic parameters. Potato plants (Solanum tuberosum, cv. Desiree) were chosen as a model to measure photosynthetically derived oxidative signals due to their global importance for food security and the availability of highly efficient transformation protocols. To monitor redox signals, agrobacterium-mediated genetic transformation was performed to obtain potato plants expressing chl-roGFP2-PrxΔC_R and chl-roGFP2-Prx (Fig. 1A). These biosensors are more sensitive to oxidative signals compared with the unfused chl-roGFP2 due to the genetic fusion of roGFP2 to Arabidopsis 2-Cys Prx A (BAS1), allowing the transfer of oxidizing equivalents from 2-Cys Prx A to the roGFP2 moiety (Morgan et al. 2016; Niemeyer et al. 2021; Lampl et al. 2022). The mutation in the resolving Cys of 2-Cys Prx A (C241A) in chl-roGFP2-PrxΔC_R impairs the thioredoxin-dependent reduction rates of 2-Cys Prx, enhancing the sensitivity of this biosensor to H₂O₂-mediated oxidation (Morgan et al. 2016; Niemeyer et al. 2021; Lampl et al. 2022). Efficient chloroplast targeting was verified by the overlap of the chl-roGFP2-PrxΔC_R, chl-roGFP2-Prx and chlorophyll fluorescence signals (Fig. 1B, Supplementary Fig. S1), as was also shown for potato plants expressing chl-roGFP2 (Fig. 1B, Hipsch et al. 2021). The overall fluorescence intensity recorded for roGFP2-PrxΔC_R and chl-roGFP2-Prx was lower than chl-roGFP2 (Fig. 1C, Supplementary Fig. S2), a phenomenon probably associated with the shortened half-life of green fluorescent protein (GFP) when fused to endogenous proteins (Li et al. 1998; Corish and Tyler-Smith 1999). No phenotypic differences between independent lines expressing the biosensors and wild type were observed (Supplementary Fig. S3); lines with the highest fluorescence intensity were selected for subsequent experiments. In vivo fluorescence ratiometric imaging of leaves treated with H₂O₂, dithiothreitol (DTT) or untreated (rest) demonstrated the sensitivity of chl-roGFP2-PrxΔC_R and chl-roGFP2-Prx toward redox changes (Fig. 1D).

Figure 1.

Generation of potato plants expressing chl-roGFP2-Prx and chl-roGFP2-PrxΔC_R, and fluorescence detection using live imaging and full-spectrum acquisition. A) Schematic diagram of the gene cassette used to introduce chl-roGFP2, chl-roGFP2-Prx, and chl-roGFP2-PrxΔCR, and a proposed model illustrating the oxidation and reduction activities influencing the oxidation state of these biosensors. Figure created using BioRender (https://biorender.com/). B) Confocal images showing the colocalization of biosensors and chlorophyll fluorescence. C) Fluorescence intensities recorded in WT and chloroplast-targeted roGFP2 variants (chl-roGFP2, roGFP2-Prx, and chl-roGFP2-PrxΔC_R) at 405 and 465 nm excitation, with 515 nm emission. Data represent mean ± SE (n = 4). Statistical significance was determined using one-way ANOVA (P < 0.05). D) Ratiometric analysis conducted by dividing pixel-by-pixel fluorescence images taken following excitation at 405 and 465 nm during rest, under fully oxidized (1000 mm H₂O₂, 10 min) and fully reduced (100 mm DTT, 60 min) conditions. The same leaf underwent all three treatments. E) Simultaneous detection of biosensor and chlorophyll fluorescence emission by wavelength-resolved acquisition. Spectra derived from chl-roGFP2-PrxΔC_R emission are shown. The presented spectrum was derived from two spectrometers tuned at different sensitivity levels. F, G) Spectral analysis of chl-roGFP2-PrxΔC_R plants during rest, under fully oxidized and fully reduced conditions following excitation at 405 nm (F) and 465 nm (G). H to J) Fluorescence ratios (R_405/465) in plants expressing chl-roGFP2, chl-roGFP2-Prx, and chl-roGFP2-PrxΔCR during rest, under fully oxidized and fully reduced conditions. Data collected from 4 to 6 repeats is presented as box plots. WT, wild type; DTT, dithiothreitol.

Redox alterations in response to light were measured using a recently developed system that allows time-resolved and wavelength-resolved fluorescence measurement while applying red actinic light at varying intensities, including a burst of single turnover saturating flashes that “close” PSII reaction centers (Nanda et al. 2024). As shown in Fig. 1E, spectrally resolved data facilitated the capture of fluorescence emission at 500–550 nm emanating from chl-roGFP2-PrxΔC_R plants excited at 405 nm, as expected for enhanced green fluorescent protein-based biosensors. Notably, resolving the entire spectra allowed for the simultaneous capture of biosensor and chlorophyll fluorescence. Resolving the entire spectra also enabled assessing and subtracting the structured autofluorescence originating from plant pigments (Supplementary Fig. S4, See Methods). Dynamic ranges (R_405/465 for fully oxidized state divided by R_405/465 for fully reduced state) of 5–6 were recorded for chl-roGFP2-Prx, chl-roGFP2-PrxΔC_R, and chl-roGFP2 (Fig. 1, F to J), consistent with the performance of the biosensors in Arabidopsis plants (Lampl et al. 2022).

