Additive effects of multiple photoprotective mechanisms drive efficient photosynthesis under variable light conditions

To cope with changing external conditions, plants undergo dynamic acclimation processes that remodel their photosynthetic machinery, optimizing energy use while minimizing damage to photosystems (PS). Key photoprotective mechanisms include non-photochemical quenching (NPQ), which dissipates excess excitation energy, and alternative electron transport (AET) pathways, which prevent over-reduction of the photosynthetic electron transport chain. This study provides a comprehensive analysis of how various photoprotective mechanisms contribute to long-term acclimation to high and fluctuating light in Physcomitrium patens, a moss that exhibits well-conserved photoprotective responses bridging algae and vascular plants. Our results demonstrate that modulation of photoprotection around PSII and PSI is critical for maintaining photosynthetic efficiency and enable acclimation to variable light conditions. P. patens mutants deficient in NPQ or AET exposed to high or fluctuating light displayed growth defects, reduced photosynthetic efficiency and unbalanced PSI and PSII activity compared to WT plants. These findings indicate that photosynthetic response under varying light conditions depends on the complementary action of multiple protective strategies, rather than a single dominant photoprotective mechanism.

In photosynthetic eukaryotes, light-harvesting complexes (LHCs) associated with photosystems (PS) play a pivotal role in capturing photons providing the energy driving linear electron flow (LEF) to produce NADPH and ATP. These energy carriers are essential for carbon fixation in the Calvin-Benson cycle. However, under natural conditions, photosynthetic organisms are continuously exposed to fluctuating light intensities due to changes in weather, cloud cover, and canopy movements. Under these conditions, LHCs can absorb more energy excitation than the plant can use for photosynthesis. Excess energy can be harmful, leading to the formation of reactive oxygen species (ROS) and photo-oxidative stress (Eberhard et al. 2008). Regulating light reactions helps optimize photosynthetic efficiency, allowing plants to meet their metabolic needs and maintain fitness under highly dynamic environment (Külheim et al. 2002). Photosynthetic organisms evolved multiple strategies to regulate photosynthetic activity and limit photodamage. Under conditions of excess illumination, a prominent photoprotective role is played by non-photochemical quenching (NPQ), which drives dissipation of excess excitation energy as heat (Li et al. 2009b). NPQ is activated by the decrease in lumenal pH occurring under strong light. In green algae and vascular plants NPQ is regulated by two distinct activators, respectively light-harvesting complex stress-related protein (LCHSR) and photosystem II subunit S (PSBS) (Peers et al. 2009; Niyogi and Truong 2013). While PSBS plays a prominent role in vascular plants, LHCSR is the primary NPQ activator in green algae and diatoms (Bailleul et al. 2010; Bonente et al. 2011; Tibiletti et al. 2016; Redekop et al. 2020). The decrease in lumenal pH also activates the xanthophyll cycle by which the violaxanthin de-epoxidase enzyme (VDE) catalyzes the de-epoxidation of violaxanthin to zeaxanthin (Arnoux et al. 2009; Simionato et al. 2015). Zeaxanthin is crucial for NPQ activation and maintenance as well as for scavenging reactive oxygen species (ROS) to protect against oxidative damage (Ruban et al. 2007; Jahns and Holzwarth 2012). A significant contribution to photoprotection is also provided by alternative electron transport (AET) mechanisms, which prevent overreduction and damage to PSI (Allahverdiyeva et al. 2015; Shikanai and Yamamoto 2017; Burlacot 2023; Hoh et al. 2024). Cyclic electron flow (CEF) recycles electrons from PSI to the plastoquinone pool (PQ) or to Cyt b 6 f , while pseudo cyclic electron flow (PCEF) is involved in oxygen photoreduction to water downstream of PSI. Two distinct CEF pathways have been described, one depending on PGRL1/PGR5, and the other one depending on NADH dehydrogenase-like complex (NDH). PCEF includes pathways such as the Mehler reaction and flavodiiron proteins (FLVs), the latter present in cyanobacteria, green algae, non-vascular plants, and gymnosperms, but absent in angiosperms (Ilík et al. 2017). Additionally, photosynthetic control at the Cyt b 6 f complex contributes to PSI photoprotection by limiting LEF when NADPH and ATP exceed the demands for CO 2 fixation (Eberhard et al. 2008; Degen and Johnson 2024). All the above-mentioned mechanisms operate short-term, activated within minutes after a change in light intensity and have been shown to be essential for the response to fast changes in illumination (Külheim et al. 2002; Yamori and Shikanai 2016; Nawrocki et al. 2019; Burlacot 2023). However, plants are often exposed to prolonged stress conditions and, in response to different environmental conditions, photosynthetic organisms also adjust their photosynthetic apparatus to optimize its efficiency and mitigate eventual photo-oxidative stress through a process called photosynthetic acclimation (Walters 2005). For example, acclimation responses include the modulation of pigment content and composition to fine-tune their light-harvesting efficiency under varying light intensities (Sukenik et al. 1987; Anderson et al. 1995; Ballottari et al. 2007). Moreover, adjustment in CO 2 assimilation plays a pivotal role in acclimation by optimizing carbon fixation pathways to prevailing environmental conditions (Retkute et al. 2015). For instance, under high light and optimal CO₂ availability, plants may increase the activity of Rubisco and other Calvin cycle enzymes to enhance photosynthetic efficiency (Miller et al. 2017). Despite significant progress, the interplay and the regulation of various photoprotective mechanisms during long-term acclimation remain under-investigated (Eckardt et al. 2024). In this study, we provide an integrated analysis of the strategies used by the moss Physcomitrium patens during long term acclimation to challenging light conditions. As a representative of bryophytes, that diverged from vascular plants early after land colonization, P. patens provide critical insights into the transition from aquatic to terrestrial life. Understanding its strategies to cope with light stress is of great interest from both biochemical and evolutionary perspectives. In P. patens, NPQ is activated by both PSBS and LHCSR proteins (Alboresi et al. 2010) and, differently from angiosperms, it also express a full set of CEF (i.e. PGRL1/PGR5, NDH complex) (Kukuczka et al. 2014; Storti et al. 2020a) and PCEF proteins (i.e. Mehler reaction, FLVs) (Allahverdiyeva et al. 2013; Gerotto et al. 2016). The availability of a wide range of multiple P. patens mutants deficient in key regulatory proteins for photosynthesis, overlooked in the study of acclimation responses, provides a unique opportunity to assess the relative contribution of these mechanisms under varying light conditions.

