Photoreceptor-induced LHL4 protects the photosystem II monomer in Chlamydomonas reinhardtii

Significance

For photoautotrophs, light is both an essential driver of photosynthesis and a potential threat when in excess. Our research unveils the photoreceptor-induced Light Harvesting complex-Like 4 (LHL4) as a protagonist in the photoprotective response against harmful effects of high light (HL) in Chlamydomonas reinhardtii. LHL4 is a critical component of the core of the photosynthetic machinery where it limits the production of damaging molecules, ensuring cell survival under HL. Thus, we provide insights into the molecular and mechanistic strategies employed by photosynthetic microalgae confronted with light stress, improving our understanding of their acclimation responses.

Abstract

Photosynthesis, the fundamental process using light energy to convert carbon dioxide to organic matter, is vital for life on Earth. It relies on capturing light through light-harvesting complexes (LHC) in photosystem I (PSI) and PSII and on the conversion of light energy into chemical energy. Composition and organization of PSI and PSII core complexes are well conserved across evolution. PSII is particularly sensitive to photodamage but benefits from a large diversity of photoprotective mechanisms, finely tuned to handle the dynamic and ever-changing light conditions. Light Harvesting Complex protein family members (LHC and LHC-like families) have acquired a dual function during evolution. Members of the LHC antenna complexes of PS capture light energy, whereas others dissipate excess energy that cannot be harnessed for photosynthesis. This process mainly occurs through nonphotochemical quenching (NPQ). In this work, we focus on the Light Harvesting complex-Like 4 (LHL4) protein, a LHC-like protein induced by ultraviolet-B (UV-B) and blue light through UV Resistance locus 8 (UVR8) and phototropin photoreceptor-activated signaling pathways in the model green microalgae Chlamydomonas reinhardtii. We demonstrate that alongside established NPQ effectors, LHL4 plays a key role in photoprotection, preventing singlet oxygen accumulation in PSII and promoting cell survival upon light stress. LHL4 protective function is distinct from that of NPQ-related proteins, as LHL4 specifically and uniquely binds to the transient monomeric form of the core PSII complex, safeguarding its integrity. LHL4 characterization expands our understanding of the interplay between light harvesting and photoprotection mechanisms upon light stress in photosynthetic microalgae.

Photosynthetic organisms encounter daily light fluctuations in their natural environments. As light is a source of energy for photosynthesis but can also be a source of photodamage, balancing light absorption and light dissipation is crucial for survival and fitness (1). Photosynthetic organisms have evolved different strategies to acclimate and respond to light fluctuations. These responses include a range of complementary mechanisms collectively known as photoprotection (2). These mechanisms, which are activated at different times in response to light changes, serve to prevent, limit, or repair photodamage (3, 4). For example, dissipation of light energy through nonphotochemical quenching (NPQ) and scavenging of reactive oxygen species (ROS) are activated within minutes, playing crucial roles for survival under excessive light (5–8). Light-Harvesting Complex (LHC) proteins and the related LHC-like proteins have evolved different functions in light absorption and photoprotection from green algae to flowering plants (9). LHC-like family members contain one to four transmembrane domains each harboring putative LHC motifs that may bind chlorophyll a/b, carotenoid, and lipids (9). In algae, including the green microalgae Chlamydomonas reinhardtii (abbreviated as Chlamydomonas hereafter), important NPQ effectors are the LHC-Like Stress Related (LHCSR) proteins LHCSR1 and LHCSR3 (10). In vascular plants, where LHCSR proteins are absent, Photosystem II Subunit S (PSBS) is a major player of NPQ (7). PSBS also contributes to NPQ in green microalgae, although to a lesser extent than the LHCSR proteins (11–14). Besides NPQ, plant photoprotection has been proposed to be modulated through the action of the LHC-like Early Light Induced Protein (ELIP) family. ELIPs possibly prevent the negative effect of free chlorophyll molecules released during the turnover of photosystem II (PSII) (15). However, the exact function of other LHC-like proteins remains unknown.

Photoprotective mechanisms are activated by photoreceptors that monitor the light environment (16). In green microalgae, expression of LHCSRs, PSBS, and ELIPs is highly light-inducible through blue light-activated phototropin (PHOT) and UV-B-activated UV Resistance Locus 8 (UVR8) photoreceptors (12, 17, 18). The early steps of UVR8 signaling are well conserved in the green lineage (19, 20), with UV-B-induced UVR8 monomerization and binding to Constitutively Photomorphogenic 1 (COP1), an E3 ubiquitin ligase playing a vital role in repressing light responses and photomorphogenesis in photosynthetic organisms (12, 21–25). UVR8 binding inhibits COP1, stabilizing transcription factors and thereby leading to transcriptional reprogramming (19, 26–28). In flowering plants, blue light gene expression relies on cryptochrome photoreceptor–COP1 mediated pathway (23, 27, 29), whereas PHOTs seem to control blue light responses independently of early transcriptional changes (30, 31). In contrast, in Chlamydomonas, PHOT induces transcription of LHCSR3 through a signaling cascade that involves COP1 (also known as LRS1/HIT1) (17, 28, 32, 33). Downstream of COP1, molecular players apparently diverged during evolution. The B-box transcription factor Constans (CrCO) controls LHCSRs expression in response to both UV-B and high light (HL) in Chlamydomonas (28, 32), whereas the bZIP transcription factor Elongated Hypocotyl 5 (HY5) plays the major role in the UV-B and blue light signaling pathway in flowering plants (34–38), but its role remains elusive in green algae (39).

Here, we identify the Chlamydomonas LHC-Like protein 4 (LHL4) as a crucial component of the photoprotection response. LHL4 expression and LHL4 accumulation are strongly induced under UV-B and HL in a UVR8- and PHOT-dependent manner, respectively. Induction involves transcriptional regulation through CrCO. Transcriptomic data corroborate the conclusion that CrCO plays a major role in UVR8-dependent transcriptional reprogramming. Finally, we propose that LHL4-mediated photoprotection prevents ROS production during HL stress by directly interacting with the PSII monomer. The LHL4 protective function is prominent during a first phase of moderate HL stress, i.e. before activation of the canonical NPQ effectors, or when they do not provide sufficient protection under strong HL stress.

Results and Discussion

LHL4 Is Highly Induced upon UV-B.

