Mechanisms of light harvesting complex proteins in photoprotection of the brown tide alga

Domain: neuroscience
Published: 2025-12-12
Editorial outcome: major_revision

Abstract
The propensity of Aureococcus anophagefferens to form harmful brown tide blooms has been linked to rapid light responses, but the underlying molecular mechanisms remain elusive. Here, we find that two glutamic residues in plastid luminal and C terminal domains in light harvesting complex (LHC) proteins are crucial to the alga’s unique photoadaptation capacity. Specifically, we demonstrate that glutamate residues contribute to the induction of non-photochemical quenching (NPQ). Protein structure analysis further indicates that these acidic residues can form stable hydrogen bonds under protonation, causing changes in the secondary structure of LHC. Our data suggest that this is the initial action of amino acids under light-induced lumen acidification, which then drives the function of NPQ through a complex process. This photoprotection mechanism, along with low light adaptation, enables this alga to thrive throughout water columns with spatially contrasting and temporally fluctuating irradiance, with implications of bloom formation.

Introduction
The ability to efficiently harvest light energy and protect against photodamage is crucial for the ecological success of phytoplankton. Aureococcus anophagefferens is widely distributed across the global oceans, causing large-scale brown tide blooms frequently observed in the coastal waters of the United States, South Africa, and China1–4. These outbreaks pose serious threats to shellfish farming, fishing, and tourism industries, adversely affecting local economies and ecosystems3. Brown tides typically erupt in relatively enclosed, shallow and turbid estuaries characterized by low light intensity, high organic matter concentrations, and abundant trace elements5.

The competitive advantage of A. anophagefferens in low-light environments is critical for its ability to form brown tide blooms and derives from its unique cellular responses and gene regulation. This species achieves maximum growth rates under low light conditions6, enabled by its large repertoire of 62 light-harvesting protein genes, which are several times more abundant than in competing species, thereby enhancing light capture efficiency5,7. These adaptations demonstrate its exceptional light-harvesting abilities.

Low-light-adaptive algae are typically more vulnerable to high-light stress8,9. A. anophagefferens, living in shallow waters, must cope with occasional oversaturated light intensities through diel cycles or vertical mixing. In algae, photoprotection is achieved by dissipating excess light energy, mostly through non-photochemical quenching (NPQ), a mechanism shared by both algae and vascular plants10. Energy-dependent fluorescence quenching (qE), triggered by the proton gradient (ΔpH) across thylakoid membranes, represents the fastest NPQ component11 and typically associates with the xanthophyll cycle12–14. In addition, qE is related to the protein function of photosystem II subunit S (PsbS) in vascular plants, which belongs to the light-harvesting complex (LHC) family15. Photosynthetic algae exhibit similar photoprotection mechanisms, such as the LhcSR protein in green algae and the Lhcx protein in diatoms16,17. However, the specific process by which the ΔpH ultimately triggers qE remains unclear. Diatoms utilize the xanthophyll cycle to respond to ΔpH but cannot directly trigger qE12,18,19. The decreased pH in the thylakoid lumen triggers qE through the protonation of proteins, a process sensed by acidic amino acids15,19.

In vascular plants, the PsbS protein with four transmembrane helices senses the ΔpH through two glutamate residues symmetrically positioned on the two loops on the lumenal side20. Although the PsbS gene is also present in green algae, its function remains unknown21. The LhcSR or Lhcx proteins in algae contain acidic amino acid residues on the lumenal side, but unlike the PsbS protein, they both have a three-transmembrane helix structure, leaving their mechanism for sensing the proton gradient unclear. In C. reinhardtii, the D117, E221 and E224 of LhcSR3 are essential for NPQ induction19. However, the equivalent residues (D95 and E205) in Lhcx1 of the diatom Phaeodactylum tricornutum are not required for inducing NPQ22. Therefore, while acidic amino acids potentially play an important role, there is no consensus on their significance.

Here, we isolate and culture A. anophagefferens from a brown tide bloom, investigating how its light-harvesting system provides competitive advantages that enable the alga to form blooms. Brown tide is one of the harmful algal blooms creating devastating impacts on the aquatic ecosystem, the coastal environment, and economy, as well as public health concerns. Focusing on the numerous LHC proteins of A. anophagefferens, we examine the roles of these proteins, particularly their association with brown tide outbreaks in estuaries, and the underlying molecular mechanisms. We discover the critical roles of two glutamic residues in the LHC proteins of A. anophagefferens. Data from multiple experiments verify their connection to thylakoid acidification. In this study, we provide insight into the proton gradient response sites and common patterns of algal LHC proteins, which suggest significant implications for understanding the effects of light on brown tide blooms.

Results
The strong adaptability of A. anophagefferens to light changes
Similar to populations in the US and Africa, A. anophagefferens frequently blooms in shallow estuaries of China, particularly in the Bohai Sea. We conducted year-round sampling in Bohai Sea from 2013 to 2014 (Fig. 1a). Surface light intensity varied considerably across sampling sites, with approximately one-third receiving less than 200 μmol photons m−2 s−1 (Fig. 1b). Light attenuation increased with depth: about two-thirds of sites recorded below 200 μmol photons m−2 s−1 at one meter depth, while 91.43% of sites fell below 100 μmol photons m−2 s−1 at two meters. Field surveys revealed that A. anophagefferens maintained a uniform vertical distribution, whereas competing phytoplankton species exhibited varying distribution patterns. Dinoflagellates tended to prefer the surface with stronger light intensity. In contrast, the light intensity preferences of diatoms varied among species, resulting in non-uniform vertical distributions (Fig. 1c). These findings demonstrate A. anophagefferens’ exceptional adaptation to light variability. Its ability to adapt to low light helps them survive in the light-deficient coastal waters5, while its uniform abundance from the sea surface to depths suggests strong photoprotection. This strong photo-plasticity is exceptional. The only comparable case, best to our knowledge, is the cyanobacterium Prochlorococcus, which differentiates into different ecotypes: low-light-adapted strains that thrive in the deep euphotic zone and high-light-adapted strains that thrive at the surface23. They adjust the light capture efficiency through special pigments (Chl b and divinyl Chl a) and proteins (Pcb proteins), enabling them to survive widely in the ocean24. However, studies have found that this niche differentiation involves genetic divergence. In coral symbiotic dinoflagellates, those adapted to high-light environments can avoid photodamage through photoprotective mechanisms, while those adapted to low-light environments are more susceptible to photoinhibition25. Among vascular plants, Hedera helix is sensitive to changes in light intensity and can rapidly induce non-photochemical quenching (NPQ) shortly after a slight increase in light intensity26. From this perspective, A. anophagefferens and H. helix share similar low-light response characteristics. Both species are well adapted to thrive in low-light environments, are sensitive to minor fluctuations in light intensity, and possess strong photoprotective mechanisms.

