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. Author manuscript; available in PMC: 2026 May 1.
Published in final edited form as: Physiol Plant. 2025 May 1;177(3):e70242. doi: 10.1111/ppl.70242

A minute-scale P515 kinetic traces light-driven proton gradient formation in dark-incubated Synechocystis sp. PCC 6803

Milena Zhivkovikj 1,#, Marcel Dann 1,2,*,#, Maysoon Noureddine 1, Martin Lehmann 1, Dario Leister 1,*
PMCID: PMC7617733  EMSID: EMS205976  PMID: 40302127

Abstract

The light-driven formation of a proton-motive force (pmf) across thylakoid membranes is crucial for ATP synthesis and photosynthesis in chloroplasts and cyanobacteria. Cyclic electron flow (CEF) around photosystem (PS) I is hypothesized to be a key contributor to pmf formation, but direct observation of CEF in vivo remains a major challenge. As one possible proxy, pmf formation can be measured on a millisecond scale using electrochromic shifts (ECS) of thylakoid pigments conventionally observed in plants through absorbance changes at a wavelength of 515 nm (P515). In this study, we describe a new P515 signal in the model cyanobacterium Synechocystis sp. PCC 6803 which can be observed on a time-scale of seconds to minutes upon red actinic light treatment. Treatments with uncouplers of electrochemical gradients and inhibitors of the photosynthetic electron transport chain indicate that the signal primarily traces proton gradient formation across the thylakoid membrane and suggest a major ECS contribution, but its precise origin remains to be deciphered. Still, the measuring routine was found to allow for phenotypic distinction between mutants with altered capacities for NDH-and PGR5-dependent CEF around PSI, thus enabling future research on both CEF pathways and photosynthetic trans-thylakoid pmf formation.

Introduction

Oxygenic photosynthesis in most cyanobacteria and all photosynthetic eukaryotes encompasses the light-driven production of both NADPH and ATP in the thylakoid membrane system. As thylakoids and the photosynthetic machinery they house have changed little in functional terms from their evolutionary origins in early cyanobacteria to their most derived phylogenetic descendants in modern plant chloroplasts, oxygenic photosynthesis is often studied back-to-back in both plants and cyanobacteria(Nelson & Ben-Shem, 2004). In the past decade, the transfer of plant-like photosynthetic machinery and biosynthetic modules into cyanobacteria for use as synthetic biology chassis has been extensively discussed as a means of studying and engineering (crop) plant photosynthesis (e.g Dann & Leister, 2017; Hitchcock et al., 2022). An ideal proxy system will comprise a plant-type photosynthetic apparatus including plant-type xanthophyll cycling (Demmig-Adams & Adams, 1996) being established within cyanobacteria (Lehmann et al., 2021), thus necessitating a suitable measurement routine for its functional characterization. Carotenoid interconversion can readily be traced by HPLC analyses of cellular pigment extracts (e.g., Schubert et al., 1994), but a non-invasive and time-resolved method for its in vivo assessment would be preferable. In plants, actinic-light induced absorbance changes at 505 nm wavelength (ΔA505 nm) on a minutes-timescale have long been used to trace zeaxanthin accumulation in leave material (Bilger et al., 1989; Ruban et al., 1993), but to our knowledge, no such experimental reports exist for cyanobacteria – likely due the lack of xanthophyll cycling. Another avenue towards such in vivo assessment can potentially be found in seconds- to minute-scale P515 spectroscopic analysis under red actinic light illumination, tracing the P515 signal through differential changes in absorbance at 550 nm and 515 nm wavelengths with the latter actually being measured at 520 nm (i.e., ΔA550-520 nm) (Schreiber & Klughammer, 2008). In such measurements, a slow kinetic of P515 in tobacco has been attributed to cellular accumulation of zeaxanthin in the wake of xanthophyll cycle activity (Schreiber & Klughammer, 2008) likely relating to changes in proton motive force (pmf) indirectly through trans-thylakoid pH gradient (ΔpH)-dependent violaxanthin de-epoxidase activation in the thylakoid lumen (Bratt et al., 1995; Wilson et al., 2021). Such P515 changes occur in conjunction with thylakoid membrane energization increasing light scattering at 535 nm (Deamer et al., 1967), presumably through altering association of xanthophylls with LHCII (Ruban et al., 1992; Ruban et al., 1993), thus rendering the P515 signal arising from minute-timescale illumination of plant leaves hard to interpret. Surprisingly, we could observe a similar light-induced P515 kinetic in Synechocystis despite the evident lack of any inherent zeaxanthin production through xanthophyll cycling and LHCII. The corresponding P515 kinetic proved highly susceptible to proton gradient uncoupler treatment, as well as inhibitory and mutational disruption of the photosynthetic electron transport chain. As no light-dependent increase of cellular zeaxanthin levels was detected over the course of the measurement routine, a different cause is likely to underlie the observed effect. Thus, the presented measurement routine may allow for non-invasive tracing of proton gradient formation by proxy, while at the same time casting doubt on previous interpretations of the corresponding signal and its origins in plants.