Plant growth. Protonemal tissue of Gransden wild-type (WT) ecotype of P. patens was grown on minimum PpNO 3 medium solidified with 0.8% Agar (Ashton et al. 1979). Plants were propagated under sterile conditions on 9-cm Petri dishes overlaid with a cellophane disk. Plates were placed in a growth chamber under controlled conditions: 22 °C, 16-h light/8-h dark photoperiod, and a light intensity of 50 μmol·photons ·m -2 ·s -1 (control conditions; CL). For excess light and fluctuating light acclimation, 4-day-old plants were moved for 6 days from control to 500 μmol·photons m -2 ·s -1 (high light; HL) and 25/800 μmol·photons·m -2 ·s -1 for 5/1 min respectively (fluctuating light; FL), maintaining temperature and photoperiod. Photoprotective mutants (Figure 1A) used in this study were previously isolated. psbs lhcsr1 lhcsr 2 KO(Alboresi et al. 2010), vde KO (Pinnola et al. 2013), pgrl1 KO (Kukuczka et al. 2014), pgrl1 ndhm KO (Storti et al. 2020b), flva/b KO (Gerotto et al. 2016), (Traverso et al. 2025). In vivo chlorophyll a fluorescence and P700 + Measurement with Dual-PAM. In vivo chlorophyll a fluorescence and oxidized P700 + absorption signal were monitored simultaneously at room temperature with a Dual PAM-100 fluorometer (Walz). Before measurements, plates were dark acclimated for 40 min. PSII and PSI parameters were calculated as following: Fv/Fm as (Fm − Fo)/Fm, NPQ as (Fm − Fm′)/Fm′, Y(II) as (Fm′ − F)/Fm′, Y(NO) as F/Fm, Y(NPQ) as F/Fm’-F/Fm, qL = (Fm′‐F)/(Fm′‐Fo’) × Fo’/F, Y(I) as 1 −Y(ND) − Y(NA), Y(NA) as (Pm − Pm’)/Pm, Y(ND) as (1 − P700 red). Actinic light intensity (850 µmol photons·m⁻²·s⁻¹) was sub-saturating for photosynthesis in WT plants grown under constant light (CL) (Gerotto et al. 2011) . Spectroscopic Analyses with Joliot-Type Spectrometer (JTS). Spectroscopic analysis was performed in vivo on 10‐day old intact tissues using a JTS‐10 spectrophotometer (Biologic). Relative amount of functional photosynthetic complexes were evaluated measuring the Electrochromic Shift (ECS) spectral change on buffer‐infiltrated plants (HEPES 20 mM pH 7.5, KCl 10 mM) in presence and absence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU 20 μM) and hydroxylamine (HA, 4 mM) as in previous studies (Bailleul et al. 2010; Gerotto et al. 2016) . PSII functional antenna size was calculated by treating the sample with 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU 20 μM). The DCMU treated sample was exposed to actinic light 150 µmol photons s -1 and PSII antenna size has been estimated from Fm saturation kinetic (1/t 2/3 ) (Cardol et al. 2008) . Total Protein Extracts. Total extracts from P. patens grown in CL, HL, FL conditions were obtained by grinding tissues in sample buffer (50 mM TRIS pH 6.8, 100 mM DTT, 2% SDS, and 10% glycerol) before SDS/PAGE. Samples were loaded at the same equivalent amount of chlorophylls. For immunoblotting analysis, after SDS/PAGE, proteins were transferred to nitro-cellulose membranes and detected with both Horseradish Peroxidase (HRP, Agrisera #AS09-60s) or Alkaline Phosphatase‐conjugated secondary antibody (Sigma, #A3562) after hybridization with specific primary homemade polyclonal antibodies (α-LHCSR, α-PSBS, α-NDHM, α-FLVB). For densitometry quantification of Western blot bands, images were processed with FIJI (https://fiji.sc/) using the ‘mean grey value’ for measurements. For each band relative background was also subtracted. Mean grey value was measured for each band and normalized to WT CL. Chl a/b and Chl/Car ratios were obtained by fitting the spectrum of 80% acetone pigment extracts with spectra of the individual purified pigments, as reported in (Chazaux et al. 2022). Growth test. Growth in PPNO 3 media under different light regimes (specific conditions are described in the text and in the figure legends) was evaluated starting from protonema colonies of 2 mm in diameter and then followed for 28 days. High-resolution images (600 dpi) were acquired using a Konica Minolta Bizhub C280 scanner. Images were processed with FIJI (https://fiji.sc/) using the ‘threshold colour’ plugin to remove the plate background. Integrated density was measured for each colony and normalized to WT CL Day 1 (Storti et al. 2019). Statistical analysis. Descriptive statistics and inferential statistics were performed using OriginPro9.1 software©. Differences between light treatments (CL, HL, FL) were statistically tested by One-Way ANOVA for WT characterization. The interaction between genotypes (WT, vde KO, psbs lhcsr KO, pgrl1 KO, pgrl1 ndhm KO, flva/b KO) and light treatments (CL, HL, FL) were analyzed by Two-Way ANOVA. TukeyHSD post-hoc test was used to evaluate pairwise differences between genotypes.