High LHC gene abundance in A. anophagefferens with unique glutamic acid residues and evidence of their role in photoprotection
The strong photoadaptation of A. anophagefferens observed from the uniform vertical abundance distribution is consistent with the high number of LHC protein genes (AaLHC) in its genome5. To examine the role of AaLHC proteins in the photoprotection, we conducted a response analysis for the 62 AaLHC found in A. anophagefferens to different irradiance environments. Two expression patterns emerged (Fig. 2a). The 11 AaLHC in Group I showed increased expression levels when transferred from low light to high light, and from darkness to light of white and blue spectra. In contrast, the 51 AaLHC in Group II exhibited the opposite trend, with expression levels decreasing under high light stress and only increasing after a longer period of transfer from darkness to a lighted environment. Among them, the four genes AaLhc27, AaLhc37, AaLhc42, and AaLhc52 in Group I were more sensitive to short-term exposure. Additionally, weighted gene co-expression network analysis (WGCNA) identified the 11 AaLHC almost all in the same module (blue module, Supplementary Tables 1, 2), which were associated with short-term high light stress and short-term exposure to white and blue light, suggesting that these genes respond rapidly to changes in the light environment (Fig. 2b). We further analyzed these four significantly responsive genes using qPCR. Results showed that their expression was upregulated immediately after cells were exposed to high light, and dropped quickly when the cultures were returned to a low light environment (Fig. 2c). These genes were also upregulated when the algal cultures were transferred from darkness to low light or from white light to blue light (light stress in comparison, which has a shorter wavelength and higher energy). This result suggests that AaLhc27, AaLhc37, AaLhc42, and AaLhc52, and potentially others in Group I AaLHC as well, likely play the same role in light response.

We further analyzed their amino acid sequences to identify the reasons behind the differences in photoprotective capabilities between the two groups of AaLHC. We noted that the AaLHC proteins in Group I all had two conserved glutamic acid (E) residues (henceforth termed AaLhc2E), while AaLHC proteins in Group II had none (in most cases) or one (Supplementary Fig. 1). Further structural simulations revealed that these two E residues were located on the lumenal loop and the C-terminal domain within the thylakoid membrane (Fig. 2d). Moreover, the two glutamic residues were located very closely to each other (<3.0 Å apart) in all the 11 AaLhc2Es. The proximity potentially facilitates structural adjustments of the protein when they are protonated, as documented for LhcSR in the chlorophyte C. reinhardtii19.

Based on the key role of the proton gradient (ΔpH) across the thylakoid membrane in NPQ, we manipulated ΔpH in A. anophagefferens by adding NH4Cl to the algal culture to disrupt ΔpH, following a previously reported protocol12. The results showed that without NH4Cl treatment, NPQ could be induced by light and relaxed in the dark. NH4Cl addition rapidly led to a NPQ decrease (Fig. 2e). This result indicates that potentially, the cross-membrane proton gradient may be modulated or sensed involving the two E residues in the lumenal loop, thereby regulating NPQ.

As the xanthophyll cycle is coupled with and crucial for NPQ11, we investigated the potential concerted action of them. We detected all xanthophyll pigments in A. anophagefferens, including trace amounts of violaxanthin (Vx), antheraxanthin (Ax), and zeaxanthin (Zx), and observed diadinoxanthin (Dd) de-epoxidation to diatoxanthin (Dt) under high light irradiation (Supplementary Fig. 2a). Furthermore, treatment with NH4Cl inhibited the de-epoxidation state (DPS), indicating that the Dd-Dt cycle, rather than the Vx-Ax-Zx cycle, responds to pH changes. In addition, the conversion of fucoxanthin (Fx) to 19′-butanoyloxyfucoxanthin (19′-BFx)7 also appeared to occur under high light but was unaffected by ΔpH (Supplementary Fig. 2b), indicating that the role of Fx-to-19′-BFx conversion in photoprotection is independent of the ΔpH-modulated NPQ pathway.

Similarity of AaLHC with glutamic acid residues to the photoprotective Lhcx/LhcSR in other algae
As Lhcx/LhcSR is the best known photoprotective protein complex and shared by Archaeplastids, Stramenopiles, Alveolates and Rhizaria27, we compared AaLHCs with it and other light-harvesting proteins. Our phylogenetic analysis results showed that the 62 AaLHC proteins were divided into three categories, which clustered with Lhcf, Lhcr, and Lhcx/LhcSR, respectively (Fig. 3a). Among them, all the 11 AaLhc2E proteins clustered with Lhcx/LhcSR. This indicates that the two E residues are ancient and play a crucial functional role.

Alignment analysis of the 11 AaLhc2E proteins and the Lhcx/LhcSR proteins (Fig. 3a) revealed that transmembrane helices 1 and 3 were relatively conserved (Fig. 3b). The two conserved E residues of the three-transmembrane Lhcx/LhcSR were located at the ends of the helices extending into the lumen. Differently, the two E residues (E122 and E226) in the four-transmembrane PsbS of vascular plants are both located on the loop in the thylakoid lumen28. So far, it has been unclear whether algal LHC proteins all possess conserved acidic residues that respond to pH and are crucial to NPQ. In C. reinhardtii, residues D117, E221, and E224 in LhcSR3 are considered essential for inducing NPQ19. In the model diatom P. tricornutum, residues with potential protonation functions (D95 and E205 in Lhcx1) appear to be unnecessary for NPQ, while tryptophan residues located in the transmembrane helices may be essential22,29. Therefore, it is necessary to experimentally demonstrate that the two conserved glutamate residues in AaLhc2E are directly involved in inducing NPQ in A. anophagefferens. Such experimental evidence is presented in the next section.

Crucial role of E residues in the function of AaLHC for NPQ
As a functional genetic system is not yet available for A. anophagefferens, we chose a diatom model for the functional demonstration of AaLhc2Es. Based on the conservation of transmembrane helices 1 and 3 of the Lhcx/LhcSR among photosynthetic species, we compared the LHC protein sequences of A. anophagefferens and the model diatom Thalassiosira pseudonana. Results showed that the transmembrane regions of LHC proteins between these two species are also highly similar (Fig. 4a). We used T. pseudonana as the model to determine the function of a AaLhc2E for photoprotection. We overexpressed AaLhc27 in T. pseudonana (Tp_AaLhc27E). To verify the function of the E residues, we mutated E102 and E210 in AaLhc27E to Q102 and Q210, respectively, and included them in the heterologous expression experiment (Tp_AaLhc27Q) (Fig. 4b, Supplementary Fig. 3). We chose to replace the glutamic acid (E) residues with glutamine (Q) to minimize biochemical alterations except for the charge change. As a result, the AaLhc27E-expressing T. pseudonana strain (Tp_AaLhc27E) exhibited a greater NPQ under high light, while the AaLhc27Q-expressing strain (Tp_AaLhc27Q) displayed a smaller change in NPQ induction compared to the wild-type (Fig. 4c), indicating that the E residues play a crucial role in the function of AaLHC27 for NPQ. The capacity of NPQ is closely related to the expression of photoprotective LHCs and xanthophyll pigments bound to them30,31. Compared to the wild-type and Tp_AaLhc27Q, the DPS of Tp_AaLhc27E increased more rapidly upon light induction (Fig. 4d), indicating that the xanthophyll cycle is involved in the AaLHC27E-based NPQ regulation.