Material and Methods

Cell growth and sample preparation

Glucose-tolerant Synechocystis sp. PCC 6803 cells were kindly provided by Himadri Pakrasi (Washington University, St. Louis, USA). Cells were initially cultivated in BG11 medium without added glucose as 50 ml precultures for each genotype at 30°C, under 40 µmol photons m-2 s-1 fluorescent white light and agitated at 150 rpm. Culture growth was monitored by measuring the optical density at a wavelength of 730 nm (OD730) of cells in 1 cm cuvettes using an Ultrospec 2100 pro UV/Visible photometer (Biochrom Ltd., Cambridge, United Kingdom). Precultures were used to inoculate 100-200 ml cultures at an initial OD730 of 0.05 into 500 ml silicone-capped Erlenmeyer flasks and maintained under identical growth conditions. Cells were harvested during the exponential growth (OD730 between 0.4 and 0.8) by centrifugation at 3,500 rpm (Eppendorf Centrifuge 5430 R) for 10 min at room temperature, using 50 ml Falcon tubes. The cell pellets were washed twice with BG11 medium and resuspended in BG11 to an OD730 of 2.0 in 2 ml to approximate normalization to cell density. The cell suspensions were incubated overnight in the dark (>16 h), agitated at 150 rpm.

DUAL-PAM 100 P515 measurements

P515 measurements were performed using a Dual PAM100 fluorospectrometer (Walz, Effeltrich, Germany) equipped with a P515/535 module. This module measures P515 as the ΔA550 – 520 nm reflecting ECS, and the absorbance at 535 nm, reflecting membrane energization (Schreiber & Klughammer, 2008). Dark-incubated (>16 h) cell suspensions (2 ml) were transferred into a 10 mm pathlength quartz cuvette and subjected to variations of the following DUAL-PAM protocol: 15 s dark OR 600 s dark > 600 s red actinic light (216 µmol m2 s-1; 635 nm) > 125 s dark OR 135 s dark. Prolonged dark steps were applied in uncoupler treatment experiments, shorter dark steps were applied in all other experiments. Data was collected at an acquisition rate of 1024000/20 ms-1 at measuring light intensity 5. Inhibitors were added to the cell suspensions at the following concentrations: 3-(3,4-dichlorophenyl)-1,1-dimethylurea [DCMU] (10 µM), methyl viologen [MV] (125 µM), 2,5-dibromo-3-methyl-6- isopropyl-p-benzoquinone [DBMIB] (10 µM), and HgCl2 (10 µM). The cell suspensions were incubated with each inhibitor for 2 min before measurement, except for MV, which required a 10 min incubation period. The electrochemical gradient uncouplers carbonyl cyanide 3-chlorophenylhydrazone [CCCP] (250 µM) and valinomycin [Val] (10 µM) (both corresponding to concentrations used by Miller and coworkers for ΔpH tracing through acridine orange fluorescence (Miller et al., 2021) were added to the cell suspension within the measuring cuvette five minutes before onset of red actinic light illumination to observe changes in the dark baseline. Average spectra of n = 3 BG11 media control measurements were subtracted, and spectra were baseline corrected for average values of initial dark steps equaling 0.

Pigment analysis

For the LC-MS/MS analysis of zeaxanthin (Zea) and chlorophyll a (Chl a) content, methanolic extracts of dark-incubated (>16 h) samples (OD730 2) were prepared before and after exposure to the light protocol (Zavrel et al., 2015). The dry pellet was resuspended in 100 µL MeOH. Pigments were analysed using a Dionex Ultimate 3000 UHPLC with diode array detector (DAD) (Thermo Fisher Scientific), in combination with a timsTOF mass spectrometer (Bruker).

A 5-µL injection volume at a 500 µL min-1 flow rate was used on a C30 reversed-phase column (Acclaim C30, 3 µm, 2.1 x 150 mm, Thermo Fisher Scientific) at 15°C. Solvents were (A) acetonitrile and (B) a methanol-ethyl acetate mix (50/50 v/v) with 0.1% formic acid. The gradient started at 14.5% B, increased to 34.5% B within 15 min, maintained for 10 min, then returned to 14.5% B for 5 min re-equilibration. Detection was by electrospray ionizstion (ESI) in positive mode, with nitrogen as the dry gas at 8 L min-1, 8 bar and 200°C. Mass spectra were acquired in MS mode from 50 m/z to 1300 m/z with 40,000 resolution, a 1 Hz scan speed, and 0.3 ppm mass accuracy. Compounds were identified using reference standards or DAD data and specific mass (m/z) in relation to retention time and isotopic pattern, and relative pigment quantification was performed as previously described (Lehmann et al., 2021; Dann et al., 2021). Data was collected with otofControl 4.0 and analysed with DataAnalysis 5.3.