Physcomitrium patens photosynthetic apparatus acclimates to different light regimes. To investigate the mechanism of light acclimation in P. patens, WT plants were grown for six days under three distinct light conditions: CL, HL and FL (Figure 1B). Figure 1. Schematic representation of P. patens photoprotective mutants and growth conditions used in this study. A) Illustration of P. patens linear electron flow components (white boxes) and key photoprotective mechanisms studied in this work. NPQ players (PSBS, LHCSR and VDE) are highlighted in orange. CEF-mediated pathway components (NDH complex, PGRL1-PGR5 complex) are highlighted in blue. FLVA/FLVB of PCEF pathway are indicated in pink. B) Plants were initially grown at control light (CL, 50 μmol photons·m -2 ·s -1 ) for 4 days before exposure to high light (HL, 500 μmol photons m -2 ·s -1 ) or fluctuating light (FL, 25/800 μmol photons ·m -2 ·s -1 5/1min) for 6 days. A control group of samples was also maintained under CL for 10 days. Maximal PSII quantum efficiency (F v /F m ) was found stable across the three conditions, indicating that plants did not present extensive photodamage and thus were able to successfully acclimate to different light regimes (Table 1). Pigment extracts of acclimated plants revealed a lower Chl/car ratio under HL conditions as compared to CL and FL, indicating that carotenoids play a prominent photoprotective role under HL. However, the Chl a/b ratio remained consistent across all three conditions. The PSII functional antenna size, estimated from fluorescence induction kinetics in the presence of DCMU (Cardol et al. 2008), was not significantly affected by the different light regimes in WT plants. This is consistent with earlier work showing limited antenna size modulation in P. patens (Gerotto et al. 2011). | Chl/Car | Chl a / b | Antenna size | Fv/Fm | | | WT CL | 3,7±0,33 | 2,5±0,15 | 0,025±0,005 | 0,78±0,01 | | WT HL | 2,67 a ±0,12 | 2,7±0,34 | 0,026±0,004 | 0,76±0,03 | | WT FL | 3,47±0,16 | 2,7±0,25 | 0,03±0,005 | 0,77±0,04 | | a One-way ANOVA, P5 | a statistically significant difference with respect to WT plants, One-way ANOVA, P5. To evaluate response of key photosynthetic parameters after plant acclimation to different light regimes, we measured Chl a fluorescence and P700⁺ absorption signals under strong actinic illumination followed by dark relaxation. The overall efficiency of PSII (Y(II)) and PSI (Y(I)) was similar for plants grown in the three conditions (Figure 2 A-B). Acclimation to HL increased the fraction of open PSII reaction centers upon actinic light, as shown by the decreased 1-qL parameter (Figure 2C). This suggests that HL acclimated plants experience less PSII saturation (Kramer et al. 2004) once exposed to strong light, indicating enhanced photosynthetic electron transport capacity. Figure 2. Photochemistry of acclimated WT P. patens at different light intensities. A) PSII and B) PSI quantum yield, and C) PSII excitation pressure (1-qL) were measured in plants acclimated to (CL), high light (HL) and fluctuating light (FL), represented by empty squares, black circles and stars, respectively. Plants were exposed to 850 µmol photons m -1 s -1 actinic illumination for 8 minutes, followed by 10 minutes of dark. Bars indicate standard deviation ( n ≥ 3). Red asterisks indicate values significantly different from the CL sample (One-way ANOVA, P<0.01; statistical analysis considered time points at 0, 2, 4, 6, and 8 minutes after actinic light exposure). Acclimation to variable light regimes is achieved through enhanced photoprotective mechanisms. To investigate the regulation of photoprotection during long-term acclimation, we assessed WT capacity to modulate NPQ and AET under different light conditions along with the biochemical accumulation of proteins involved in these mechanisms. NPQ activity varied depending on growth conditions, with HL-acclimated plants showing NPQ levels twice as high as those grown under CL conditions. Similarly, plants exposed to FL showed an increased NPQ compared to CL (Figure 3A). The different electron transport rates between PSII and PSI was used to evaluate CEF around PSI. HL-acclimated plants displayed higher CEF than those grown under CL and FL, as estimated from the difference between estimated ETRI and ETRII (Figure 3B). Immunoblot analysis showed that both PSBS and LHCSR, the molecular activators of NPQ in P. patens (Alboresi et al. 2010), were more abundant in HL compared to CL. PSBS was also more abundant in FL compared to CL (Figure 3C-D), in agreement with the enhanced NPQ during acclimation to HL and FL (Figure 3A). To investigate the regulation of alternative electron transporters, we examined the accumulation of the NDHM subunit (Storti et al. 2020a), essential component of the NADH dehydrogenase-like complex I. PGRL1 and PGR5 were not quantified because Arabidopsis thaliana antibodies did not recognize well P. patens proteins, while FLVB was chosen as a representative of PCEF (Gerotto et al. 2016). Both NDHM and FLVB accumulated more in HL acclimated plants