Notably, the centric diatom T. pseudonana exhibits lower DPS values than the pennate diatom P. tricornutum32. Apart from the influence of growth conditions and light history33, this difference may result from the distinct topologies of NADPH-dehydrogenases (type-1 and type-2)32. The suppression of DPS elevation upon DCMU addition (Fig. 4d) suggests that T. pseudonana likely possesses a non-membrane-spanning type-2 NADPH dehydrogenase, which is probably not involved in generating the lumenal proton gradient that facilitates DDE-mediated de-epoxidation. Whether this enzyme releases protons on the stromal side and, together with abundant NADPH, promotes the epoxidation of Dt also warrants further investigation. Furthermore, our growth measurements indicated that the promoted high light adaptation by AaLHC27E enabled the AaLhc27E-expressing T. pseudonana to grow better under higher light conditions (200 and 800 μmol photons m−2 s−1) compared to the AaLhc27Q-expressing strain and the wild type strain (Fig. 4e). Given that T. pseudonana was transformed with only one AaLhc2E, whereas A. anophagefferens possesses eleven AaLhc2Es, this suggests that the multiple AaLhc2Es in A. anophagefferens may provide greater advantages in dissipating excess energy and growing well under intense and fluctuating light conditions.

To shed light on how proton gradient dynamics may alter AaLhc2E structures and modulate NPQ, we conducted fourier transform infrared (FTIR) spectroscopy analysis of AaLHC27 under varying pH conditions. D2O (D=deuterium) detergent buffer was used to avoid interference from O–H bending absorption, which overlaps with the Amide I region corresponding to the C=O vibrations in the protein backbone. Therefore, the FTIR results will be discussed in terms of pD instead of pH (pD=pH + 0.4). At pD 7.5, the FTIR spectrum of AaLHC27E exhibited a prominent band at 1570 cm−1, which originated from the stretching vibrations of the deprotonated carboxylic acid in the deprotonated E residues (Fig. 5a)34. Notable alteration in the FTIR spectrum was detected at pD 5.0, where a broad positive band in the region of 1700 to 1750 cm−1 was observed, indicative of the presence of deuterated carboxyls (COOD). The characteristic of protonated (deuterated) carboxyls suggests that nearly all E residues were protonated at pD 5.0. In addition, comparing the FTIR spectra at pD 5.0 and pD 7.5 revealed changes in the Amide I region from 1600 to 1700 cm−1, where a negative band at 1640 cm−1 and a broad positive band at 1690 cm−1 indicated a change in the Amide I band, and hence a conformational change in the secondary structure of the AaLHC27E. In contrast, only a minimal change was detected in the FTIR spectra of the AaLHC27Q mutant under different pD conditions. Clearly, mutating the two E residues to Q abolished proton response sensitivity while the general secondary structure of the protein was not altered.

At acidic pH, the E102 and E210 of AaLHC27 were in close proximity to I217 and I116 (2.4 Å), respectively, which likely contributes to the formation of stable interactions (Fig. 5b). Similarly, the two E residues of AaLhc2E proteins could form stable hydrogen bonds with amino acid residues on the lumenal side, and these residues are located in the C-terminal domain and the lumenal loop, respectively (Fig. 5c). Therefore, in an acidic lumenal environment, the glutamic residues on helix 1 and helix 3 become protonated and can easily approach the amino acid residues of the other within a very short range to establish stable interactions, thereby leading to changes in the protein’s secondary structure (Fig. 5d). Through 1000 ns of constant pH molecular dynamics (CpHMD) simulations, the dynamic stability changed little from a neutral environment to an acidic environment (Supplementary Fig. 4, Supplementary Tables 3–6). To further observe the major motion states of the four systems (AaLHC27E_pH_4.5, AaLHC27E_pH_7, AaLHC27Q_pH_4.5, and AaLHC27Q_pH_7), the slowest motions were captured using time-lagged independent component analysis (tICA), and Markov state models (MSMs) were built to examine the differences in state transitions across the systems. The MSM models showed that each state transition varied in time and conformation across the four systems. Specifically, in the AaLHC27E system, both neutral and acidic conditions led to the formation of three metastable states, while the AaLHC27Q system formed four metastable states under both conditions. The most significant structural difference among the four systems was in the lumenal side (e.g., residues 118-130). Under both acidic and neutral conditions, the AaLHC27Q system exhibited a clear helix secondary structure. In contrast, the AaLHC27E system exhibited a partial loop structure along with the helix structure under both conditions (Supplementary Figs. 5, 6).

Our results clearly indicate the crucial role of the two luminal E residues in AaLHC27 for NPQ in A. anophagefferens. Given the highly similarity in sequence and simulated structure, including the spatial proximity of the two glutamic residues, the other 10 AaLhc2E proteins likely serve the same function. However, experimental proof remains to be obtained in future research. Further research is also required to understand if the structurally conserved E residues in LHCs in other algae play the same role. It is worth noting that the total of 11 AaLhc2E proteins (and 62 total LHC proteins) in A. anophagefferens far exceeds that of many other algae but is comparable to some lineages living in highly light-variable environments. The intertidal brown alga Ectocarpus siliculosus and the polar ocean-adapted diatom Fragilariopsis cylindrus both contain 11 Lhcx-like/Lhcx proteins35,36. The intertidal environment undergoes extreme light changes daily. The polar environment experiences complete light to complete dark annually, but F. cylindrus flourishes and forms blooms there37. Emiliania huxleyi, which thrives and form large-scale blooms in habitats ranging from the equator to the subarctic, possesses even more (17) LHCs with photoprotective functions38, although the apparent plasticity within species can potentially be attributed to genetic differentiation into distinct geographic populations. Our findings provide insights into a molecular mechanism by which LHCs mediate photoprotection in A. anophagefferens, with implications in other variable light-adapted lineages.