Results

Long-term dark incubated Synechocystis cells accumulate a proton-gradient associated P515 signal upon exposure to red actinic light

Synechocystis wildtype (WT) cells dark-incubated overnight and subsequently subjected to 216 µmol photons m-2 s-2 of red actinic light (AL; 635 nm) displayed a distinct P515 signal accumulation kinetic (Fig 1A) closely resembling those described for long-term AL exposed tobacco leaves (Schreiber & Klughammer, 2008). The signal proved highly susceptible to singular treatment with the proton gradient uncoupler CCCP, but largely insusceptible to singular treatment with the potassium cation (K+) gradient uncoupler valinomycin (Fig 1B). This indicates primary dependence on trans-thylakoid proton gradient formation and thus the ΔpH component of the pmf. CCCP treatment caused a rapid decline in dark P515 signal upon addition to the cell suspension (inhibitor injection indicated by black arrowhead; Fig 1 B, C), resulting in a lowering of the P515 dark baseline value before onset of illumination. Meanwhile, valinomycin injection caused a miniscule downward shift in dark P515 baseline (Fig 1C), indicating that the K+ gradient does not contribute much to the observed dark signal. Combinatory treatment with CCCP and valinomycin, meanwhile, caused a super-additive decline in dark P515 (Fig 1C). Upon onset of AL, CCCP-treated cells displayed a rapid decline in P515 below the dark signal baseline, which was exacerbated by combinatory treatment with valinomycin (Figure 1B). Thus, the observed signal does not appear to be fully depleted in the dark, and light-driven processes appear to enhance or further diminish it in the absence and presence of a proton gradient uncoupler, respectively. Finally, the accumulated P515 signal showed a very slow decay upon offset of AL (Fig 1A, B), indicating slow re-equilibration the causative agent underlying light-driven P515 signal accumulation. This notion is substantiated by a near-complete absence of (additional) AL-induced P515 accumulation in previously light-incubated cells (Fig 1A inset).

Figure 1. Dark-incubated Synechocystis WT cells display a pronounced P515 kinetics under red actinic light.

Figure 1

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A, P515 spectra of dark-incubated (>16 h) Synechocystis WT cells exposed to 216 µmol photons m-2 s-1 of red actinic light (AL; wavelength λ = 635 nm). On- and offset of AL exposure is indicated by arrowheads. Inset: Analogous P515 spectra of growth-light incubated WT cells. B, P515 spectra of dark-incubated Synechocystis WT cells exposed to 216 µmol photons m-2 s-1 of AL after addition of the proton gradient uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the K+ gradient uncoupler valinomycin (Val). C, P515 signal response to addition of CCCP and/or Val in the dark (enlarged section of P515 spectra as indicated in (B)). On- and offset of AL exposure and inhibitor injection are indicated by arrowheads as labelled. Graphs shown represent averages (lines) ± standard deviations (shaded areas) for A: n = 9, inset: n = 6, B and C: n = 5 biological replicates.

AL-driven P515 signal accumulation does not stem from zeaxanthin accumulation

To assess light-driven zeaxanthin accumulation as a possible cause for changes in P515 signal, we compared cellular zeaxanthin levels of untreated and CCCP/valinomycin treated WT cells before and after subjection to the AL illumination protocol. 10 minutes of AL treatment did not increase cellular zeaxanthin-to-chlorophyll ratios in either sample but rather resulted in an average decrease of 7,8% (p = 0.003) and 10,4% (p = 0.0007) in relative zeaxanthin abundance in untreated and CCCP/valinomycin treated WT, respectively (Fig 2). A significant decline in cellular zeaxanthin relative to chlorophyll a was observed in WT treated with CCCP/Val previous to AL onset (-6.1%; p = 0.04), indicating the trans-thylakoid electrochemical gradient to affect cellular zeaxanthin levels in the dark (Fig 2A). Cellular chlorophyll a levels remained unaltered among all samples, while average cellular zeaxanthin levels declined due AL treatment (-15.1%; p= 0.14), Val/CCCP treatment (-15.0%; p = 0.12) and combinatory treatment (-24.6%; p = 0.007). Still, consistent AL-driven zeaxanthin depletion in both AL control and CCCP/Val-treated samples suggests a proton- and K+-gradient independent cause to the observed light-driven P515 kinetics, corroborating that the slow rise in Synechocystis P515 under AL does not stem from light-induced cellular accumulation of zeaxanthin.

Figure 2. Red actinic light treatment during P515 measuring routine does not increase Synechocystis zeaxanthin-to-chlorophyll ratios.

Figure 2

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A, Cellular zeaxanthin (Zea)-to-chlorophyll a (Chl a) ratios represented as signal intensity quotients of peaks corresponding to zeaxanthin and Chl a for n = 5 biological replicates, respectively. Methanolic extracts of cell suspensions (OD730nm = 2.0) of Synechocystis WT were analysed by HPLC/MS. Extracts were obtained from dark incubated (>16 h) cells or from cells that were dark-incubated and then subjected to 10 minutes of red actinic light (λ = 635 nm) for P515 measurement. Data is presented as box plots with medians (bold horizontal lines), 25th and 75th percentiles (boxes), and whiskers extending 1.5× the interquartile range. Averages are indicated by “+” signs and are provided above the corresponding box plots. pre/post signifying samples taken prior to and after red actinic light treatment, respectively. B, HPLC/MS signal intensity representing cellular abundance of Zea and Chl a derived from the same samples presented in (A). Columns represent averages, error bars represent standard deviations. Individual data points are indicated. C, Representative UV/Vis absorbance spectra of eluate fractions identified as Zea and Chl a prior to MS injection.