We analyzed the effect of UV-B on the membrane-enriched proteome of Chlamydomonas and identified several proteins potentially involved in photoprotection among those accumulating under 16 h UV-B (Fig. 1A). After UV-B exposure, 46 proteins showed a significant increase in abundance, and nine proteins showed a significant decrease in abundance. Several proteins that showed increased accumulation in response to UV-B are involved in PSII biogenesis, stability, or repair [Filamentous Temperature-Sensitive-like H (FTSH-like) and Degradation of periplasmic proteases (40), and members of the High Chlorophyll Fluorescence (HCF) family (41)], as well as in NPQ (the two LHC-like proteins LHCSR1 and LHCSR3) (SI Appendix, Table S1). Interestingly, we identified a third LHC-like protein, namely LHL4, enriched more than seven times under UV-B compared to control (Fig. 1A and SI Appendix, Table S1). In a parallel RNA-Seq analysis, LHL4 was also identified as highly induced at the transcript level in response to 1 h exposure to UV-B, in a UVR8-dependent manner, along with 831 genes (Fig. 1B and SI Appendix, Fig. S1 and Table S2). While LHCSRs were detected in both experiments, PSBS was identified among the top-induced genes in our transcriptome dataset (SI Appendix, Table S2), as expected (12, 25), but showed only limited accumulation at the protein level in the UV-B-treated samples (SI Appendix, Table S1), consistent with its reported limited stability over time (11, 14). In total, 44 out of the 46 proteins accumulating in response to UV-B were also found to be transcriptionally induced (Fig. 1C and SI Appendix, Table S3). LHL4 is a 285-amino acid protein with three predicted transmembrane domains that differs from the other LHC-Like proteins (including the ELIP family) because it contains an exceptionally long predicted loop between the second and third transmembrane domains (Fig. 1D) (42, 43). LHL4 homologs are uniquely found in green microalgae (44, 45), where they are closely related to PSBS, but belong to a different clade (Fig. 1E).

Fig. 1.

LHL4 is induced by UV-B in Chlamydomonas. (A) Mass spectrometry (MS)-based quantitative comparison of membrane-enriched proteomes from Chlamydomonas exposed to 16 h of supplemental UV-B [Low Light (LL, 20 µmol photons m⁻² s⁻¹) + UV-B (0.2 mW cm⁻²)] compared to untreated control (LL). Volcano plot displaying the differential abundance of proteins in the membrane proteomes analyzed by MS-based label-free quantitative proteomics. The volcano plot represents the −log₁₀ (P-value), (limma P-value, y axis) plotted against the log₂FC (LL+UV-B vs. LL, x axis) for each quantified protein. Green and red dots represent proteins significantly enriched in LL+UV-B and in LL samples, respectively (log₂FC ≥ 1 and −log₁₀(P-value) ≥ 2.11, corresponding to a Benjamini–Hochberg FDR < 1%). Dots representing LHL4, LHCSR1, and LHCSR3 proteins are indicated. (B) RNA-Seq analysis of Chlamydomonas exposed to 1 h LL+UV-B compared to LL. Volcano plot displaying the differential abundance of transcripts by representing the −log₁₀ (FDR), (y axis) plotted against the log₂FC (LL+UV-B vs. LL, x axis). Dots representing LHL4, PSBS1/PSBS2 (encoding PSBS), LHCSR1, and LHCSR3.1/LHCSR3.2 (encoding LHCSR3) transcripts are indicated. (C) Venn diagram showing the overlap of proteins (blue) and transcripts (orange) significantly enriched in LL+UV-B (SI Appendix, Tables S1 and S2). (D) Scaled schematic representation of LHC-Like protein sizes and the locations of predicted chlorophyll a/b binding domains (green, predicted using the Superfamily database) and transmembrane domains (gray motifs) in LHL4 and in consensus Chlamydomonas ELIP, LHCSR, and PSBS like protein sequences. The red arrow indicates the presence of an exceptionally long loop in LHL4 compared to the other proteins. aa, amino acids. (E) Phylogenetic tree generated from aligned protein sequences of LHC-Like LHCSRs, LHL4s, PSBSs, and ELIPs homologs. Proteins from Chlamydomonas and Arabidopsis are approximatively emphasized by green and gray dots, respectively. Due to their proximity, the two ELIP dots for Arabidopsis overlap.

LHL4 Expression Is Controlled by UVR8 and PHOT Photoreceptors and Depends on CrCOP1 and CrCO.

LHL4 gene expression was found to be rapidly and transiently induced in response to both UV-B and HL (Fig. 2A) (25, 42, 43). LHL4 showed a higher induction by UV-B than HL at both transcript and protein levels. The LHL4 protein level rapidly increased up to 4 h and remained stable for 8 h upon both light treatments (Fig. 2B). However, the LHL4 abundance rapidly decreased when cells were returned to low light (LL), unlike LHCSR1 and LHCSR3, which level remained stable for at least 8 h posttreatment (Fig. 2C) (46).

Fig. 2.

LHL4 expression is controlled by UVR8 and PHOT photoreceptor signaling pathways. (A) RT-qPCR analysis of LHL4 expression in WT 137C cells grown under LL (20 µmol photons m⁻² s⁻¹) and then transferred to LL supplemented with UV-B (0.2 mW cm⁻², LL+UV-B), or HL (300 µmol photons m⁻² s⁻¹) for the indicated times. The data were normalized to the LHL4 levels at time 0 for each condition. Individual data points of biological replicates and means ± SD are shown (n = 4). (B) Immunodetection of LHL4 in cells exposed for up to 8 h under LL, LL+UV-B, or HL. ATP synthase beta subunit (ATPB) levels were used as loading control. (C) Comparative stability of LHL4 and LHCSRs proteins. Immunodetection of LHL4, LHCSR1, LHSCR3, and ATPB in cells sequentially exposed to LL (20 μmol photons m⁻² s⁻¹) supplemented with UV-B (0.2 mW cm⁻²) for 16 h and then HL (300 μmol photons m⁻² s⁻¹) for 4 h. Cells were finally placed under LL (20 μmol photons m⁻² s⁻¹) for the indicated times. ATPB was used as loading control. (D–F) RT-qPCR analysis of LHL4 expression in (D) uvr8 and phot, (E) cop1^hit1, and (F) crco and crblz3 cells grown under LL (0), or exposed for 1 h to LL+UV-B, or HL. Data are normalized to levels in respective WTs (strains 137C, CC124, CC5325) under LL (“0”). Values of independent measurements and means ± SD are shown (n = 3). (G–I) Immunodetection of LHL4 in uvr8 and phot, (H) cop1^hit1, (I) crco and crblz3, as well as their respective WT cells grown under LL (0), or 6 h LL+UV-B, or HL. ATPB was used as loading control.