Our results on AaLhc2Es also have significant ecological implications for the brown tide alga A. anophagefferens, which thrives and forms massive blooms in turbid coastal waters with dynamic light radiation, high organic matter and metals concentrations5. Harmful algal blooms occur when the causative alga outgrows co-existing species due to competitive advantages in photo energy harvesting (E), nutrient acquisition (N), defense against environmental stress, grazing, and microbial attacks (D), and sometimes sexual reproduction (S)-known as ENDS drivers-with photoprotection being one significant D component39. While the high number of LHC genes in A. anophagefferens have been linked to the species’ ability to grow in a dim light environment5, we demonstrate here that at least some are crucial for protection against photodamages. Combined with the substantial genetic capacity for utilizing organic nutrients, trace metal, and vitamins5,40,41, the large LHC protein repertoire means A. anophagefferens is strongly equipped in “END” and poised to thrive in its coastal habitat and form brown tides.

Methods
Sampling design and algal cultivation
A total of 20 phytoplankton sampling sites were established in the brown tide outbreak area (the northwestern part of the Bohai Sea, China) (Fig. 1a, Supplementary Table 7). Samples were collected monthly from June 2013 to May 2014. At each site, phytoplankton samples were collected from the surface layer (1m) and the bottom layer (1m above seafloor), and preserved in 1.5% Lugol’s solution. Phytoplankton species identification and counting were conducted under a microscope (Olympus CX31, Japan). Dominant species was classified based on the dominance index (Y ≥ 0.02). Light intensity was measured for 35 sampling sites at the water surface, 1 meter below the water surface, and at the bottom. Light intensities were measured with a PAR sensor (Onset HOBO MX2202, USA).

A. anophagefferens used in this study was isolated from the coastal waters of Qinhuangdao in the Bohai Sea in July 2012 during a brown tide event. It was preserved in the algal collection of the Research Center of Harmful Algae and Marine Biology at Jinan University, China. Cultures were grown in artificial seawater supplemented with f/2 nutrients42 at 20 °C, under a light intensity of 50 μmol photons m−2 s−1 with a 12:12 light:dark cycle, and experiments were initiated during the logarithmic growth phase. T. pseudonana (CCMP1335) was cultivated under the same conditions. Wild-type and two genotypes of T. pseudonana were maintained in semi-continuous cultures under 50, 200, and 800 μmol photons m−2 s−1. Cultures were diluted daily according to their respective growth rates. Steady state was reached at final dilution rates of 0.37, 0.60 and 0.50 d−1 under 50, 200 and 800 μmol photons m−2 s−1, respectively. To accurately calculate growth rate, one-mL samples were collected daily before and after dilution from each culture and fixed using Lugol’s solution. Cells were counted using a hemocytometer under an inverted microscope (BX53, Olympus, Tokyo, Japan). Growth rates (μ, in d−1) were calculated as: μ=(ln N2 – ln N1)/(t2 – t1), where N1 and N2 represent the cellular concentrations at t1 (before dilution on day 2) and t2 (after dilution on day 1), respectively.

Experiment with different light conditions and fluorescence analyses
Different light environments were used for the study of algal responses to light, including low light (LL, 50 μmol photons m−2 s−1), high light (HL, 800 μmol photons m−2 s−1), white light (WL, 100 μmol photons m−2 s−1), blue light (BL, 100 μmol photons m−2 s−1 at 450 nm), and darkness (D). The growth of T. pseudonana and its AaLhc-overexpressing strains were conducted under an irradiance of 50, 200, and 800 μmol photons m−2 s−1. The determination of NPQ capacity was conducted using a Phyto-PAM Phytoplankton Analyzer (Walz, Germany). After different algal strains were calibrated to the same chlorophyll a concentration, kinetic NPQ measurements were initiated. In the experiment on A. anophagefferens, NPQ was induced by 10 min of 764 μmol photons m−2 s−1, followed by a 10-min recovery phase in darkness to provide information on the relaxation kinetics. After 5 min high light exposure, 1 mM and 20 mM NH4Cl were added, respectively. In the experiment on T. pseudonana and its AaLhc-overexpressing mutants, NPQ was induced by 30-min exposure to a photon flux of 764 μmol photons m−2 s−1, followed by a 30-min recovery phase in the dark. The experiment was conducted with 3–5 parallel samples. NPQ was calculated as Fm/Fm’−1.

Pigment analysis
A. anophagefferens in the exponential growth phase was collected for pigment detection at 0 h, 0.5 h, and 1 h under HL and LL irradiation. NH4Cl was added 25 min after HL exposure. Samples of T. pseudonana and its mutants in the exponential growth phase were taken at 0 h and 0.5 h under HL irradiation and after an additional 0.5 h in darkness. Under identical experimental conditions, 40 μM DCMU was added to inhibit linear electron transport. The samples obtained by filtration (25 mm GF/F membrane, 0.7 μm pore size) were immediately flash-frozen in liquid nitrogen and stored at −80 °C. Pure methanol was added for pigment extraction. Pigment analysis was performed using a 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with an automatic sampler (Agilent 1100 series G1329A). The system was fitted with a reversed-phase Inertsil C8 column (150 × 4.6 mm, 5 μm particle size; GL Science) and eluted with mobile phase A (methanol: acetonitrile: 0.25 M pyridine solvent, 50: 25: 25, v: v: v) and mobile phase B (methanol: acetonitrile: acetone, 20: 60: 20, v: v: v)43. Elution was performed following the method described in ref. 44 with a flow rate of 0.9 mL min-1.

RNA-seq
In the high-light induction experiment, algal cells cultured under LL were transferred to a HL environment for 1 h, 3 h, and 6 h. Additionally, cells that had been kept in darkness for 12 h were transferred to WL (1 h and long-term) and BL (1 h and long-term). Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, US). After the total RNA was qualified, RNA-seq libraries was sequenced in BGI-tech (Shenzhen, China) using an Illumina NovaSeq 6000 (San Diego, CA, USA) at Sangon Biotech (Shanghai, China). Sequencing quality was assessed using FastQC and MultiQC43,45. For the significance of gene expression difference, a cutoff of: qValue < 0.05 and |log2FC| ≥ 1 was used. RNA-seq data were uploaded to the NCBI Sequence Read Archive (SRA) database (PRJNA680884 and PRJNA787041).

WGCNA
The gene expression matrix, which contained HL and LL (1 h, 3 h, and 6 h), was employed to perform the gene co-expression network analysis using WGCNA46, followed by the identification of the modules that clustered with LHC-encoding genes. Similarly, the gene co-expression network analysis was also performed within BL (1 h and long-term), WL (1 h and long-term) and D (darkness).