compared to CL plants. Interestingly, FL acclimated plants showed a higher amount of FLVB than CL grown plants, but similar NDHM accumulation compared to CL (Figure 3C-D). While the accumulation of proteins involved in photoprotection changed during acclimation, the same amount of Light Harvesting Complexes II (LHCII) was detected across the three conditions, consistently with pigment assessment and antenna size estimations. Figure 3. Functional and biochemical analysis of photoprotection related proteins in P. patens plants. A) NPQ B) ETRI-ETRII. Plants were exposed for 8 minutes to actinic light (850 µmol photons m -1 s -1 ) followed by 10 minutes of darkness. Data are presented as empty squares (CL), black circles (HL), and stars (FL). Error bars indicate standard deviation ( n ≥ 3). Red asterisks indicate values significantly different from the CL sample (One-way ANOVA, P<0.01. Minutes 0, 2, 4, 6, 8 after switching on the actinic light were considered for statistical analysis). C) Immunoblot analysis of photoprotection-related proteins in WT plants grown under CL, HL and FL. The proteins analyzed included LHCSR, PSBS, FLVB, NDHM and LHCII. Protein loading corresponded to different chlorophyll equivalents as follows: 1× is equivalent to 1 µg of chlorophylls, and 2× and 4× indicating two- and fourfold amounts, respectively. D) Quantification of PSBS, LHCSR, NDHM, FLVB, LHCII protein levels was densitometric analysis of immunoblots. Band intensities were normalized to CL-grown plants present on the same membrane. Bar plots indicate mean (empty square) and median (horizontal lines within bars), with black circles represent individual replicate measurements (n ≥ 3). Red asterisks indicate statistically significant differences compared to CL condition (One-way ANOVA, * p-value NPQ and AET mechanisms are critical for optimal growth during acclimation to different light regimes. Functional and biochemical analyses of WT P. patens plants revealed enhanced photoprotective mechanisms under challenging light environments. To better understand the roles of these mechanisms in acclimation, a set of photoprotective mutants was grown under the same CL, HL and FL conditions alongside WT plants. The mutants included those defective in NPQ and xanthophyll cycle (i.e. vde KO, psbs lhcsr KO) and in AET pathways (i.e. pgrl1 KO, pgrl1 ndhm KO, flva/b KO). Plant growth was monitored over a 28-day period to assess growth differences, allowing the plant colonies to reach a diameter of growth under HL, while under FL, their growth decreased by 30% compared to CL (Figure 4). Under CL, all tested mutants, except vde KO, exhibited growth rate comparable to WT plants. In all tested conditions, the vde KO showed more dispersed protonemata development compared to WT, and a lack of response to HL and FL, highlighting crucial role of zeaxanthin for plant growth and light acclimation. Under HL, all mutants, except psbs lhcsr KO, grew less than WT plants, with the mutant pgrl1 ndhm KO showing the most pronounced decrease (Figure 4). In FL conditions, psbs lhcsr KO, pgrl1 KO, pgrl1 ndhm KO and flva/a KO lines displayed a significant growth reduction compared to CL. Moreover, in FL conditions flva/b KO showed a clear and significant growth impairment as compared to WT plants. Overall, these findings confirmed that both NPQ and AET mechanisms are critical for optimal growth under different light regimes, with specific roles depending on the nature of the light stress encountered. Figure 4. Growth phenotypes of P. patens plants after 28 days of growth under different light conditions. A) Representative images of plants grown under CL, HL and FL conditions for 28 days. B) Quantification of plant growth under each condition. Bar plots indicate mean (empty square) and median (horizontal lines within bars), circles represent individual replicate measurements (n ≥ 3). WT, vde KO, psbs lhcsr KO, pgrl1, pgrl1 ndhm, flva/b KO are indicated in black, light blue, orange, green, pink and blue respectively. Two-Way ANOVA indicated significant difference between explanatory variables (genotypes, light treatments) and significant interaction between the two (p < 0.0001). Values marked with different letters are significantly different between treatments (TukeyHSD post-hoc test, p < 0.05). NPQ and AET mechanisms provide critical photoprotection in variable light environments. To evaluate the impact of compromised photoprotection on photosynthetic efficiency, we measured key parameters in 10-day-old protonema of WT and photoprotective mutants grown under CL, HL and FL. The F v /F m ratio, reflecting the maximum efficiency of PSII, remained stable across the three light regimes in WT plants, revealing its photosynthetic acclimation capacity. Under CL conditions all mutant genotypes were similar to WT (Figure 5A). Interestingly, acclimation to HL and FL reduced F v /F m across all mutants relative to WT plants grown under the same conditions suggesting the presence of light induce damage. Notably, mutants deficient in NPQ ( psbs lhcsr KO, vde KO) and in CEF ( pgrl1 KO, pgrl1 ndhm KO) displayed the lowest PSII efficiency under HL, whereas PCEF mutant flva/b KO exhibited the most significant reduction under FL (Figure 5A). We also evaluated the relative activity of the two photosystems using electrochromic shift (ECS) signal analysis following a single flash of light (Bailleul et al. 2010) (Figure 5B). Under CL conditions, all lines showed PSI/PSII ratios like WT. However, under HL and FL conditions, while NPQ mutants retained a PSI/PSII ratio comparable to WT plants, the AET mutants showed reduced ratios, likely as the result of PSI inactivation. In particular, the double mutant pgrl1 ndhm KO showed a marked reduction in PSI/PSII ratio under HL. Under FL conditions, the PSI/PSII ratio also decreased in the pgrl1 ndhm KO mutant, though the reduction was less severe than under HL. Conversely, pseudo-cyclic flva/b KO mutant displayed the most significant decline in the PSI/PSII ratio under FL conditions. Figure 5. PSII maximal efficiency and PSI/PSII ratio in P. patens plants. A) PSII maximal quantum efficiency (F v /F m ) of WT and mutant lines ( vde KO, psbs lhcsr KO, pgrl1 KO, pgrl1 ndhm KO, flva/b KO) grown under CL, HL and FL. B) PSI/PSII ratio quantified from ECS signals following the application of a flash of light on the samples. For each condition, mean and median are shown respectively with empty square and horizontal lines in the bar blot. Different replicates are indicated with points. WT, vde KO, psbs lhcsr KO, pgrl1, pgrl1 ndhm, flva/b KO are indicated in black, light blue, orange, green, pink and blue respectively. Genotypes conditions are reported at the bottom of panel B. Two-Way ANOVA indicated significant difference between explanatory variables (genotypes, light treatments) and significant interaction between the two (p < 0.0001). Values marked with different letters are significantly different between treatments (TukeyHSD post-hoc test, p < 0.05). Efficient energy partitioning between photosystems requires effective photoprotection during light acclimation. Given that impaired photoprotection can affect photosystem efficiency and activity, we investigated the contributions of NPQ and AET to energy allocation within PSII and PSI. Using a DUAL-PAM-100, we simultaneously monitored Chl a fluorescence and P700 + absorption signals during 8-minute exposure to actinic light, followed by 10 minutes of dark relaxation as in figure 2. Our analysis focused on two critical time points: i) the dark-to-light transition, to assess the impact of NPQ and AET upon a sudden increase in illumination (Figure 6A), and ii) steady-state photosynthesis after 5-8 minutes of illumination, when the Calvin-Benson Cycle is fully activated (Figure 6B). At the onset of light, in the WT around 14% of energy was involved in photochemistry (Y(II)), 6% was dissipated as heat (Y(NPQ) and 80% of the energy was dissipated at the level of reaction centers in non-regulated manner (Y(NO)). Moreover, 57% of PSI were donor-side limited (Y(ND)) and 18% were acceptor-side limited (Y(NA)) resulting in 24% of PSI that could be photo-oxidised with a saturating pulse (YI). When the light was switched on, NPQ mutants showed no significant differences in energy allocation between the photosystems compared to WT, likely because NPQ requires several minutes to become fully activated (Figure 6A, Figure S1-S2). In contrast, AET mutants exhibited marked differences during the dark-to-light transition. Specifically, pgrl1 ndhm KO and flva/b KO mutants demonstrated reduced Y(II) efficiency in all growth conditions (0-5%) compared to approximately 12% in WT, with pgrl1 KO mutants under HL showing similar reductions (around 2%). Furthermore, these AET mutants enhanced energy dissipation via either Y(NO) or Y(NPQ), depending on the growth conditions (Figure 6A, Figure S3). Impaired photoprotection also impacted PSI energy allocation during the light-to-dark transition in AET mutants (Figure S4). pgrl1 ndhm KO lines displayed significantly higher Y(NA) values under both CL (87%) and HL (96%) conditions compared to WT plants (CL: 18%, HL: 15%). Although this difference persisted under FL, it was less pronounced (WT: 16%, pgrl1 ndhm KO: 43%). Notably, the single pgrl1 KO mutant showed a milder phenotype, with high Y(NA) values observed only under HL (81%). These results indicate that under high light conditions, both the NDH the and PGRL1/PGR5 pathway are required to maintain proper PSI functionality upon light exposure. In the flva/b KO mutant, Y(NA) reached maximal levels at the onset of light under CL (95%) and HL (94%). In plants grown under FL, the absence of FLVs resulted in such severe PSI impairment that accurate energy allocation measurements were not possible, and overall yield was drastically reduced (Figure 6A). During steady state photosynthesis, when the Calvin-Benson Cycle is fully active, WT plants displayed maximal NPQ activation (Y(NPQ), (Y(NO), ~23%), while no difference in energy allocation within PSI were observed compared to dark to light transition consistent with the very fast