As LHL4 expression is induced in response to UV-B and HL, we examined the accumulation of LHL4 transcripts and LHL4 proteins in uvr8 and phot mutants. UV-B-dependent induction of LHL4 expression was abolished in the uvr8 mutant, but not affected in a phot mutant (Fig. 2D). Consistently, UV-B-induced LHL4 protein accumulation was absent in uvr8 but comparable to wild type (WT) in phot cells (Fig. 2G). Under HL, a comparably weak LHL4 induction was observed in both photoreceptor mutants and WT cells (Fig. 2D). However, accumulation of LHL4 protein was reduced in response to HL in the phot mutant compared to WT and uvr8 (Fig. 2G), similar to LHCSR3 (SI Appendix, Fig. S2A) (17). Altogether, our data demonstrate that the induction of LHL4 accumulation by UV-B and HL is mediated by UVR8 and at least partially PHOT, respectively. We thus examined the involvement of downstream signaling components on LHL4 induction and LHL4 accumulation. We first used the cop1^hit1 loss-of-function mutant strain that contains an Arg-1256-to-Pro mutation (CrCOP1^R1256P) in the C-terminal WD40 domain (12, 33). Under both UV-B and HL, the cop1^hit1 mutant exhibited a much weaker induction of LHL4 transcripts and reduced LHL4 protein accumulation in comparison to WT (Fig. 2 E and H). This result is similar to the reduced accumulation of LHCSR1 and LHCSR3 (SI Appendix, Fig. S2B), as well as PSBS (12), confirming that the UV-B and HL signaling pathways converge at the level of COP1. It is also of note that cop1^hit1 is more strongly affected in HL-induced LHCSR3 and LHCSR1 accumulation than phot (SI Appendix, Fig. S2 A and B), suggesting that additional HL-induced signaling pathway(s) converge at COP1.

We next investigated transcription factors potentially involved in LHL4 regulation upon UV-B and HL by comparing LHL4 levels in crco (28, 32) and crblz3 [CrBLZ3 is a putative AtHY5 ortholog, (39)] mutants with WT. At the transcriptome-wide level, we found that both CrCO and CrBLZ3 are required for UV-B-regulated gene expression with CrCO playing a major role (SI Appendix, Fig. S3 A and B and Table S2). In agreement, accumulation of LHL4 mRNA and LHL4 protein in response to both UV-B and HL was severely impaired in crco but was similar between crblz3 and WT (Fig. 2 F and I and SI Appendix, Fig. S2C and Table S2). We conclude that UV-B- and HL-dependent accumulation of LHL4 depends on UVR8 and partially on PHOT, respectively, and in both cases involves CrCO-dependent transcriptional activation of LHL4 expression.

LHL4 Binds the PSII Monomer upon UV-B Exposure.

To elucidate the integration of LHL4 within the Chlamydomonas photosynthetic apparatus, we performed biochemical analysis under nondenaturing conditions by Blue native polyacrylamide gel electrophoresis (BN-PAGE) of solubilized thylakoid complexes isolated from WT cells exposed to UV-B. We found that LHL4 was only detected upon UV-B exposure and migrated in the LHCII trimer region, as well as in two different spots in proximity to the PSII monomer/cytochrome b₆f bands (Fig. 3A). We then used complexome profiling (47) to analyze the protein composition of these two spots by cutting this specific region of the gel into six slices and examining their protein content using mass spectrometry (MS) (Fig. 3B). LHL4 was found to be localized in two different organizations of the PSII monomer. The lower band (band 5) corresponded to a PSII monomer containing only one of the two core antennae, specifically CP47, known as the RC47 intermediate complex during PSII assembly (48). The upper band (band 1) consisted of the full PSII monomer with both core antennae, CP43 and CP47. LHL4 was also detected in LL-acclimated samples with the same accumulation pattern (Fig. 3B), albeit at a much lower level compared to UV-B-acclimated samples. This suggests that a basal level of the protein is also constitutively present within the PSII monomer in nonstressed samples. To identify potential interactions of LHL4 within the PSII monomer, we then used a combination of BN-PAGE and cross-linking MS (XL-MS) analysis. This approach allows capturing protein–protein interaction by creating covalent bonds between amino acids in close proximity and can be directly applied to complexes previously separated via BN-PAGE (49). Importantly, the chemical cross-linking reaction did not produce any noticeable difference on the presence of major photosynthetic complexes (SI Appendix, Fig. S4A). XL-MS data acquired on these specific gel bands were first validated by mapping the cross-links on known PSII and cytochrome b₆f structures (SI Appendix, Fig. S4 B–D), where most fall within an acceptable 20 Å distance cut-off (SI Appendix, Fig. S4C) (50). We detected two cross-links of LHL4 with both CP43 and CP47, these are compatible with two putative positions predicted by using AlphaFold2 multimer (49) (SI Appendix, Fig. S5). The distances of these two reproducible cross-links with CP43 and CP47 are of ~12 and ~20 Å, respectively (SI Appendix, Fig. S5), thus validating both structural models. Taken together, these data suggest that LHL4 binds to a fully assembled monomer on both inner antennae (Fig. 3C), producing a complex at higher molecular weight detected by immunodetection (Fig. 3A) and complexome profiling (band 1, Fig. 3B). Alternatively, LHL4 may bind to a partially assembled RC47 on CP47 only, resulting in a smaller complex (Fig. 3A and band 5 in Fig. 3B). In both cases, LHL4 binds to the site where the second PSII monomer attaches, suggesting that the protein may need to be removed prior to dimer assembly. We propose that LHL4 acts as or is a part of a specific antenna for the PSII monomer, capable of binding to the complex early in its assembly process by a direct connection to the two core antenna proteins CP43 and CP47.

Fig. 3.