Real-time quantitative PCR (RT-qPCR)
In the high-light induction experiment, the process involves transferring from LL to HL for 0.5 h, 1 h, 2 h, and 3 h, then exposing to LL again (0.5 h, 1 h, 2 h, 3 h), followed by HL exposure (0.5 h, 1 h, 2 h, 3 h). For low light experiments, the transfer is from D to LL for 1 h, 2 h, and 3 h, with the LL+D group receiving LL exposure for 1 h followed by D for 1 h and 2 h. In the blue light induction experiment, the transfer is from WL to BL for 1 h of exposure, followed by 1 h of WL exposure. For the validation of the exogenous genes AaLhc27E and AaLhc27Q in T. pseudonana, HL exposure was applied to the algae cells cultured under LL conditions for 0.5 h, 1 h, and 3 h. Algal cells were collected by centrifugation and performed RNA extraction. RNA was reverse transcribed using the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme-Biotect, China). Reverse transcription PCR was performed using 50 ng cDNA with the AceQ qPCR SYBR Green Master Mix (Vazyme-Biotect, China). RT-qPCR was performed with the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). The 18S rRNA and ACT4 were used as reference genes for experiments with A. anophagefferens and T. pseudonana, respectively. The gene-specific primers used for this study were listed in Supplementary Table 8. Experiments were repeated three times independently.

Prediction of the LHC protein structure
AlphaFold3 [https://alphafoldserver.com/] was used to predict the three-dimensional structures. The 50 AaLHC prediction models are selected based on predicted template modeling (pTM) score (range from 0.62 to 0.92) for the best ranking. A pTM score above 0.5 indicates that the predicted fold might be the true structure47. The structures were visualized using PyMOL [http://www.pymol.org].

Phylogenetic analysis
A total of 151 amino acid sequences of LHC proteins were obtained by the NCBI database (https://www.ncbi.nlm.nih.gov/) and JGI genome portal (https://genome.jgi.doe.gov/) from A. anophagefferens, Bigelowiella natans, Chlamydomonas eugametos, C. reinhardtii, Cyclotella cryptica, I. galbana, Karlodinium micrum, Mesostigma viride, Micromonas sp., Ostreococcus lucimarinus, Ostreococcus tauri, P. tricornutum, Physcomitrella patens, Scenedesmus obliquus and T. pseudonana (Supplementary Table 9)48. These sequences were aligned using MUSCLE (v5.1)49 and Clustal Omega (v1.2.4)50 with default parameters. The maximum likelihood (ML) phylogenetic tree was estimated using IQ-TREE (v1.6.5)51. The recommended best-fitting model LG+F+R5 was selected by using ModelFinder in IQ-TREE (v1.6.5). ML analysis was run with 1000 replicates via a bootstrap test. WebLogo was used to build sequence logos from LHCs52.

Molecular dynamics (MDs) simulations
MDs simulations were performed using the Gromacs constant pH program53, with the CHARMM36m force field selected, and the TIP3P water model was employed. The pH builder tool was used to generate topology files for CphHMD, with pH values set to 4.5 and 7, respectively, to determine all titratable protein residues in the protein. A cubic box with a distance of 1.5 nm from the protein was established, and water molecules were added. The pH builder tool was used to add an appropriate number of positive/negative ions (sodium ions/chloride ions) and buffer to ensure the system is a net-neutral system, and to generate the corresponding structural and topology files for the system. Four systems were constructed, namely AaLHC27E_pH_4.5, AaLHC27E_pH_7, AaLHC27Q_pH_4.5, and AaLHC27Q_pH_7, respectively. Each protein was solvated in a cubic box (1.5 nm buffer) with water molecules, followed by ion addition (Na⁺/Cl⁻) and buffer components to ensure charge neutrality. Energy minimization utilized the steepest descent algorithm (5000 steps) with Verlet cutoffs, PME electrostatics (rcoulomb=1.2 nm, fourier spacing=0.14), and force-switch vdW treatment (rvdw=1.2 nm). Bond constraints were applied via LINCS. Equilibration comprised NVT (100 ps, leap-frog integrator, V-rescale thermostat at 300 K, tau_t=0.5 ps) and NPT phases (100 ps, C-rescale barostat at 1 bar, isotropic coupling, tau_p=5.0 ps, refcoord_scaling = com). Production runs employed CpHMD with identical parameters to NPT, excluding refcoord_scaling. During the 1000 ns production simulations, conformational snapshots were recorded at 1 ns intervals for all systems. System stability was assessed using built-in modules for root-mean-square deviation (RMSD), residue fluctuations (RMSF), radius of gyration (Rg), and solvent-accessible surface area (SASA). Markov state models (MSMs) and time-lagged independent component analysis (tICA) were computed via PyEMMA54. To encode secondary structural integrity, protein backbone root-mean-square deviation (RMSD) relative to the energy-minimized reference structure was employed as the primary feature for tICA dimensionality reduction. This RMSD-based metric explicitly captures global conformational variations while preserving secondary structure signatures.

Construction of vectors for Lhc and overexpression
pTHa-K1 vector, containing the fcp8 promoter from T. pseudonana, was used for overexpression55. The encoding gene sequence of AaLhc27 was cloned into the pTHa-K1 vector to generate a pTHa-AaLhc27E plasmid. The Mut Express II Fast Mutagenesis Kit V2 (Vazyme-Biotect, China) was used for site-directed mutagenesis of E102 and E210 to Q102 and Q210, respectively, to generate another pTHa-AaLhc27Q plasmid. Plasmids were transformed into wild-type T. pseudonana cells by microprojectile bombardment using the Bio-Rad Biolistic PDS-1000/He Particle delivery system (Bio-Rad, CA, USA)56. With the 100 μg mL−1 nourseothricin resistance screening, colonies appeared after 14 days. The colonies were verified for successful transformation through PCR sequencing.

Plasmid construction, expression, purification and refolding of AaLHC27E and AaLHC27Q
The pET-28a-AaLHC27E and pET-28a-AaLHC27Q vectors containing a C-terminal His6-tag were transformed into Escherichia coli BL21 (DE3) cells, respectively. All transformants were incubated in LB medium with 100 mg/L kanamycin at 37 °C. To induce protein expression, isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM when the bacterial solution was cultured to OD600=0.6. Cells were incubated at 37 °C for 5 h, collected by centrifugation at 4 °C and resuspended in lysis buffer (50 mM Tris-base, 500 mM NaCl, 8 M Urea, pH 7.4, Sangon-Biotect, China). After sonication and centrifugation (10,000 × g, 4 °C, 30 min), the supernatant was collected. His-tag Protein Purification Kit (Beyotime, China) was used to purify the unfolded AaLHC27E and AaLHC27Q. Then exchange buffer to 50 mM Hepes, 0.1% LDS, pH 5.0 (Dialysis Membranes, 14 kDa, Beyotime, China) for removing the high concentration of urea and adjusting the pH. Recombinant proteins of AaLHC27E and AaLHC27Q were refolded following the method described in refs. 57, 58. SDS-page gel electrophoresis analysis (Sangon-Biotect, China) was carried out to check the yield of recombinant protein at each step (Supplementary Fig. 7).