activation of AET. In these conditions, both NPQ and AET mutants displayed some differences in energy partitioning between photosystems compared to WT (Figure 6B). Under CL conditions, the Y(NPQ) was reduced in vde KO plants (37%) compared to WT (63%), and even further decreased in psbs lhcsr KO plants (21%) (Figure 6B, Figure S1). This reduction was compensated by a concomitant increase in the constitutive loss process Y(NO) in these mutants. Notably, this energy distribution pattern was maintained in WT and vde KO lines acclimated to HL and FL. In contrast, psbs lhcsr KO mutants experienced a further decrease in Y(NPQ) accompanied by a significant increase in Y(NO) under HL and FL conditions (Figure 6B, Figure S1). Regarding PSII energy partitioning, the pgrl1 and pgrl1 ndhm mutants displayed profiles similar to WT under all conditions. However, under FL, the absence of FLVs in the flva/b KO mutant significantly reduced the Y(II) efficiency (2%) and caused alterations in the development and relaxation of Y(NPQ) (Figure 6B, S3). At PSI, impaired photoprotection also led to altered energy allocation during steady state photosynthesis. NPQ mutants showed increased donor side limitation during actinic light exposure, with maximal increase observed in HL acclimated plants (Figure 6B, Figure S2). In these mutants, inefficient thermal energy dissipation impaired linear electron transport, reducing the number of electrons reaching PSI and thereby limiting the availability of electrons to PSI. Moreover, under HL conditions, at steady state photosynthesis, the pgrl1 and pgrl1 ndhm KO lines exhibited high values of acceptor side limitation (40% in pgrl1 KO and 63% in pgrl1 NDHM KO, compared to 11% in WT) and minimal donor side limitation (39% in pgrl1 KO and 11% in pgrl1 ndhm KO, compared to 61% in WT) (Figure 6B, S4). Under other growth conditions, these CEF mutants displayed values similar to WT. In contrast, the flva/b KO mutants showed no differences in PSI energy partitioning under steady-state across all tested conditions (CL, HL), suggesting that when the Calvin-Benson cycle is fully functional, alternative mechanisms help to safeguard PSI. Figure 6. PSII and PSI energy partitioning in WT and photo-protective mutants acclimated to different light conditions. (A) Parameters measured during the light-to-dark transition at the onset of actinic illumination, and (B) parameters measured during steady-state photosynthesis (minutes 5–8 of actinic illumination) in WT and mutant plants acclimated to control light (CL), high light (HL), and fluctuating light (FL). Acclimated plants were exposed to 850µmol photons m -1 s -1 actinic light illumination using a DUAL-PAM chlorophyll fluorometer. Data represent the mean of at least 3 replicates ± SD. Parameters include Y(II), PSII quantum yield (white); Y(NPQ), non-photochemical quenching (grey); Y(NO), non-regulated energy dissipation (green); Y(I), PSI quantum yield (orange); Y(NA), PSI acceptor-side limitation (yellow); Y(ND), PSI donor-side limitation (blue). N.D.= not detectable.

Boosting photoprotection to maintain photosynthetic efficiency This study explored the strategies that P. patens plants employ to sustain photosynthetic efficiency during long-term acclimation to HL and FL. Our findings highlight that the activation of distinct mechanisms enables P. patens to cope with adverse light regimes, thereby optimizing photosynthetic efficiency and energy distribution along the electron transport chain. We showed that upon exposition to HL and FL, WT P. patens retained F v /F m and efficient functioning of both photosystems, demonstrating that it is able to effectively acclimate to those challenging conditions (Table I, Figure 2). The conditions chosen were effectively stressful for the moss, as shown by the growth defects of mutant lines (Figure 5) indicating that acclimation is indeed effective in WT plants. Unlike other photosynthetic organisms (Walters and Horton 1994; Ballottari et al. 2007; Jia et al. 2016; Meneghesso et al. 2016; Štroch et al. 2022), P. patens does not primarily rely on antenna size adjustment for light acclimation. This observation aligns with previous studies in P. patens under HL and low temperature (Gerotto et al. 2011), in C. reinhardtii under HL (Bonente et al. 2012) and in Picea abies (Štroch et al. 2022). HL-acclimated plants exhibited increased carotenoid accumulation, a conserved strategy to prevent photo-oxidative damage (Havaux 2014). Conversely, no significant changes in pigment composition were observed between FL- and CL-grown plants, consistent with A. thaliana responses to fluctuating light (Gollan et al. 2023) (Table I). Long-term acclimation to both HL and FL induced an increased accumulation of proteins involved in photoprotection (Figure 3). Both light regimes triggered NPQ upregulation, with HL-acclimated plants exhibiting the highest NPQ levels. Up-regulation of NPQ during excess light exposure it is well-conserved in the green lineage and have been extensively observed and studied in many organisms (Peers et al. 