LHL4 localizes with PSII monomers at the dimerization interface interacting with the inner PSII antenna proteins CP43 and CP47. (A) BN-PAGE of thylakoids extracted from cells exposed to LL (20 µmol photons m⁻² s⁻¹) or LL supplemented with UV-B (LL +UV-B; 0.2 mW cm⁻²). Second dimension blots indicate LHL4 (red circles), and its absence in LL cells. A blue circle indicates a nonspecific band detected by the LHL4 antibody in both WT and lhl4. (B) The middle section of the gel was divided into six parts, as shown in the Left image. The relative abundance of LHL4 (Left graph) and the main components of the PSII monomer (Right graph; UV-B acclimated cells) were assessed by MS. n = 4 (C) LHL4 interactors CP43 and CP47 uncovered by cross-linking and in-gel digestion of the BN-PAGE band. The two AlphaFold2 pairwise predictions of LHL4-CP43/47 are shown overlaid on the PSII monomer structure (PDB 6KAD, light blue) and both are localized at the dimerization interface.

LHL4 Prevents PSII Photoinhibition upon Exposure to HL.

The sequence homology of LHL4 with LHCSR and PSBS, its accumulation in response to both UV-B and HL, as well as its location within the PSII monomer may suggest a role for LHL4 in photoprotection. To investigate this possibility, we generated lhl4 knock-out mutants using CRISPR-Cas9 and evaluated their tolerance to HL compared to WT. LL-acclimated cells were exposed to HL (900 µmol photons m⁻² s⁻¹) for 1 h, followed by 1 h of recovery under LL. Their photosynthetic capacity was monitored as the maximum quantum yield of PSII (F_v/F_m). Lower F_v/F_m values indicate, but are not limited to, PSII damage due to photoinhibition (51). lhl4 mutants showed no difference in PSII antenna size compared to WT (SI Appendix, Fig. S6) but exhibited impaired recovery from light stress (Fig. 4 A and B). To further characterize the role of LHL4, we conducted parallel experiments using lincomycin to inhibit the synthesis of chloroplast-encoded proteins of PSII, effectively blocking its repair system. Upon lincomycin treatment, we assume that recovery is linked to reversibly photodamaged PSII, as suggested before (52). On the contrary, the additional recovery observed without the inhibitor likely reflects irreversible photodamaged PSII, which can only be repaired via assembly of de novo synthesized PSII proteins (52). As expected, we observed a significant reduction in the recovery of both strains compared to untreated samples upon addition of lincomycin. While the inhibition rate was the same, a large difference in the recovery between WT and lhl4 became evident (Fig. 4 A and B). This finding rules out a direct role for LHL4 in the repair process, in agreement with a parallel report (53). Instead, it implies a role of LHL4 in PSII recovery from light-mediated degradation, which would stem from increased pool of reversibly photodamaged PSII. By binding to the PSII monomer at the core antenna subunits CP43 and CP47, we propose that LHL4 might protect the PSII monomer pool from degradation during HL. This could help preserve its structural integrity and facilitate the assembly of functional PSII dimers, leading to a more efficient recovery.

Fig. 4.

LHL4 protects PSII and facilitates cell survival under HL. WT and lhl4 cells were grown under LL (20 µmol photons m⁻² s⁻¹). (A) Cells were exposed to HL (900 µmol photons m⁻² s⁻¹) for 1 h and then placed under LL for 1 h and their maximum PSII quantum yield (Fv/Fm) was monitored after 1 min of dark relaxation. The data presented are representative of four biological replicates. Errors bars represent the SE of the means of three technical replicates. (B) The rate of recovery was calculated using the data from panel A by determining the slope of the first 30 min of recovery under LL and 1 min of dark relaxation. n = 4. The asterisk indicates statistical difference (***P < 0.001, ****P < 0.0001). (C) WT and lhl4 cells were exposed to HL (600 µmol photons m⁻² s⁻¹, red light) and ¹O₂ accumulation was monitored over 80 min of exposure. n = 3. Asterisks indicate statistical differences (*P < 0.05). (D) Photographs of WT and lhl4 cultures were taken after LL acclimation (0) and then after 1, 1.5, and 2 h under 2,000 µmol photons m⁻² s⁻¹.

A defect in photoprotection generally leads to ROS production during photosynthesis, mainly singlet oxygen (¹O₂) at the PSII level (8). We measured ¹O₂ levels in WT and lhl4 during HL exposure (Fig. 4C). During early HL exposure (20 min), i.e., when LHL4 expression is still very low (Fig. 3B), ¹O₂ levels are comparable in both strains. However, we observed a significant overaccumulation of ¹O₂ in the lhl4 strain compared to the WT under prolonged stress, while LHL4 accumulated at a higher level in the WT (Fig. 4C). Our data suggest that the protection of PSII monomers by LHL4 facilitates PSII renewal and helps limiting photoinhibition and ¹O₂ production. Accordingly, in case of higher (2,000 µmol photons m⁻² s⁻¹) and prolonged light stress exposure, in contrast to WT, lhl4 cultures were unable to survive, resulting in complete bleaching within 2 h of exposure (Fig. 4D).

To cope with the harmful effects of light stress, photosynthetic organisms can activate NPQ. We investigated the interplay between the NPQ-driven and LHL4-mediated protection. First, we monitored the phenotype of LHL4 during the first 3 h of HL exposure. During this phase, cells begin to accumulate the LHCSR3 protein, which is responsible for NPQ activity (54, 55). We observed a stable difference in the recovery between WT and lhl4 cells during the first 2 h of HL (SI Appendix, Fig. S7A). At this point, NPQ became activated to a similar extent in both strains (SI Appendix, Fig. S7B). This observation suggests that LHL4 does not directly contribute to this process in Chlamydomonas. Alternatively, NPQ capacity could compensate, at least partially, the absence of protection conferred by LHL4 in the mutant. After 3 h of exposure, NPQ was highly activated, leading to a higher Fv/Fm and similar fluorescence recovery between the two strains. Overall, these results suggest that LHL4 primarily catalyzes a recovery from light stress when cells have not yet developed their NPQ capacity.