FTIR spectroscopy
Samples of AaLHC27E and AaLHC27Q were loaded in a Specac Omni Cell demountable cell with calcium fluoride (CaF2) windows with a 6 μm path length Mylar spacer. Infrared spectra were recorded using an FTIR spectrometer (IRTracer-100, Shimadzu, Japan) equipped with a Deuterated L-Alanine Triglycine Sulfate detector. Samples were solubilized in D2O detergent buffer. Measurements for the pD 5.0 and 7.5 samples were conducted sequentially, and differential FTIR spectra were generated by normalizing to the integrated absorbance of the amide I band (1550–1750 cm−1).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Aureococcus anophagefferens is a brown alga, which has attracted research interest for its ability to form brown tidal blooms. This ability has been suggested previously to arise from adaptation to low light environments, possibly aided by a large proliferated set of 62 light harvesting protein genes (Gobler et al 2011), which may provide it with a competitive advantage under turbit low light conditions. Low-light adaptation typically enhances vulnerability to high light exposure. Non-photochemical quenching can be induced to protect against photodamage, but it is unclear what molecular mechanisms are involved in inducing NPQ in this alga and how important this is under its niche conditions. A sampling campaign in a coastal area of the Bohai sea was used to show that A. anophagefferens was uniformly distributed in upper and lower depts. Transcript level analysis of LHC genes in response to a range of light regimes showed two groups of genes with contrasting responses. 11 genes seems to increase under light stress, others go down. Phylogenetic analysis seems to suggest that some of these 11 are similar to LHCSR/LHCX, especially with regards to the protonatable glutamate residues. Overexpression of one of the 11 light inducible genes, Lhc27, in the diatom Thalassiosira pseudonana gave rise to a mild stimulation of NPQ induction, which was not observed when both Es were mutated to Qs. The Lhc27E overexpression strain also seemed to show slight growth enhancement under high light. FTIR analysis of Lhc27E and Lhc27Q shows that the former undergoes changes in secondary structure between pH 7.5 and pH 5, whereas these are abolished in the mutated form. This was also concluded from molecular dynamics simulations performed at pH 7 and 4.5 (unclear why pHs are not matching with the FTIR experiments). Altogether, this is an interesting and multi-faceted body of work on molecular mechanism of photoprotection in a relevant brown algal species, but there are a number of issues with the current manuscript.\n1. The narrative is very hard to follow and jumps around a lot between analyses/findings on A. anophagefferens versus other species as well as between different types of analyses which is really hard to follow. As a result, the manuscript is lacking coherence. It also ends very abruptly (I even searched to make sure I hadn’t overlooked part of the text). In addition, the different experiments are only loosely connected, without clear causal relationships linking from the molecular to the organismal scale, which means that the relevance of findings across scales is hard to assess.\n2. The paper could differentiate better between statements of NPQ mechanisms in other photosynthetic organisms, versus those that apply to the species under investigation. The following section is one example of a rather generic description of things important for NPQ, which may or may not apply here: (L157) “The proton gradient (ΔpH) across the thylakoid membrane is known to trigger the activation of specific proteins within the thylakoid membrane, which is essential for photoprotection mechanisms. One notable example is the concerted action of photoprotective proteins and xanthophyll cycle, involving the interconversion of violaxanthin to antheraxanthin and zeaxanthin, or the conversion of diadinoxanthin to diatoxanthin. Xanthophyll cycle is crucial for NPQ11.” In addition, in section L207-229, it is not always clear whether the authors are referring to their own analyses or those done by others, especially since the narrative suddenly broadens to include discussion of several other algal species.\n3. Decoupling may also affect the pigment composition, which in turn may also play a functional role in the observed quenching patterns. Did the authors assess how the pigment composition changed in response to decoupling with NH4Cl and whether the composition was similar in their Tp mutant strains expressing the different Lhc27 forms?\n4. How did the 62 gene expression profiles differ between the different sampling sites? Was there any relation with the prevailing light levels at the sampling sites?\n5. While the mechanistic work on Lhc27 is very interesting and seems sound (as far as I can judge), the extrapolation of the same mechanism to other genes in the same group and even across to other species seems to be much more speculative.\n6. This is also especially true for the suggestion that “due to the higher number of genes encoding photoprotective LHCs with two E residues than competing algae, A. anophagefferens can maintain photosynthetic efficiency and reduce oxidative damage throughout the water column and through fluctuating light conditions. This has a high potential to promote formation of brown tides”. This seems highly speculative on the basis of functional evaluation of one single protein in a heterologous system. In addition, A. anophagefferens may be specifically adapted to withstand microbe predation (Gobler et al. 2011), which could provide an alternative explanation for its competitive advantage in waters with high levels of dissolved organic carbon.\n7. It is not clear how unique the LHC proliferation and associated mechanism (as suggested by the title) really is since other diatoms have also been found to contain 11 LhcX genes (eg Mock et al Nature 541: 536-640).\n8. L155 “Moreover, the two glutamic residues were located very closely to each other (<3.0 Å apart) in all these 11 AaLHC proteins, indicating the potential that these two E residues serve as a cascade of proton flux.” Not sure what is meant by this?\n9. I find the schematic in Fig 3b a bit confusing. What are we supposed to learn from the LCHSR/PsbS depictions underneath the Green algae/Mosses/vascular Plants?\n10. “Light intensity was measured for 25 sampling sites at the water surface, 1 meter below the water surface, and at the bottom. Light intensities were measured with a PAR sensor (Onset HOBO MX2202, USA). “ As far as I know, this sensor measures relative light levels, how were these converted to the absolute numbers shown? Additionally, light stress is often associated with abrupt increases, rather than constant high light. Did the authors look at the temporal profile of light intensity?\n11. The experimental section lacks detail about the experimental conditions for the various light environments and phytoPAM instrument settings.\n12. “Recombinant protein of AaLhc27E and AaLhc27Q were refolded in ref. 52,53.” Rephrase, I guess the protocol from these references was used?", "reviewer_2": "Comments\nThe authors present new and original data on the function of LHC genes in A. anophagefferens, a boom forming brown tide alga. This species contains an unusual high number of LHC genes. The authors could identify a couple of genes which have a similar function as LHCx genes in green algae or diatoms playing a central role in LHC aggregation. The latter is of crucial importance in the formation of the non-photochemical quenching under high light stress. An efficient NPQ is part of an effective photoprotection under excessive light exposure. The authors show on the basis of gene expression upon light shift experiments and sequence comparison with LHCx genes from other algal classes that at least ten of the AaLhc genes cluster within the LHCx group. A detailed analysis of the AaLhc27 genes shows the presence of two glutamic acid residues exposed to the lumen which is a uniform structure element of pH dependent protein aggregation in LHCs. Using FTIR spectroscopy of wildtype and modified heterologous proteins samples clearly prove the expected function of AaLhc27 . This part of the word is sound and merits publication.