Letters signify statistically significant differences with p ≤ 0.05 according to post-hoc Bonferroni-Holm simultaneous comparison of all measurements after significant among-group differences were detected by one-factorial ANOVA (two-sided). Val, valinomycin; CCCP, carbonyl cyanide 3-chlorophenylhydrazone.

Effects of electron transport chain inhibitors on AL-induced P515 kinetics

Light-driven P515 accumulation was indicative of involvement of the photosynthetic electron transport chain (ETC) in shaping the observed signal kinetics. To detail the relative contributions of different ETC factors, we performed a series of ETC inhibitor treatments on WT cells prior to AL exposure and found P515 kinetics to be sensitive to various disruptions of the photosynthetic ETC (Fig 3). Suppressing light-driven plastoquinone (PQ) pool reduction through PSII by treatment with 10 µM 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to abolish AL-induced P515 signal accumulation (Fig 3A). Treatment with 125 µM methyl viologen (MV) acting as competitive electron acceptor for PSI was found to suppress the late phase of the signal development (Fig 3A). Treatment with 10 µM 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) inhibiting the Cyt b6f complex through blocking of the Qo site of subunit IV was found to suppress the early phase of the signal (Fig 3A). This indicated that PSII water splitting and subsequent linear electron transport through Cyt b6f are essential for initial AL-driven P515 signal accumulation in Synechocystis WT (early phase, approximately 0-5 min AL treatment). Meanwhile, later P515 signal accumulation (late phase, approximately 5-10 min AL treatment) appears to co-depend on PSI activity and thus possibly CEF. The latter was corroborated by abolition of late phase P515 signal accumulation through cell treatment with 10 µM mercury chloride (Hg2+; Fig 3A), a potent inhibitor of the NDH-1 complex (Mi et al., 1992) through which an estimated 80% of Synechocystis CEF around PSI is directed (Yeremenko et al., 2005). These observations are thus in line with expectations towards a signal tracing proton gradient formation across the thylakoid membrane as a result of photosynthetic electron transport. Accumulated P515 signal remained stable in the dark in control samples, while minor drops in signal intensity could be observed upon offset of AL illumination in DCMU, MV, and Hg2+ treated cells. Meanwhile, DBMIB caused complete abolition of P515 signal decay after light-to-dark transition (Fig 3B).

Figure 3. Synechocystis WT P515 signal accumulation under red actinic light is differentially affected by ETC inhibitor treatment.

Figure 3

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A, P515 spectra of dark-incubated (>16 h) Synechocystis WT cells treated with electron transport chain inhibitors prior to exposure to 216 µmol photons m-2 s-1 of red actinic light (AL; wavelength λ = 635 nm). Dashed lines indicate respective spectra dark baselines. B, Continuation of P515 spectra upon offset of AL. On- and offset of AL exposure are indicated by arrowheads as labelled. Graphs shown represent averages (lines) ± standard deviations (shaded areas) for n = 9 biological replicates, except for MV and DBMIB where n = 8 and n = 6, respectively. P515 absorbance spectra are presented in units ΔI/I ×103 of 550 – 520 nm difference signal (see scale bar at top left).

Effects of genetic disruption of CEF pathways on AL-induced P515 kinetics

CEF around PSI has long been hypothesized to be a major contributor to trans-thylakoid proton gradient and thus pmf formation (Tagawa et al., 1963; Wang et al., 2015). A lack of non-invasive measuring techniques directly probing or tracing CEF in vivo is still obstructing the study of this alternative photosynthetic electron pathway, however. To scrutinize the applicability of the described P515 kinetics as a probe for light-driven proton gradient formation, we examined a set of previously established CEF mutant strains with altered PGR5-dependent CEF activity (Dann & Leister, 2019; Yeremenko et al., 2005) and disrupted NDH-CEF (Ogawa, 1991) for expected changes in P515 signal formation (i.e., impaired P515 signal accumulation in CEF-deficient mutants, and increased P515 signal accumulation in CEF-stimulated mutants).

In both single Δpgr5 and ΔndhB mutants, AL-driven P515 signal accumulation was severely impaired, and in ΔndhB Δpgr5 double knockout mutants, near-complete signal abolition highly similar to Hg2+-treated WT cells was observed (Fig 4A). Intriguingly, the Δpgr5 single mutant displayed an early depression of the AL-induced P515 signal below dark-baseline levels, as well as a reproducible transient P515 signal increase upon offset of AL exposure, setting it apart from both WT and ΔndhB mutants (Fig 4A, B).

Figure 4. CEF-deficient Synechocystis mutants show compromised P515 signal accumulation under red actinic light.

Figure 4

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A, P515 spectra of dark-incubated (>16 h) Synechocystis mutants compromised in PGR5-dependent (Δpgr5) and/or NDH-dependent (ΔndhB) cyclic electron flow (CEF) around photosystem I exposed to 216 µmol photons m-2 s-1 of red actinic light (AL; wavelength λ = 635 nm). Dashed lines indicate respective spectra dark baselines. B, Continuation of P515 spectra upon offset of AL. On- and offset of AL exposure are indicated by arrowheads as labelled. Graphs shown represent averages (lines) ± standard deviations (shaded areas) for n = 9 biological replicates of WT and Δpgr5, and n = 5 biological replicates of ΔndhB and Δpgr5 ΔndhB, respectively. P515 absorbance spectra are presented in units ΔI/I ×103 of 550 – 520 nm difference signal (see scale bar at top left).