We further dissected the impact of NPQ on LHL4-mediated protection, focusing on UV-B acclimated cells that had fully developed their NPQ capacity (SI Appendix, Fig. S7C). We observed that LHL4 accumulation in UV-B-acclimated cells was significantly lower after 1 h of HL exposure compared to LL-acclimated cells (SI Appendix, Fig. S7D). Additionally, the lhl4 strain showed no significant difference in Fv/Fm compared to the WT during the first hour of light stress and subsequent recovery, which was significantly higher than in nonacclimated cells (SI Appendix, Fig. S7 E and F). These results confirm that when cells have acquired NPQ capacity, energy dissipation via NPQ prevents photoinhibition, reducing PSII damage and turnover, and limits LHL4 induction, which is not essential under these conditions. We further evaluated the impact of LHL4 under long-term exposure and stronger HL intensity (2,000 µmol photons m⁻² s⁻¹) in UV-B acclimated samples. Under these conditions, LHL4 levels are maintained at a very high level (SI Appendix, Fig. S7G). Both WT and lhl4 cells retained their green pigmentation longer than LL-acclimated cells (SI Appendix, Fig. S7H vs. Fig. 4D). However, after 8 h of prolonged exposure, lhl4 failed to endure the stress and bleached, whereas the WT cells remained green (SI Appendix, Fig. S7H). These results suggest that under prolonged and extremely high irradiance, NPQ alone is no longer sufficient to prevent photodamage. Therefore, the cumulative effect over time of the absence of PSII monomers-driven LHL4 protection affects cell survival. This complementary role of NPQ and LHL4 in these extreme conditions underscores the importance of LHL4 in priming UV-B-induced photoprotection in Chlamydomonas, alongside LHCSRs (12).

Conclusion

Our study reveals a key role for the LHL4 protein in photoprotection. LHL4 protects PSII monomers by associating with the core complex via two antenna proteins, CP43 and CP47. This protection takes place during the early stages of photodamage upon HL exposure, but also under prolonged stress conditions when light intensity is too high. Protection conferred by LHL4 occurs following cell exposure to UV-B and HL, which concomitantly activate the UVR8 and PHOT photoreceptor signaling pathways that converge likely at the COP1 level in Chlamydomonas. Our phylogenetic analysis indicates that LHL4 is exclusively present in green microalgae. However, its crucial function in photoprotection of these organisms suggests the existence of similar actors in other phototrophs. Consistently, specific High-Light-Inducible Proteins (HLIPs) have been identified in cyanobacteria, where they associate with CP47 and intermediates of PSII assembly modules (56). Remarkably, cyanobacteria lacking HLIPs are viable but experience extreme light-induced stress levels, suggesting the presence of protective mechanism in prokaryotic photosynthetic organisms similar to the one conferred by Chlamydomonas LHL4. Investigating potential candidates in other microalgae and flowering plants may unlock a deeper understanding of photoprotection mechanisms across diverse photosynthetic life forms.

Materials and Methods

Algae Strains.

Chlamydomonas mutant strains lhl4 (LHL4^K34*; ^* indicating a translation stop codon), uvr8 (UVR8^S86*), and phot (PHOT^C32*) were generated in the WT137C parental strain (55) (SI Appendix, Fig. S8) by introducing a TAA STOP codon by homology-directed repair using CRISPR-Cas9 (57, 58). The following target sequences were used: 5′-CGTGTCAACGCCGAGAAAAG-3′ for lhl4, 5′-CGAGGACAGATCTACAGCTG-3′ for uvr8, and 5′-CACGCTTCCGGACTGTCCGC-3′ for phot. The following templates for DNA repair were used: 5′-CGCAGTGATTGCCCGTGTCAACGCCGAGAAtataaggaccacgacatcgactacaaggacAAGTGGCTTTGCAAAGGTAAGCTGAGAACT-3′ for lhl4, 5′-GGTCGCGTCGTCACGAGGACAGATCTACAGtataaggaccacgacatcgactacaaggacCTGGGGCTGGTGGGTGGCGTTGGGGCGCAG-3′ for uvr8, and 5′-TCGTCGCAGATGCCACGCTTCCGGACTGTCtataaggaccacgacatcgactacaaggacCGCTGGTCTACGCCAGCGAGGGGTGAGCGG-3′ for phot. The cop1^hit1 mutant is in the CC124 WT background (33). crco (LMJ.RY402.149321) (28, 32) and crblz3 (LMJ.RY402.194448) (39) mutants were obtained from the Chlamydomonas Library Project (CLiP) (59) together with their corresponding WT (CC5325). uvr8 (LMJ.RY40202.156289) (12) from the CLiP library was used for the RNA-seq experiment.

Algae Culture and Light Treatment.

Algae cells were grown in Tris Acetate Phosphate growth medium (60) under continuous light (20 µmol photons m⁻² s⁻¹, from white Light Emitting Diode (LED) panels), 25 °C, and shaking (110 rpm). In all experiments, cells were harvested during the exponential phase (2 to 3 × 10⁶ cells per mL), washed, and resuspended in High Salt Medium minimal growth medium at 4.5 × 10⁶ cells per mL, with the exception of Fig. 4D where they were resuspended at 6 × 10⁶ cells per mL. Cells were placed for at least 1 h under dim light (20 µmol photons m⁻² s⁻¹) before applying specific light treatments. UV-B-exposed samples (+UV-B) were treated by Philips TL20W/01S narrowband UV-B tubes filtered with a WG filter (Schott Glaswerke) with half-maximal transmission at 311 nm (0.07 or 0.2 mW cm⁻²) (61). Control (−UV-B) samples were placed under a WG filter with half-maximal transmission at 360 nm to block UV-B. In both conditions, samples were concomitantly exposed to dim light (20 µmol photons m⁻² s⁻¹) provided by fluorescent white light tubes (Osram Dulux L). Cells were exposed to UV-B for 6 h, with the exception of Fig. 1A and SI Appendix, Fig. S7 C–H, where the treatment duration was extended to 16 h to fully induce the UV-B protection of the photosynthetic machinery, as detailed in ref. 12. For HL treatment, cells were placed under white LED panels (SL 3500, Photon Systems Instruments) at the indicated light intensity.

Phylogenetic Analysis.

The sequences of LHL4, PSBS1, LHCSR3.1, and ELIP1-9 were subjected to a blastp analysis using the online tool available at https://www.ncbi.nlm.nih.gov/ with a percent identity range of 35 to 100% and a query coverage range of 50 to 100%. Protein sequences were then aligned using the Multiple Alignment using Fast Fourier Transform tool (auto strategy, https://mafft.cbrc.jp/alignment/server/). To enhance the accuracy of the alignment, manual curation was performed using Jalview to eliminate any duplicate or misaligned sequences. The phylogenetic tree was constructed using IQ-TREE, employing the LG+F+I+G4 model. The generated tree was then visualized graphically using iTOL v7, a web-based tool accessible at https://itol.embl.de/.