\nHowever, the author conclude from their experiments that the LHCx triggered NPQ formation explains the ability of the species to form blooms by better growth performance along light gradients. I think that the data do not allow to justify this conclusion, because of the following arguments\n1. This conclusion is based on the hypothesis that solely protein aggregation alone determines NPQ capacity and the latter solely determines growth. This is obviously wrong. The efficiency of NPQ formation is dependent on the amplitude and its kinetic. The latter is controlled by the activity of the de-epoxidase enzyme. If this enzyme operates with higher activity in A. anophagefferens, it would also explain in parts the advantange in species competition. In Fig 4 the initial value of NPQ is much higher than in Fig 2. Why? Also the control should show strong NPQ formation because the necessary components are not inactivated. The data would also indicate that the presence of a changed AaLHC27E gene inactivates the native NPQ mechanism in the diatom.\n2. The assumption that NPQ is the only component that determines growth is in contrast to the literature. The growth experiment in Fig 4 clearly shows that at the beginning of the experiment at low cell density, when the cells are exposed to higher light than in the later growth stages due to self shading, no growth difference is observed. The data do not give any hint to the operation by ROS (no data given and therefore, the idea is only speculation) but clearly indicate that not photoprotection by light acclimation is different between both cell lines. During light acclimation also the dark reaction capacities are increased leading to a higher CO2 fixation capacity under saturating light. This is exactly the condition when the cells are exposed to 200 µE/m² sec. However, these parameters are not analysed in the study.\n3. The authors show in figure 1 the uniform vertical distribution of . A. anophagefferens and conclude from this observation that this species species specifically equally performes in all light climates leading to population dominance. This observation can also explained by optimized nutrient uptake in different layers by different ecotypes or by optimized C-allocation in the cells of A. anophagefferens compared to competing species. Therefore, the number of LHC genes and the specific functions of LHC aggregation is only one component which may explain bloom formation.\nMinor points\nFig 1 What is the depth of the sea flor?\nFig 4 Please explain why a competitive growth experiment between the WT diatom and . A. anophagefferens has not been performed?\nIn conclusion\nThe paper must be revised with re-review with including the state of the art of light acclimation and revision of the conclusion.", "reviewer_3": "This study investigates the critical role of the glutamic acid (E) residues located in the plastid luminal and C-terminal domains of the light-harvesting complex (LHC) protein gene AaLhc27 from Aureococcus anophagefferens, in its capacity to adapt to rapid light responses. To evaluate the function of these glutamic acid residues, the authors mutated the E102 and E210 residues to glutamine (Q102 and Q210), respectively, in the AaLhc27 protein. The results showed that proton gradient sensitivity was completely lost in the mutants, as determined through Fourier transform infrared (FTIR) spectroscopy.\nTo further investigate this effect, the authors performed constant pH molecular dynamics (CpHMD) simulations. Using AlphaFold, they predicted the 3D structure of the protein and modelled at two distinct pH values (pH 4.5 and pH 7.0) for both the E and Q variants. The authors then conducted a 100 ns CpHMD simulation using the Gromacs software. Their analysis revealed significant changes in the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and solvent-accessible surface area (SASA) for the glutamate (E) variant at different pH levels. In contrast, these changes were minimal in the glutamine (Q) mutants, suggesting that the glutamic acid residues play an important role in structural adaptations to pH variations.\nGiven that the observed differences in the computed properties were relatively small and very basic, I would suggest extending the simulation length into the microsecond timescale. Longer simulations would allow for a more comprehensive characterization of the conformational dynamics, providing a clearer understanding of how the protein adapts over longer timescales.\nAdditionally, I recommend that the authors consider using Markov State Modeling (MSM) and time-lagged independent component analysis (tICA) as complementary tools for analyzing the conformational dynamics of the protein. These approaches would be particularly valuable for capturing slow, pH-induced structural transitions, as well as identifying key conformational states and transitions. MSM could help to model the state space and transition dynamics of the protein, while tICA could facilitate the identification of slow collective motions that are crucial for understanding the pH-dependent structural changes in AaLhc27. These analyses could provide deeper insights into the role of the glutamic acid residues and enhance the overall understanding of how AaLhc27 adapts to rapid light conditions.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response to Editor’s and Reviewers’ Comment\nWe thank all reviewers for their valuable comments and suggestions, which are very\nhelpful for improving our manuscript. We carefully addressed them and revised the manuscript\naccordingly. As per the reviewer’s suggestions, revisions are marked by using blue color font in the\nrevised manuscript.\nReviewer #1 (Remarks to the Author):\nAureococcus anophagefferens is a brown alga, which has attracted research interest for its ability to form\nbrown tidal blooms. This ability has been suggested previously to arise from adaptation to low light\nenvironments, possibly aided by a large proliferated set of 62 light harvesting protein genes (Gobler et\nal 2011), which may provide it with a competitive advantage under turbit low light conditions. Low-light\nadaptation typically enhances vulnerability to high light exposure. Non-photochemical quenching can be\ninduced to protect against photodamage, but it is unclear what molecular mechanisms are involved in\ninducing NPQ in this alga and how important this is under its niche conditions. A sampling campaign in\na coastal area of the Bohai sea was used to show that A. anophagefferens was uniformly distributed in\nupper and lower depts. Transcript level analysis of LHC genes in response to a range of light regimes\nshowed two groups of genes with contrasting responses. 11 genes seems to increase under light stress,\nothers go down. Phylogenetic analysis seems to suggest that some of these 11 are similar to\nLHCSR/LHCX, especially with regards to the protonatable glutamate residues. Overexpression of one\nof the 11 light inducible genes, Lhc27, in the diatom Thalassiosira pseudonana gave rise to a mild\nstimulation of NPQ induction, which was not observed when both Es were mutated to Qs. The Lhc27E\noverexpression strain also seemed to show slight growth enhancement under high light. FTIR analysis\nof Lhc27E and Lhc27Q shows that the former undergoes changes in secondary structure between pH 7.5\nand pH 5, whereas these are abolished in the mutated form. This was also concluded from molecular\ndynamics simulations performed at pH 7 and 4.5 (unclear why pHs are not matching with the FTIR\nexperiments). Altogether, this is an interesting and multi-faceted body of work on molecular mechanism\nof photoprotection in a relevant brown algal species, but there are a number of issues with the current\nmanuscript.