The Δpgr5-specific early P515 depression could not be observed in any other mutant strain, and no analogous effect could be observed upon treatment of WT with ETC inhibitors. However, it resembled an inversion of the residual early P515 spike observed in Δpgr5 ΔndhB (Fig 4A) which could also be observed in WT+MV but was abolished in WT+DCMU and WT+DBMIB (Fig 3A), indicating a possible correspondence to PSII-LEF activity through Cyt b6f. To further investigate this Δpgr5-specific phenomenon, we assessed the isolated and combined effects of DCMU and MV on Δpgr5 mutant cells. We found the early AL-induced P515 depression to be insensitive to MV, but sensitive to DCMU (Fig 5A), thus corroborating the postulated PSII-dependency. Similarly, the transient P515 rise post AL offset was suppressed by DCMU treatment but persisted upon MV treatment (Fig 5B). Moreover, MV treatment of Δpgr5 resulted in AL-induced P515 signal depletion, similar to but less severe than that observed in WT treated with CCCP (Fig 5A; compare Fig 1B). Meanwhile, combinatory treatment of Δpgr5 with DCMU and MV recapitulated the effect of DCMU, indicating PSII activity and corresponding PQ pool reduction to underlie the observed phenomena.

Figure 5. P515 signal depression upon red actinic light onset and increase upon actinic light offset in Synechocystis Δpgr5 mutants is sensitive to DCMU treatment.

Figure 5

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A, P515 spectra of dark-incubated (>16 h) Synechocystis Δpgr5 cells exposed to 216 µmol photons m-2 s-1 of red actinic light (AL; wavelength λ = 635 nm) treated with electron transport chain inhibitors prior to AL exposure. Dashed lines indicate respective spectra dark baselines. B, Continuation of P515 spectra upon offset of AL. On- and offset of AL exposure are indicated by arrowheads as labelled. Graphs shown represent averages (lines) ± standard deviations (shaded areas) for n = 9 biological replicates. P515 absorbance spectra are presented in units ΔI/I ×103 of 550 – 520 nm difference signal (see scale bar at top left).

Overexpression of pgr5 renders AL-induced P515 signal accumulation insensitive to ETC inhibitors

The unique effects observed upon mutational disruption of pgr5 were considered highly intriguing and commanded further investigation. An opposite effect on AL-induced P515 signal accumulation was expected to occur upon overexpression of pgr5, and indeed, a pgr5 overexpression strain (pgr5OE) showed rapid formation and early saturation of its P515 signal (Fig 6A). Surprisingly, ETC inhibitor treatments were found not to affect pgr5OE cells in the same way as WT material. Indeed, we observed a linear P515 signal rise to be maintained in pgr5OE cells upon treatment with DCMU, while MV suppressed late-stage signal accumulation to a lesser degree than in WT (Fig 6A; compare Fig 3A). Similarly, Hg2+ treatment failed to suppress AL-induced P515 signal accumulation, particularly in later phases of the kinetics, while the early-stage effects of DBMIB were entirely suppressed in pgr5OE, resulting in a continuous and linear P515 signal accumulation (Fig 6A). Near-complete suppression of P515 signal accumulation was achieved by combinatory treatment with DCMU and MV, indicating that linear P515 signal accumulation in presence of DCMU is likely dependent on electron flow through PSI (Fig 6A). Finally, P515 signal decay upon light-to-dark transition was found enhanced in untreated pgr5OE cells (Fig 6B). Surprisingly, this effect was suppressed by all ETC inhibitor treatments (Fig 6B), thus indicating a dependence of fast PGR5-mediated dark decay of P515 on an otherwise undisrupted photosynthetic electron transport chain. In summary, these observations indicate a stabilizing effect of increased cellular pgr5 gene expression levels on AL-induced P515 signal accumulation even upon disruption of the photosynthetic ETC through inhibitors. The differential effects of NDH-CEF and PGR5-CEF disruption on the observed P515 signal moreover indicate its potential suitability for the detailed study of CEF effects in vivo.

Figure 6. Mutational stimulation of PGR5-CEF in Synechocystis renders P515 signal accumulation under red actinic light insensitive to ETC inhibitors.

Figure 6

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A, P515 spectra of dark-incubated (>16 h) Synechocystis cells overexpressing the endogenous pgr5 gene (pgr5OE, Dann and Leister, 2019) exposed to 216 µmol photons m-2 s-1 of red actinic light (AL; wavelength λ = 635 nm) treated with electron transport chain inhibitors prior to AL exposure. Dashed lines indicate respective spectra dark baselines. B, P515 signal decay in inhibitor-treated pgr5OE cells upon offset of AL exposure. On- and offset of AL exposure are indicated by arrowheads as labelled. Graphs shown represent averages (lines) ± standard deviations (shaded areas) for for n = 9 biological replicates, except DCMU where n = 8, and DBMIB and Hg2+ where n = 4, respectively. P515 absorbance spectra are presented in units ΔI/I ×103 of 550 – 520 nm difference signal (see scale bar at top left).