Protein Extraction and Immunoblot Analysis.

Cells were pelleted and resuspended in 80% (v/v) acetone. Proteins were precipitated by centrifugation at 4 °C and resuspended in lysis buffer [100 mM Tris-HCl, pH 6.8, 4% (v/v) SDS, 20 mM Ethylenediaminetetraacetic acid (EDTA)] supplemented with protease inhibitor (cOmplete, Roche). 10 to 30 µg of proteins were separated by SDS-PAGE in Tris-Glycine buffer (25 mM Tris, 190 mM glycine, 0.05% Sodium Dodecyl Sulfate (SDS)), transferred onto nitrocellulose membrane using the same buffer supplemented with 20% (v/v) ethanol for 80 min at 110 V. Anti-CrUVR8 (25), anti-PHOT (17), anti-LHCSR3 (AS142766; Agrisera), anti-LHCSR1 (AS142819; Agrisera), anti-LHL4 (AS07250, Agrisera), anti-ATPB (AS05085; Agrisera), anti-CP43 (AS111787; Agrisera), and anti-PSAH (AS06143; Agrisera) were used in this study. Detection was carried out with an Horseradish Peroxydase -conjugated secondary antibody, and the signal was developed using the Clarity Western ECL Substrates kit (Biorad). Images of the blots were captured using a Charge-Coupled Device imager.

RNA Extraction.

Cells were prepared as described above. 20 million cells were then pelleted and frozen in liquid nitrogen. RNA were extracted using the RNeasy Plant Mini Kit (Qiagen) including a DNase treatment to remove residual genomic DNA (RNase-Free DNase Set, Qiagen).

Quantitative Real-Time PCR (qRT-PCR).

cDNA synthesis was performed with the TaqMan Reverse Transcription Reagents kit (Applied Biosystems). Amplification by RT-qPCR was performed using complementary DNA (cDNA) in the presence of SYBR Green (Master Mix PCR Power SYBR™, Applied Biosystems) and specific primers (0.3 µM) for the amplification of the gene of interest, using a CFX Connect Real-Time PCR Detection System (Biorad). Data were analyzed using the ΔΔCt method (62) and the Cre06.g278222_4532 reference gene (25, 63). The following primers were used: 5′- TACGGTGTGGATGACGTGAC-3′ and 5′- AGGTGATAATCTGGCGGATG-3′ for LHL4 and 5′- CTTCTCGCCCATGACCAC-3′ and 5′- CCCACCAGGTTGTTCTTCAG-3′ for Cre06.g278222_4532.

RNA-seq Analysis.

Cells of WT CC-5325, uvr8, crco, and crblz3 were exposed for 1 h to LL (20 µmol photons m⁻² s⁻¹; −UV-B control samples) or LL supplemented with 0.2 mW cm⁻² of UV-B (+UV-B). Total RNA was extracted from 20 million cells of three independent biological replicates. The RNA quality control, library preparation using TruSeqHT Stranded mRNA (Illumina), and sequencing on an Illumina HiSeq 4000 System using 100-bp single-end reads protocol were performed at the iGE3 genomics platform of the University of Geneva. Quality control was performed with FastQCv.0.11.9. Reads were mapped to the Chlamydomonas genome CreinhardtiiCC_4532_707_v6.1 (PhytozomeV13) using STARv.2.7.10b software (64), with average alignment of 91.65%. Raw counts were obtained using HTSeq v.0.11.3 (65). Filtering out lowly expressed genes (13,613 genes were kept), normalization and differential expression analysis were performed with the R/Bioconductor package edgeR v.3.42.4 (66), and statistical significance was assessed with a general linear model, negative binomial distribution, and quasi-likelihood F test. Genes with a fold change ≥2 and P-value ≤ 0.05 [with a multiple testing Benjamini and Hochberg False Discovery Rate (FDR) correction] were considered differentially expressed. Annotations (v6.1) were obtained from the Phytozome database (v13). The RNA‐Seq data reported have been deposited in the NCBI Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series accession number GSE255943.

ROS Quantification.

¹O₂ production was estimated using the Singlet Oxygen Sensor Green (SOSG, Invitrogen) dye (67). Cultures at 3 × 10⁶ cells/mL were placed in 24-well plates. 0.5 µM of SOSG was added to each well, before exposure for 80 min to red light (630 nm, 600 µmol photons m⁻² s⁻¹, equivalent to 900 µmol photons m⁻² s⁻¹ white light in our condition). This wavelength minimizes photobleaching of the dye, which is sensitive to blue light and prolonged exposure to high-intensity illumination (68). The fluorescence of each well was then measured with a plate reader (Infinite M1000; Tecan) (λ excitation: 504 nm; λ emission: 525 nm ± 5 nm).

NPQ, Photoinhibition, and Antenna Size Measurements.

Chlorophyll fluorescence was imaged in 100 µL of cell suspension in a 96-well plate. Fluorescence was quantified using a Speedzen III fluorescence imaging setup (JBeam Bio). Maximum fluorescence in the dark (F_m) or during actinic light exposure (F_m’, 900 µmol photons m⁻² s⁻¹) was measured using saturating red pulses (250 ms, 3,000 µmol photons m⁻² s⁻¹ at 630 nm) followed by blue light (470 nm) detection pulses (10 µs). NPQ was calculated as (F_m − F_m’)/F_m’ (69). For photoinhibition experiments, cells were treated in 24-well plates for 1 h with HL (white light, 900 µmol photons m⁻² s⁻¹) and then placed under LL for 1 h (20 µmol photons m⁻² s⁻¹). Lincomycin was added at a final concentration of 1.2 mM to inhibit chloroplast translation when needed. The chlorophyll fluorescence was then measured before (F₀) and right after the saturating pulse (F_m), and the maximum quantum yield of PSII was calculated as F_v/F_m = (F_m − F₀)/F_m. Antenna size was measured under different light intensity after addition of 10 µM of [(3-(3,4-dichlorophenyl)-1,1-dimethylurea] and estimated as the half-time to reach F_m level (70, 71). Samples were dark adapted for at least 10 min for NPQ (55) and antenna size experiments, 1 min for photoinhibition experiments (52).