2009; Ware et al. 2015; Flannery et al. 2021). In P. patens NPQ up-regulation under HL is associated with enhanced accumulation of LHCSR and even higher accumulation of PSBS (Gerotto et al. 2011). Our data indicate that FL triggers a similar response, characterized by elevated NPQ and increased PSBS levels, suggesting that rapid NPQ activation protects plants from sudden light excess—a response that appears to be conserved in angiosperms. For instance, A. thaliana plants deficient in NPQ photoprotection exhibit growth defects under fluctuating light conditions in controlled environment (Külheim et al. 2002). NPQ also protects leaves of N. tobacco plants when exposed to natural sunlight and thus shaded by clouds or other leaves (Kromdijk et al. 2016). Our findings indicated also enhanced PSI photoprotection during acclimation to both HL and FL by modulation of AET mechanisms. However, while the levels of FLVB protein are increased in both conditions, NDHM subunit representative of NDH is more accumulated only under HL, suggesting the major role of CEF-mediated pathway under this light regime. This finding are consistent with earlier analyses of P. patens mutants (Storti et al. 2019). Overall, our results indicate that acclimation to HL and FL boosts photoprotection, allowing the maintenance of photosynthetic efficiency under different light conditions. Under all tested conditions, photoprotection of PSII depended on enhanced NPQ, while PSI photoprotection revealed an interplay between cyclic and pseudo-cyclic electron flow, with the former dominating under HL and the latter under FL conditions. Effective photosynthesis and growth depend on the interplay of multiple photoprotective mechanisms. The use of different P. patens photoprotective mutants highlighted the role and the complementary effect of multiple strategies during acclimation to safeguard both, PSII and PSI functionality. Indeed, P. patens mutants allowed to evaluate the contributions of NPQ and AET to growth and photosynthesis under different light conditions. The vde KO mutant showed severe growth defects even at CL (Figure 5A) (Pinnola et al. 2013) likely due to the absence of zeaxanthin rather than solely due to impaired NPQ. Growth defects were not previously reported in vde KO mutants of other plant species such as A. thaliana (Li et al. 2009a). This highlights a specific dependence on zeaxanthin, likely related to an altered xanthophyll cycle that might impact carotenoid biosynthesis and hormone metabolism (Fujita et al. 2013; D’Alessandro and Havaux 2019), leading to an alteration of moss growth. Indeed, the psbs lhcsr KO mutant, which completely lacks NPQ, displayed less severe phenotype compared to the vde KO in all analyzed conditions. The lack of NPQ efficient mechanism could be compensated by higher PSII repair rate (Roach and Krieger-Liszkay 2012; Barbato et al. 2020) which also allows the maintenance of PSI/PSII values in NPQ mutants similar to WT plants. Both the NPQ mutants exhibited elevated donor side limitation (Y(ND)) at steady state during acclimation to HL and FL (Figure 6). This increase likely results from mutants’ inability to effectively manage excitation energy at the level of PSII, which can lead to alterations in linear electron flow (LEF) and boosted electron transfer to PSI. We also observed several interplays between alternative electron transport and photosynthesis regulation during acclimation to different light conditions. Consistent with the biochemical analysis on WT plants, we observed that lack of CEF caused the most pronounced acceptor side limitation in plants acclimated to HL. In contrast, the damage to PSI due to the absence of PCEF under fluctuating light (FL) was so severe that it was not possible to detect its activity (Figure 6). Additionally, the lack of PCEF also led to altered NPQ dynamics, impairing both its activation and relaxation, suggesting that proper electron flow downstream PSI is also essential for optimal energy management and dissipation at the level of PSII (Figure S4). Overall, our findings suggest that the efficient photosynthesis under varying light conditions is not dependent on a single mechanism but rather on the additive effect of multiple protective strategies. During long-term acclimation, removing any one of these strategies leads to reduced energy efficiency and compromised growth. Indeed, in mutants defective in either NPQ ( vde KO, psbs lhcsr KO) or AET ( pgrl1 KO, pgrl1 ndhm KO, flva/b KO) growth (Figure 4) and photosynthesis were impaired (Figure 5) under challenging light conditions. The biochemical and functional characterization of WT and mutants during acclimation underscored that different photoprotective mechanisms operate in an additive manner to ensure proper energy management with no single mechanism fully able to compensate for the absence of the other.

We thank Silvia Ferrando for helping with preliminary experiments. TM acknowledges funding from MUR PRIN2022PNRR - IPERAFIX (P2022Z498J). AA acknowledges funding from MUR PRIN2022PNRR - IRONCROP (P2022ZXWLK).