Discussion

Our findings show that a hitherto undescribed AL-induced P515 kinetic can be observed in Synechocystis on a seconds- to minutes-timescale after prolonged dark incubation. The observed signal falls into the spectral range of electrochromic shift (ECS) of photosynthetic pigment absorbance in response to changes in the electric field across the thylakoid membrane as described in plants (i.e., 515 – 520 nm) (Junge & Witt, 1968) although ECS in Synechocystis was recently described to mostly occur at shorter wavelengths of 480 – 485 and 500 – 505 nm, respectively (Viola et al., 2019). The same study also reported a broader ECS peak at 510 – 540 nm, with an amplitude comparable to the established ECS readout regions (480-485 nm and 500-505 nm), however. This peak may correspond to the here-described P515 signal whose pronounced CCCP sensitivity (Fig. 1) strongly indicates a substantial contribution of ECS. The observed kinetic superficially resembles long-term AL illumination effects on P515 observed in tobacco leaves previously attributed to xanthophyll-cycle related zeaxanthin accumulation (Schreiber & Klughammer, 2008) which likely mirrors AL-driven zeaxanthin accumulation described in Arabidopsis thaliana (Wilson et al., 2021). In Synechocystis, however, no xanthophyll cycling has been described to date. Instead, the AL-induced P515 appears to trace changes in ΔpH formation through a different mechanism, as suggested by light-induced signal depletion below dark-baseline levels through CCCP (Fig 1), and since no corresponding rises in cellular zeaxanthin-to-chlorophyll ratio could be observed in response to illumination (Fig 2). Pronounced CCCP sensitivity of the P515 signal intensity in the dark (Fig 1B, C) is consistent with a trans-thylakoid proton gradient being upheld in the dark through, e.g., respiratory activity (Lea-Smith et al., 2013). Potassium (K+) has been proposed to act as a major counterion to H+ in the thylakoids of spinach (Tester & Blatt, 1989) and facilitation of its membrane transversion through the thylakoid K+ channel SynK has been shown to promote ΔpH formation in cyanobacteria (Checchetto et al., 2012; Zanetti et al., 2010). Miniscule Val sensitivity of P515 implies a low overall contribution of the trans-thylakoid K+ gradient to the measured P515 signal, however (Fig 1B, C). Still, combinatory treatment of WT with CCCP and Val resulted in an exacerbation of the effect of CCCP treatment both in the dark and under AL exposure (Fig 1B, C). This may suggest that effects of uncoupling of the K+ gradient are largely counteracted by compensatory H+ movement and raises questions regarding the effects of further counterion gradients such as Mg2+ (Barber et al., 1974; Chow et al., 1976; Pohland et al., 2024) on the measured P515 signal. Still, under the described experimental conditions (i.e., after prolonged dark incubation of >16 hours), the observed P515 signal appears to trace the electric field across the thylakoid membrane, with proton gradient formation being the primary contributor. Cells previously incubated under 50 µmol photons m-2 s-1 of white fluorescent light did not accumulate P515 signal over the course of the measuring routine (Fig 1A), possibly due to a steady-state trans-thylakoid proton gradient already being established at the time of AL onset. The accumulated P515 signal displayed pronounced dark persistence for several minutes in upon offset of AL treatment (Fig 1) may suggest such a gradient to persist through 15 seconds of dark baseline recording for AL-acclimated cells. This suggests a relatively low rate of dark depletion of ΔpH as compared to plant chloroplasts (near-complete depletion within 5 minutes (Heldt et al., 1973) likely due to respiratory activity in cyanobacterial thylakoids. Alternatively, an enzymatic conversion of a yet unidentified causal pigment giving rise to the observed P515 signal may not be reversed at a high rate in the dark.

Different inhibitor treatments revealed distinct effects of ETC disruption on AL-induced P515 signal accumulation. For the first ~300 seconds after AL onset, the observed P515 kinetic appears to largely depend on LEF (via proton gradient formation by PSII water splitting and Cyt b6f Q-cycle) and NDH activity (Fig 3A), whereas PSI activity appears to maintain and increase P515 signal accumulation from ~300 seconds past AL onset on, possibly due to CEF activity around PSI. The suppression of early-phase and recovery of later-phase P515 signal accumulation in DBMIB-treated cells (Fig 3A) may indicate a switch between LEF and CEF as primary contributor to P515, possibly due to a larger capacity of CEF to drive proton gradient formation. A possible mechanism underlying such a switch may lie in PSII- (and possibly NDH-) dependent plastoquinone pool overreduction driving the reduction of Q0-bound DBMIB to its quinol form, allowing for a subsequent replacement of DBMIB-OH by PQH2 (Velthuys, 1981). This may result in partial reconstitution of LEF and CEF capacities, resulting in increased proton gradient formation and subsequent rise in P515 signal. Alternatively, a delay in the onset of large-scale CEF activity may be due to delayed formation of a PSI-NDH supercomplex (Gao et al., 2016). The apparent co-dependence of early P515 signal accumulation on LEF and NDH activity warrants further investigation, although it parallels previous findings obtained through acridine-orange based tracing of thylakoid lumen acidification (Miller et al., 2021).