MS-Based Proteomic Analyses.

Three biological replicates of Chlamydomonas cells exposed or not to UV-B for 16 h were prepared. The Chlamydomonas cells were disrupted in a tube containing glass beads using a Precellys instrument (Bertin Technologies) at 7,500 rpm and 4 °C for two cycles of 30 s each, with a 30-s pause in between. Subsequently, the samples were centrifuged at 20,000 g for 5 min at 4 °C. The resulting pellets, enriched with membrane proteins, were resuspended in a solution containing 50 mM Tris-HCl pH 6.8, 2% SDS, 10 mM EDTA, and protease inhibitor (cOmplete, Roche) to maintain the integrity of the proteins during further processing. Proteins were then solubilized in buffer (72) and heated for 10 min at 95 °C. They were then stacked in the top of a 4 to 12% NuPAGE gel (Invitrogen), stained with Coomassie blue R-250 (Bio-Rad) before in-gel digestion using modified trypsin (Promega, sequencing grade) as previously described (73). The resulting peptides were analyzed by online nanoliquid chromatography coupled to MS/MS (Ultimate 3000 RSLCnano and Q-Exactive HF, Thermo Fisher Scientific) using a 120-min gradient. For this purpose, the peptides were sampled on a precolumn (300 μm × 5 mm PepMap C18, Thermo Scientific) and separated in a 75 μm × 250 mm C18 column (Reprosil-Pur 120 C18-AQ, 1.9 μm; Maisch GmbH). The MS and MS/MS data were acquired using Xcalibur version 2.9 (Thermo Fisher Scientific).

Peptides and proteins were identified by Mascot (version 2.8.0, Matrix Science) through concomitant searches against the Chlre5_6 databases [downloaded from JGI Genome Portal, (74), 19,526 sequences], the mitochondrion and chloroplast protein sequences (downloaded from NCBI, respectively 69 and 8 proteins), and a homemade database containing the sequences of classical contaminant proteins found in proteomic analyses (human keratins, trypsin, … 126 sequences). Trypsin/P was chosen as the enzyme and two missed cleavages were allowed. Precursor and fragment mass error tolerances were set respectively at 10 and 20 ppm. Peptide modifications allowed during the search were Carbamidomethyl (C, fixed), Acetyl (Protein N-term, variable), and Oxidation (M, variable). The Proline software [version 2.2.0, (75)] was used for the compilation, grouping, and filtering of the results [conservation of rank 1 peptides, peptide length ≥ 6 amino acids, FDR of peptide-spectrum-match identifications < 1% (76), and minimum of one specific peptide per identified protein group]. Proline was then used to perform a MS1 label-free quantification of the identified protein groups based on razor and specific peptides.

Statistical analysis was performed using the ProStaR software (77) based on the quantitative data obtained with the three biological replicates analyzed per condition. Proteins identified in the contaminant database, proteins identified by MS/MS in less than two replicates of one condition, and proteins quantified in less than three replicates of one condition were discarded. After log2 transformation, abundance values were normalized using the variance stabilizing normalization method, before missing value imputation (Sparse Low-Rank Sequence Alignment algorithm for partially observed values in the condition and DetQuantile algorithm for totally absent values in the condition). Statistical testing was conducted with limma, whereby differentially expressed proteins were selected using a log₂FC cut-off of 1 and a P-value cut-off of 0.00776, allowing to reach a FDR inferior to 1% according to the Benjamini–Hochberg estimator. Proteins found differentially abundant but identified by MS/MS in less than two replicates or detected in less than three replicates in the condition in which they were found to be more abundant were manually invalidated (P-value = 1).

Thylakoid Isolation and BN-PAGE.

400 million cells were broken in a 2 mL tube containing glass beads using a Precellys instrument (Bertin Technologies) at 7,500 rpm and 4 °C for two cycles of 30 s each, with a 30-s pause in between. The lysate was diluted in 25 mM HEPES pH 7.5, 5 mM MgCl₂, 0.3 M sucrose, and protease inhibitor (cOmplete, Roche) and then centrifuged at 20,000 g for 5 min at 4 °C. The pellet was resuspended in 1 mM HEPES pH 7.5, 5 mM EDTA, and 0.3 M sucrose and centrifuged again at the same speed. Thylakoids were isolated using a discontinuous three-step sucrose gradient (1.8 M, 1.3 M, 0.5 M) after centrifugation (76,000 g, 1 h, 4 °C). The chlorophyll concentration was estimated, and 20 µg of chlorophyll was solubilized in 1% α-dodecylmaltoside for 5 min at room temperature. For the BN-PAGE analysis, a loading buffer (0.5 M aminocaproic acid, 30% sucrose, 100 mM Bis-Tris HCl pH 7, and 50 mg/mL blue Coomassie G-250) was added to the sample. The thylakoid extract was then loaded onto a 4 to 16% NativePAGE (InVitrogen), and gel electrophoresis was conducted with increasing voltage intensity (ranging from 75 V to 200 V) in an anode buffer (50 mM Bis-Tris HCl pH 7) and a blue cathode buffer (50 mM Tricine, 15 mM Bis-Tris HCl pH 7, and 0.01% blue Coomassie G-250). When the migration reached the middle of the gel, the blue buffer was replaced with fresh cathode buffer devoid of Coomassie blue.

Cross-Linking MS and Structural Modeling.