To analyze the dynamics of reducing and oxidative signals at various light intensities, dark-adapted biosensor-expressing plants were exposed to a series of incremental light intensities with a 2-fold change between two consecutive light steps (0, 20, 40, 80, 160, 320, and 640 µmol m⁻² s⁻¹), covering the low to high light intensity range (Tendler et al. 2018). Each illumination step lasted for 10 min to allow for short-term acclimation. Full spectra recording was used to measure biosensor signals and chlorophyll fluorescence-derived parameters under the exact conditions (Fig. 2, A to C, Supplementary Fig. S5). Saturating pulses were applied to measure photosystem II (PSII) operating efficiency (ΦPSII), nonphotochemical quenching (NPQ), and electron transport rate (ETR).

Figure 2.

Spectra capturing biosensor and chlorophyll fluorescence in response to increases in actinic light intensity. A to C) 3D plots displaying time-dependent spectra of chl-roGFP2 plants excited at 405 nm (A), 465 nm (B) for biosensor detection, and 600 nm (C) for chlorophyll fluorescence. D to G) Chlorophyll fluorescence-derived parameters, including normalized fluorescence (D), ΦPSII (E), NPQ (F), and ETR II (G) extracted from spectra analysis and following the application of saturating pulses. Light intensity is indicated in the bars. Values represent means (n ≥ 3) ± Se obtained from at least three independent experiments. Se values are represented by shaded areas. H to J) Biosensors ratio (405/465) values extracted from spectra, in response to light intensity increments. Values represent means ± Se obtained from at least three independent experiments. Shaded areas represent Se values. K) Linear relationships between electron transport and biosensor signals are represented as a heatmap. The scale bar refers to correlation coefficient values. L to O) Correlation between various photosynthetic parameters and chl-roGFP2-PRX-ΔC_R redox state. ΦPSII, quantum yield of photosystem II; NPQ, nonphotochemical quenching; ETR, electron transport rate.

Increasing light intensities induced rapid chlorophyll fluorescence peaks, reflecting the reduction of the primary electron acceptor Q_A of photosystem II (PSII), followed by adaptation of the photosynthetic machinery to the newly experienced light intensity either by induction of Qa oxidation or activation of NPQ (Fig. 2D). Decreasing ΦPSII values ranging from 0.81 to 0.56, at 20 µmol m⁻² s⁻¹ and 640 µmol m⁻² s⁻¹, respectively, accompanied by increasing NPQ values ranging from 0.08 to 0.75, were recorded (Fig. 2, E and F). As expected for light intensities below light saturation, increasing in ETR II, ranging from 6.8 to 150 at 20 µmol m⁻² s⁻¹ and 640 µmol m⁻² s⁻¹, respectively, was also documented (Fig. 2G).

The overall response of chl-roGFP2 and chl-roGFP2-Prx to the increasing light intensities up to 160 µmol m⁻² s⁻¹ was similar and involved a light-dependent reduction in the redox state. Higher light intensities induced chl-roGFP2-Prx oxidation, while no change occurred in chl-roGFP2 in response to 320 µmol m⁻² s⁻¹, followed by a slight increase in the last light step of 640 µmol m⁻² s⁻¹ (Fig. 2, H and I). No such pattern was observed in plants exposed to the same sequence of measurements but kept in darkness (Supplementary Fig. S6), emphasizing the light-dependency of the observed patterns. The reduction of chl-roGFP2-Prx under low-light conditions, which turns into oxidation when light intensities increase (Fig. 2I), reflects the predominant action of reductive signals in response to incremental light intensities in low to medium light intensity range and the higher activity of oxidative signals at higher light intensities. The different turning points toward oxidation observed in chl-roGFP2-Prx compared to chl-roGFP2 reflect the prevalence of Prx-associated oxidative signals at 320 µmol m⁻² s⁻¹, which attenuate the reduction activity of TRXs and GSH/GRXs.