CEF around PSI is hypothesized to contribute to trans-thylakoid proton gradient formation in both plants (Joliot & Johnson, 2011; Shikanai & Yamamoto, 2017) and cyanobacteria (Miller et al., 2021; Nogales et al., 2012), presumably helping to poise ATP:NADPH ratios for efficient CO2 fixation (Allen, 2002). To assess the applicability of the presented measurement routine to direct investigation of CEF-dependent ΔpH formation in Synechocystis, P515 analyses were performed on mutants with disrupted NDH- and PGR5-dependent CEF pathways. Notably, for mutational CEF overstimulation, PGR5-dependent CEF represented the only practical target, as one single protein (PGR5), encoded by ORF ssr2016, needs to be overexpressed (Dann and Leister, 2019; Yeremenko et al., 2005), while the CEF-involved NDH complex isoforms NDH-1L and NDH-1L’ comprise at least 16 structural subunits (Battchikova et al., 2005; Gao et al., 2016b; Prommeenate et al., 2004; Zhang et al., 2014). CEF mutant analyses revealed a strong effect of the ΔndhB mutation especially on early phase P515 signal accumulation, while a combination of ΔndhB and Δpgr5 mutations abolished AL-induced P515 signal buildup following the initial PSII-dependent rise, thus strongly resembling the effect of Hg2+ treatment on WT cells (Fig 3A, Fig 4A). This may underpin the relevance of CEF for trans-thylakoid proton gradient formation and generally aligns with a reportedly high relative importance of the NDH-route to overall CEF activity in Synechocystis WT (Miller et al., 2021; Yeremenko et al., 2005). For PGR5-CEF, P515 analyses indicated a pronounced depression of P515 signal accumulation formation in Δpgr5 (Fig 4A), and an inverse effect upon PGR5 overexpression (Fig 6A), resulting in accelerated attainment of maximum P515 signal intensity. While generally aligning with P700-derived implications for PGR5-CEF activity in Synechocystis (Dann and Leister, 2019), the severe P515 phenotype observed in Δpgr5 as compared to ΔndhB is puzzling given the presumably low relative contribution of PGR5 to overall CEF in Synechocystis (estimated ~20% of overall CEF; (Yeremenko et al., 2005). However, in the same study, growth of the pgr5 knockout mutant was found to be more severely impaired under elevated light intensities of 200 µmol photons m-2 s-1 than that of ndhB deficient mutants, while the opposite was true under low and intermediate light intensities of 15 and 50 µmol photons m-2 s-1, respectively (Yeremenko et al., 2005). This may indicate an increased physiological significance of PGR5-CEF under light intensities close to the AL intensity applied in this study (i.e., 216 µmol photons m-2 s-1). The light-induced drop of the Δpgr5 P515 signal below the dark baseline (Fig 4, 5) is puzzling, resembling the effect of proton-gradient uncoupler treatment on WT cells (Fig 1), with MV treatment of Δpgr5 resulting in severe P515 signal depletion in the later (i.e., presumably PSI-dependent) phase of the kinetic (Fig 5). This may suggest that in presence of NDH but absence of PGR5 light-induced proton extrusion from the thylakoid system can take place, possibly paralleling light-induced proton extrusion from the cytoplasmic membrane (Katoh et al., 1996; Sonoda et al., 1997) mediated through an unknown mechanism.

As expected, overexpression of the pgr5 gene strongly affected AL-induced P515 signal accumulation kinetics. The accelerated attainment of maximum P515 signal intensity might correspond to increased CEF activity fostering light-induced proton gradient formation (Nawrocki et al., 2019; Suorsa, 2015). Meanwhile, the reduction of final signal intensity may be explained through a competitive suppression of NDH-dependent CEF resulting in lower overall proton pumping capacity. Cyt b6f is expected to translocate 2 H+ per Fd-derived electron across the membrane(Schultze et al., 2009), presumably via a PQH2 mediator, while the NDH complex likely pumps two H+ per electron transferred to plastoquinone akin to respiratory complex I (Brandt, 2017), followed by PQH2 oxidation by Cyt b6f. Thus, overstimulation of PGR5-CEF may result in reduction of ΔpH generation capacity by ~50 %, aligning well with the observed reduction in final P515 signal intensity (Fig 2, 5).