UV-B-treated and untreated (control) cells were disrupted in a tube containing glass beads using a Precellys instrument (Bertin Technologies) at 7,500 rpm and 4 °C for two cycles of 30 s each, with a 30-s pause in between. Cell lysates were chemically cross-linked with 8 mM 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM) for 30 min at 4 °C in the darkness). We used the water-soluble DMTMM reagent because of the scarce availability of exposed Lys residues in the loops of LHL4. DMTMM is considered as a “short-range” cross-linker, lacking a spacer arm and with reactive groups targeting carboxylic acids and primary amines (aspartic and glutamic acids to lysine and amino termini of proteins). This reagent provides distance constraints between 0 and ~25 Å considering the flexibility of the side chains (7 and 5 Å for lysine and carboxylic acids, respectively), the α-carbon backbone (6 Å), and the overall protein flexibility (50, 78). Cross-linked thylakoids were purified according to established protocols and then solubilized for BN-PAGE as described above. Comparison of cross-linked and non-cross-linked thylakoids showed no noticeable differences in the band patterns, we thus assumed the XL reaction did not produce artifacts (SI Appendix, Fig. S4). The entire region where the LHL4 antibody was giving a signal on the second dimension from three technical replicates was excised (Fig. 3A) and carefully divided into six pieces of ~1 mm. The bands were then subjected to in-gel digestion and MS acquisition as for the proteomic analyses in order to precisely quantify the protein comigrating in these different regions. For the in-gel cross-linking, the entire region was washed rapidly in milliQ water, and soaked for 30 min in 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes) pH 7.5, 10 mM EDTA pH 8, and 1.3 M sucrose supplemented with 16 mM DMTMM cross-linker. The cross-linking reaction was then quenched with 10 mM Tris-HCl for 10 min and in-gel digested with the same procedure as for the proteomic analysis (73). Briefly, Liquid Chromatography (LC) separation gradients were of 180 min with elution gradient profiles as follows: 0 to 10% solvent B (0.1% (v/v) formic acid in 80% (v/v) acetonitrile) over 10 min, 12 to 35% solvent B over 125 min, 36 to 44% solvent B over 20 min, 45 to 100% solvent B over 10 min, and finally 100% B for 15 min. Full-scan MS spectra were collected in a mass range of m/z 350 to 1,300 Th in the Orbitrap at a resolution of 60,000 at m/z = 200 Th after accumulation to an Automatic Gain Control (AGC) target value of 1e6 with a maximum injection time of 50 ms. In-source fragmentation was activated and set to 15 eV. The cycle time for the acquisition of MS/MS fragmentation scans was set to 2 s. Charge states accepted for MS/MS fragmentation were set to 3 to 8. Dynamic exclusion properties were set to n = 1 and to an exclusion duration of 15 s. Stepped Higher energy Collision Dissociation fragmentation (MS/MS) was performed with increasing normalized collision energy (27, 30, 33%) and the mass spectra acquired in the Orbitrap at a resolution of 30,000 at m/z = 200 Th after accumulation to an AGC target value of 1e5 with an isolation window of m/z = 1.4 Th and maximum injection time of 120 ms. Additionally, LHL4-containing BN-PAGE bands were acquired with a MS method similar to that used for normal tryptic peptides as described above in order to screen proteins present in the BN-PAGE band.

Raw data were searched using pLink2 (79) for cross-linked peptide pairs or as described above for normal peptide search. A minimal peptide length of six and two miss cleaved sites was allowed. Cysteine carbamidomethylation was set as fixed modification. Methionine oxidation, protein N-term acetylation, and lysine acetylation were set as dynamic modifications. For cross-linked peptides, the reference database containing only the proteins identified from the normal peptide was used, but with an increased number of missed cleavages allowed of 3. Identified cross-links were only accepted through at 1% FDR filter and if present in both replicates.

Cross-links identified were mapped using the ChimeraX plugin XMAS (80) on the PSII monomer (extrapolated from PDB #6KAD) and Cyt b6f (PDB #1Q90) structures. The predicted LHL4 AlphaFold2 model downloadable from UniprotKB was used. For additional validation of the interaction, we docked in parallel LHL4 for the core complex on the HADDOCK webserver (81), using the identified cross-links as distance constraints and predicting the pairwise interaction between CP43/CP47 and LHL4 with Alphafold2 multimer algorithm in ColabFold (82).

For BN-PAGE complexome profiling, the raw LC–MS/MS data were processed using the MS-Fragger (v4.0) (83) integrated into the FragPipe GUI (v21.1). The search was performed against the Chlre5_6 databases [downloaded from JGI Genome Portal, (74), 19,526 sequences]. An integrated contaminant database was used to assign common contaminant proteins. For search, enzyme specificity was set to Trypsin/P (C-terminal cleavage of lysine and arginine) with up to two missed cleavages. The minimal peptide length was set to seven and the maximal peptide mass was set to 6,000 Da. For peptide search, fixed carbamidomethylation of cysteine and up to two variable modifications per peptide were allowed, namely: methionine oxidation and protein N-terminal acetylation. For the database search, precursor and fragment ions mass error was set to 10 and 20 ppm, respectively. Match between runs was allowed within a retention time window of 0.5 min. A FDR of 1% for peptide spectrum matches and proteins was applied. The output result file was further processed to normalize protein abundances on the total intensity of the identified peptides in each run.

Accession Numbers.

Sequence data from this article can be found in the Joint Genome Institute Phytozome data libraries (https://phytozome.jgi.doe.gov/pz/portal.html) under the accession numbers (Chlamydomonas genome v6.1) LHL4, Cre17.g740950_4532. UVR8, Cre05.g230600_4532. PHOT, Cre03.g199000_4532. COP1, Cre02.g085050_4532. CrCO, Cre06.g278159_4532. CrBLZ3, Cre06.g310500_4532. LHCSR1, Cre08.g365900_4532. LHCSR3, Cre08.g367500_4532 and Cre08.g367400_4532. PSBS, Cre01.g016600_4532 and Cre01.g016750_4532, ELIP1, Cre14.g626750_4532. ELIP2, Cre16.g679250_4532. ELIP3, Cre02.g143550_4532. ELIP4, Cre07.g320400_4532. ELIP5, Cre04.g211850_4532. ELIP7, Cre08.g384650_4532. ELIP8, Cre09.g393173_4532. ELIP9, Cre09.g394325_4532. RACK1, Cre06.g278222_4532.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

Dataset S04 (XLSX)

Data, Materials, and Software Availability

All MS raw data files, including the search results (spectral matches and the cross-link tables), are deposited to the ProteomeXchange Consortium via the PRIDE partner repository (dataset identifier PXD048496 (84) for the bottom–up proteomics data, PXD055508 (85) for the BN-PAGE complexome profiling data, and PXD049352 (86) for the XL-MS data). The RNA-Seq data reported in this article have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE255943 (87). The two AlphaFold2 predictions and the combinatory PDB model showing the two predicted interacting positions of LHL4 are available as a Zenodo public repository deposition (https://zenodo.org/records/14262714) (88).