A contrasting oxidation dynamic was observed for the chl-roGFP2-PrxΔC_R, which underwent oxidation as light intensities increased, showing a step increase in the generation of 2-Cys Prx-related oxidative signals at light intensities above 80 µmol m⁻² s⁻¹ (Fig. 2J). The difference between the patterns observed for chl-roGFP2-Prx and chl-roGFP2-PrxΔC_R may reflect the reductive activity of TRXs on 2-Cys Prx and demonstrated the higher sensitivity of chl-roGFP2-PrxΔC_R to oxidative signals. The observed chl-roGFP2-PrxΔC_R oxidation was likely attenuated by the reductive activity of the reduced form glutathione/glutaredoxins (GSH/GRXs), as observed in the chl-roGFP2 line (Fig. 2H). Accordingly, the chl-roGFP2-PrxΔC_R was normalized to the chl-roGFP2 (Supplementary Fig. S7, oxidation index [OI]), refining the oxidative activity of 2-Cys Prx. An increase in OI was observed in response to the incremental rise in light intensities in the low to medium light range (from dark to 320 µmol m⁻² s⁻¹), with no response in the last step (from 320 to 640 µmol m⁻² s⁻¹). These results demonstrate the generation of 2-Cys Prx-related oxidative signals at habitual light intensities, and the oxidation of the GSH pool at moderate to high intensities. In light of the suggested inhibitory effects of 2-Cys Prx-related oxidative signals on Calvin-Benson cycle enzymes (Lampl et al. 2022), these results highlight the limitations of photosynthesis imposed by oxidative signals in one of the world’s most important crops.

Electron flow to photosystem I (PSI) was shown to be the prominent source of H₂O₂ in chloroplasts, with the acceptor-side capacity of PSI suggested to regulate the rate of superoxide formation through the Mehler reaction (Mehler 1951). Notably, fully reduced PSI acceptors and overwhelming antioxidant pathways have been suggested to play a role in chloroplast-derived ROS signaling resulting from H₂O₂ accumulation (Fitzpatrick et al. 2022). Nonetheless, the quantifiable oxidative signals under light levels below stressful levels suggest that while H₂O₂ export from the chloroplast only occurs under stress conditions associated with PSI damage, oxidative signals transmitted to target proteins through 2-Cys Prx are delivered “below the radar” of the antioxidant systems. Notably, the exceptional catalytic efficiency of 2-Cys Prx, reaching up to 10⁸ M⁻¹ s⁻¹, enables the transmission of oxidative signals even in the presence of a highly efficient antioxidant system. This is demonstrated by the higher sensitivity of chl-roGFP2-PrxΔC_R to light-induced oxidative signals compared to chl-roGFP2.

The tight association between the electron transport rate and generation of oxidative signals was further demonstrated by the strong correlation between ETR and chl-roGFP2-PrxΔC_R oxidation state (r = 0.91, Fig. 2, K to O). A significant negative correlation (r = −0.73) was also found between chl-roGFP2 and ETR (Fig. 2K). These results demonstrate that reductive and oxidative signals are tightly connected to electron transfer. On the other hand, the unique dynamics of chl-roGFP2-Prx redox state (Fig. 2I), which was dominated by reductive activity under low light and controlled by oxidative activity under high light intensities, results in no correlation between chl-roGFP2-Prx redox state and ETR (Fig. 2K).

The tight correlation between the reductive and oxidative activity and ETR demonstrates that either the activation of CBC enzymes by reduction or their inhibition by oxidation is initiated by the electron transport chain, with increasing light intensities amplifying both signals. While it is possible that the dominant reductive activity largely attenuates the oxidative signals at a steady state, differences in the kinetics of both signals can introduce a new layer of regulation during the light transition, underlining the importance of oxidative signals. This context-dependent modulation can be harnessed to achieve precise responses to changes in light intensities.

Taken together, parallel quantification of chlorophyll and genetically encoded redox biosensors fluorescence using spectrally resolved emission data highlights a strong correlation between reductive and oxidative signals and electron transport rates, indicating the simultaneous generation of light-dependent opposing signals. Given the importance of redox signaling in dictating photosynthetic efficiency, integration between classical spectroscopic photosynthetic measurements and genetically encoded biosensors is likely to provide perspective on how light reactions are regulated under dynamic environmental conditions.

Author contributions

M.H., N.L., R.L., and S.R. performed the research and analyzed the data. S.R. wrote the manuscript with all authors providing valuable feedback and comments on the draft.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. Confocal laser scanning micrographs of chloroplast-targeted chl-roGFP2, roGFP2-Prx, and chl-roGFP2-PrxΔC_R.

Supplementary Figure S2. Pixel intensity distribution in wild-type and transgenic potato leaves.

Supplementary Figure S3. Phenotypic comparison of wild-type (WT) and transgenic potato plants expressing biosensor variants under different light intensities.

Supplementary Figure S4. Fluorescence spectra derived from WT plants excited at 405 nm.

Supplementary Figure S5. A diagram of the measuring unit and measurement frequency applied in Fig. 2.

Supplementary Figure S6. Biosensors redox state under constant dark control.

Supplementary Figure S7. Changes in the oxidation index in response to light intensity increments.

Supplementary Materials and Methods.

Funding

This research was supported by the European Research Council (ERC-COG, AGRIREDOX, grant no. 101086608) and the Israel Science Foundation (grant no. 1779/21).

Data availability

All data supporting the findings of this study are available within the figures of the published article.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• BAS1 Gramene: AT2G26710

• BAS1 Araport: AT2G26710