Surprisingly, PGR5 overexpression induced P515 kinetics and thus presumably trans-thylakoid proton gradient formation to turn partially insensitive to inhibitors of the photosynthetic electron transport chain, with MV treatment effects being ameliorated, DCMU and Hg2+ treatment being rendered largely inconsequential, and DBMIB treatment effects being overcompensated as compared to WT material (Fig 3, 6). As most current models of PGR5-dependent CEF activity assume this electron transport route to bypass PSII (e.g., Ma et al., 2021) signal insensitivity to DCMU may represent a predictable hallmark of PGR5-mediated CEF around PSI. Furthermore, the late-phase P515 signal increase (~300 seconds past illumination onset) in pgr5OE under Hg2+ treatment (Fig 6A) may reflect NDH-independent proton gradient formation, thus suggesting a contribution of PGR5-dependent CEF activity. Reduced sensitivity of pgr5OE P515 signal accumulation to MV (Fig 6A) may point towards formation of a partially MV-insensitive CEF supercomplex involving PSI and possibly Cyt b6f, possibly paralleling observations in Chlamydomonas reinhardtii (Iwai et al., 2010; Joliot et al., 2022). In this context, insensitivity to Cyt b6f inhibition (DBMIB) may result from PGR5 directly influencing PQ/DBMIB binding to Cyt b6f, possibly through its previously demonstrated physical interaction with the Cyt b6f complex (DalCorso et al., 2008; Wu et al., 2021), or stem from fostered PGR5-dependent switching from a LEF- to a CEF-specific Q cycle mode of Cyt b6f as proposed for Chlamydomonas renhardtii (Buchert et al., 2020). Either interpretation could be compatible with a tentative PSI-Cyt b6f-CEF supercomplex being formed. The inferred comparative effects of ETC inhibitor treatment on AL-induced P515 signal accumulation WT and pgr5 overexpression cells are summarized in Fig 7.

Figure 7. Targets of photosynthetic inhibitors and possible causes of altered proton gradient formation patterns in pgr5OE.

Figure 7

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A, Schematic illustration of light-driven photosynthetic electron transport and accompanying proton gradient formation ins Synechocystis wildtype (WT). Targets of inhibitors of the photosynthetic electron transport. DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone; Cyt b6f, cytochrome b6f; Fd, ferredoxin; Hg2+, mercury(II) (from Hg2+ mercury chloride); MV, methyl viologen; NDH-1; NADH dehydrogenase-like complex; OEC, oxygen-evolving complex; PBS, phycobilisome; PC, plastocyanin; PQ/PQH2, plastoquinone/plastoquinol; PS, photosystem. Red arrows indicate photosynthetically active radiation. B, Model illustrating the altered light-driven electron flow and proton translocation in pgr5OE and corresponding changes in sensitivity of P515 signal accumulation to DCMU, Hg2+, DBMIB, and MV. Disrupted and stimulated electron transport pathways are indicated by dotted and bold lines/arrows, respectively. Minor and major changes in inhibitor sensitivity are indicated through dashed and dotted lines/arrows, respectively.

Possible PGR5 effects on PSI-NDH and PSI-Cyt b6f supercomplex formation are inferred from corresponding reports in Synechocystis (Gao et al., 2016) and Chlamydomonas reinhardtii (Iwai et al., 2010; Joliot et al., 2022), respectively.

In conclusion, our observations in inhibitor-treated WT material and CEF mutants suggest that the described P515 kinetics is suitable to track the light-induced formation of a trans-thylakoid membrane proton gradient, as well as the effects of altered CEF and possibly ATP synthase activity. Importantly, an observed transient rise in P515 signal intensity upon offset of AL illumination proved unique to the Δpgr5 mutant (Fig 3B), with a reciprocal accelerated post-offset drop in P515 being observable in pgr5OE (Fig 6B). Unlike optoacoustic measurements (Yeremenko et al., 2005) and spectroscopic observation of P700 redox kinetics previously used to probe CEF activity in Synechocystis (Dann & Leister, 2019; Zhao et al., 2014) AL-induced P515 kinetics allow to draw a clear phenotypic distinction between NDH-CEF and PGR5-CEF affected mutants and indicate PGR5 to be a dosage-dependent regulator of photosynthetic electron transport and proton gradient formation in Synechocystis, much like in Arabidopsis thaliana (Rühle et al., 2021). As the P515 signal in cyanobacteria may be composed of different processes affecting absorption around 520 nm and/or 550 nm, future studies aiming at the detailed spectral deconvolution will be crucial to elucidate the physical basis of the changes measured in the P515 signal (i.e., ΔA550–520 nm). Moreover, precise normalization on chlorophyll a content or cell count rather than approximation of the latter through OD730 will further benefit comparability of future studies. Still, despite many open questions regarding the precise origin of the observed P515 signal remaining, the measurement routine presented in this study can be expected to enable non-invasive and inhibitor-free investigation of light-driven trans-thylakoid proton gradient formation in cyanobacteria by proxy.

Supplementary Material

Supporting Data File 1. Raw data underlying all graphs and charts presented in this study.

Acknowledgements

The authors thank Sabine Jarzombski for technical assistance.

Funding

The authors thank the European Research Council (ERC) for funding (Action Acronym: PhotoRedesign, Action number: 854126, Action Title: Redesigning the Photosynthetic Light Reactions) for financial support.

Footnotes

Author Contributions

M.D. and D.L. designed the study. M.Z., M.N. and M.D. performed P515 measurement. M.L. analysed pigment contents. All authors contributed to paper writing.

Data Availability Statement

The raw data underlying the findings of this study are provided in the Supplementary Data file. More data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Data File 1. Raw data underlying all graphs and charts presented in this study.
EMS205976-supplement-Supporting_Data_File_1.xlsx (19.8MB, xlsx)

Data Availability Statement

The raw data underlying the findings of this study are provided in the Supplementary Data file. More data supporting the findings of this study are available from the corresponding authors upon reasonable request.

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