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Schematic representation of P. patens photoprotective mutants and growth conditions used in this study. A) Illustration of P. patens linear electron flow components (white boxes) and key photoprotective mechanisms studied in this work. NPQ players (PSBS, LHCSR and VDE) are highlighted in orange. CEF-mediated pathway components (NDH complex, PGRL1-PGR5 complex) are highlighted in blue. FLVA/FLVB of PCEF pathway are indicated in pink. B) Plants were initially grown at control light (CL, 50 μmol photons·m -2 ·s -1 ) for 4 days before exposure to high light (HL, 500 μmol photons m -2 ·s -1 ) or fluctuating light (FL, 25/800 μmol photons ·m -2 ·s -1 5/1min) for 6 days. A control group of samples was also maintained under CL for 10 days. Figure 2. Photochemistry of acclimated WT P. patens at different light intensities. A) PSII and B) PSI quantum yield, and C) PSII excitation pressure (1-qL) were measured in plants acclimated to (CL), high light (HL) and fluctuating light (FL), represented by empty squares, black circles and stars, respectively. Plants were exposed to 850 µmol photons m -1 s -1 actinic illumination for 8 minutes, followed by 10 minutes of dark. Bars indicate standard deviation ( n ≥ 3). Red asterisks indicate values significantly different from the CL sample (One-way ANOVA, P<0.01; statistical analysis considered time points at 0, 2, 4, 6, and 8 minutes after actinic light exposure). Figure 3. Functional and biochemical analysis of photoprotection related proteins in P. patens plants. A) NPQ B) ETRI-ETRII. Plants were exposed for 8 minutes to actinic light (850 µmol photons m -1 s -1 ) followed by 10 minutes of darkness. Data are presented as empty squares (CL), black circles (HL), and stars (FL). Error bars indicate standard deviation ( n ≥ 3). Red asterisks indicate values significantly different from the CL sample (One-way ANOVA, P<0.01. Minutes 0, 2, 4, 6, 8 after switching on the actinic light were considered for statistical analysis). C) Immunoblot analysis of photoprotection-related proteins in WT plants grown under CL, HL and FL. The proteins analyzed included LHCSR, PSBS, FLVB, NDHM and LHCII. Protein loading corresponded to different chlorophyll equivalents as follows: 1× is equivalent to 1 µg of chlorophylls, and 2× and 4× indicating two- and fourfold amounts, respectively. D) Quantification of PSBS, LHCSR, NDHM, FLVB, LHCII protein levels was densitometric analysis of immunoblots. Band intensities were normalized to CL-grown plants present on the same membrane. Bar plots indicate mean (empty square) and median (horizontal lines within bars), with black circles represent individual replicate measurements (n ≥ 3). Red asterisks indicate statistically significant differences compared to CL condition (One-way ANOVA, * p-value Figure 4. Growth phenotypes of P. patens plants after 28 days of growth under different light conditions. A) Representative images of plants grown under CL, HL and FL conditions for 28 days. B) Quantification of plant growth under each condition. Bar plots indicate mean (empty square) and median (horizontal lines within bars), circles represent individual replicate measurements (n ≥ 3). WT, vde KO, psbs lhcsr KO, pgrl1, pgrl1 ndhm, flva/b KO are indicated in black, light blue, orange, green, pink and blue respectively. Two-Way ANOVA indicated significant difference between explanatory variables (genotypes, light treatments) and significant interaction between the two (p < 0.0001). Values marked with different letters are significantly different between treatments (TukeyHSD post-hoc test, p < 0.05). Figure 5. PSII maximal efficiency and PSI/PSII ratio in P. patens plants. A) PSII maximal quantum efficiency (F v /F m ) of WT and mutant lines ( vde KO, psbs lhcsr KO, pgrl1 KO, pgrl1 ndhm KO, flva/b KO) grown under CL, HL and FL. B) PSI/PSII ratio quantified from ECS signals following the application of a flash of light on the samples. For each condition, mean and median are shown respectively with empty square and horizontal lines in the bar blot. Different replicates are indicated with points. WT, vde KO, psbs lhcsr KO, pgrl1, pgrl1 ndhm, flva/b KO are indicated in black, light blue, orange, green, pink and blue respectively. Genotypes conditions are reported at the bottom of panel B. Two-Way ANOVA indicated significant difference between explanatory variables (genotypes, light treatments) and significant interaction between the two (p < 0.0001). Values marked with different letters are significantly different between treatments (TukeyHSD post-hoc test, p < 0.05). Figure 6. PSII and PSI energy partitioning in WT and photo-protective mutants acclimated to different light conditions. (A) Parameters measured during the light-to-dark transition at the onset of actinic illumination, and (B) parameters measured during steady-state photosynthesis (minutes 5–8 of actinic illumination) in WT and mutant plants acclimated to control light (CL), high light (HL), and fluctuating light (FL). Acclimated plants were exposed to 850µmol photons m -1 s -1 actinic light illumination using a DUAL-PAM chlorophyll fluorometer. Data represent the mean of at least 3 replicates ± SD. Parameters include Y(II), PSII quantum yield (white); Y(NPQ), non-photochemical quenching (grey); Y(NO), non-regulated energy dissipation (green); Y(I), PSI quantum yield (orange); Y(NA), PSI acceptor-side limitation (yellow); Y(ND), PSI donor-side limitation (blue). N.D.= not detectable. 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Authors Metrics & Citations Metrics Article Usage 542views 209downloads Citations Download citation Claudia Beraldo, Chiara Toffanin, Tomas Morosinotto, et al. Additive effects of multiple photoprotective mechanisms drive efficient photosynthesis under variable light conditions. Authorea. 21 March 2025. DOI: https://doi.org/10.22541/au.174254782.27774145/v1 DOI: https://doi.org/10.22541/au.174254782.27774145/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.