\nResponse: We would like to thank the reviewer for the positive evaluation of our work. The reason for\nthe mismatch between the pD and pH values is that the relationship between pD and pH is given by the\nequation pD = pH + 0.4. We have revised the sentence as follows to make this clear:\nL287-288: “Therefore, the FTIR results will be discussed in terms of pD instead of pH (pD = pH + 0.4).”\n1. The narrative is very hard to follow and jumps around a lot between analyses/findings on A.\nanophagefferens versus other species as well as between different types of analyses which is really hard\nto follow. As a result, the manuscript is lacking coherence. It also ends very abruptly (I even searched to\nmake sure I hadn’t overlooked part of the text). In addition, the different experiments are only loosely\nconnected, without clear causal relationships linking from the molecular to the organismal scale, which\nmeans that the relevance of findings across scales is hard to assess.\nResponse: We have revised the descriptions in the manuscript to enhance their coherence. We have\nfocused the narrative on A. anophagefferens, reducing comparative analyses with other species and\nadding more details to the final section.\n2. The paper could differentiate better between statements of NPQ mechanisms in other photosynthetic\norganisms, versus those that apply to the species under investigation. The following section is one\nexample of a rather generic description of things important for NPQ, which may or may not apply here:\n(L157) “The proton gradient (ΔpH) across the thylakoid membrane is known to trigger the activation of\nspecific proteins within the thylakoid membrane, which is essential for photoprotection mechanisms.\nOne notable example is the concerted action of photoprotective proteins and xanthophyll cycle, involving\nthe interconversion of violaxanthin to antheraxanthin and zeaxanthin, or the conversion of\ndiadinoxanthin to diatoxanthin. Xanthophyll cycle is crucial for NPQ11.” In addition, in section L207-\n229, it is not always clear whether the authors are referring to their own analyses or those done by others,\nespecially since the narrative suddenly broadens to include discussion of several other algal species.\nResponse: We have revised these two sections separately, focusing on the response of A. anophagefferens\nto the proton gradient and the specific amino acid sites involved. We have reduced speculative\ndescriptions of other algae to make the manuscript more coherent.\n3. Decoupling may also affect the pigment composition, which in turn may also play a functional role in\nthe observed quenching patterns. Did the authors assess how the pigment composition changed in\nresponse to decoupling with NH4Cl and whether the composition was similar in their Tp mutant strains\nexpressing the different Lhc27 forms?\nResponse: We have supplemented the manuscript with experiments on pigment content changes in A.\nanophagefferens under light stress, as well as the effects of increased Lhc27 on pigment content in T.\npseudonana. The revised narrative is as follows:\nLines 170-179: We detected all xanthophyll pigments in A. anophagefferens, including trace amounts of\nviolaxanthin (Vx), antheraxanthin (Ax), and zeaxanthin (Zx), and observed diadinoxanthin (Dd) de-\nepoxidation to diatoxanthin (Dt) under high light irradiation (Supplementary Fig. 2a). Furthermore,\ntreatment with NH Cl inhibited the de-epoxidation state (DPS), indicating that the Dd-Dt cycle, rather\n4\nthan the Vx-Ax-Zx cycle, responds to pH changes. In addition, the conversion of fucoxanthin (Fx) to\n19′-butanoyloxyfucoxanthin (19′-BFx) 7 also appeared to occur under high light but was unaffected by\nΔpH (Supplementary Fig. 2b), indicating that the role of Fx-to-19′-BFx conversion in photoprotection is\nindependent of the ΔpH-modulated NPQ pathway.\nSupplementary Fig. 2 | The response of pigments to increasing light intensity. The de-epoxidation state\nof the diadinoxanthin-diatoxanthin cycle (a) and the conversion rate of fucoxanthin to 19′-\nbutanoyloxyfucoxanthin (b) under high light irradiation (800 μmol photons m−2 s−1), with 20 mM NH Cl\n4\nadded 25 min after high light exposure. The control group was maintained under low light conditions (50\nμmol photons m−2 s−1).\nLines 256-258: Compared to the wild-type and Tp_AaLhc27Q, Tp_AaLhc27E also exhibited more Dd\nde-epoxidation to Dt (Fig. 4d), indicating that the xanthophyll cycle is involved in the AaLHC27E-based\nNPQ regulation.\nFig. 4d | Changes in diadinoxanthin (Dd) and diatoxanthin (Dt) concentrations of T. pseudonana and its\ntwo mutants.\n4. How did the 62 gene expression profiles differ between the different sampling sites? Was there any\nrelation with the prevailing light levels at the sampling sites?\nResponse: Unfortunately, we did not collect samples from the field for transcriptomic analysis, missing\nthe opportunity to reveal the true situation in the complex natural environment. If a brown tide occurs\nagain in the future, we will collect in situ samples for analysis. In this study, based on the range of light\nintensity measured in the field area (Fig. 1b), we set up high light (800 µmol photons m−2 s−1) and low\nlight (50 µmol photons m−2 s−1) in the laboratory, as well as white light (100 µmol photons m−2 s−1),\nblue light (100 µmol photons m−2 s−1), and darkness (0 µmol photons m−2 s−1)(Fig. 2a). These conditions were\ndesigned to simulate the possible light irradiance and light fluctuations in the field area. We aimed to\nexplore the expression characteristics of 62 genes in A. anophagefferens under different light\nenvironments.\n5. While the mechanistic work on Lhc27 is very interesting and seems sound (as far as I can judge), the\nextrapolation of the same mechanism to other genes in the same group and even across to other species\nseems to be much more speculative.\nResponse: We agree with your opinion that the mechanism by which Lhcx senses proton changes to\ninduce NPQ is complex and may vary between species. More evidence is needed to confirm whether the\nresponse mechanism identified in A. anophagefferens is universal among algae. Therefore, we have\nremoved the discussion extrapolating this mechanism to other species and focused solely on the\ndescription of A. anophagefferens. We also toned down the potential that other group I Lhc members\nhave the same function as Lhc27.\n6. This is also especially true for the suggestion that “due to the higher number of genes encoding\nphotoprotective LHCs with two E residues than competing algae, A. anophagefferens can maintain\nphotosynthetic efficiency and reduce oxidative damage throughout the water column and through\nfluctuating light conditions. This has a high potential to promote formation of brown tides”. This seems\nhighly speculative on the basis of functional evaluation of one single protein in a heterologous system.\nIn addition, A. anophagefferens may be specifically adapted to withstand microbe predation (Gobler et\nal. 2011), which could provide an alternative explanation for its competitive advantage in waters with\nhigh levels of dissolved organic carbon.\nResponse: We agree with your suggestion. We conducted additional experiments on growth to\ndemonstrate that the inclusion of AaLhc27 protein containing two E residues helps maintain population\ndensity under high light irradiation. In the last paragraph of the manuscript, we discussed the positive\nrole of LHCs in light response. We now have stated that photoprotection by Lhc-E, along with advantages\nin acquiring organic nutrients and trace elements as well as microbial defense, contribute to the superior\ngrowth potential of A. anophagefferens and the formation of brown tides.\n7. It is not clear how unique the LHC proliferation and associated mechanism (as suggested by the title)\nreally is since other diatoms have also been found to contain 11 LhcX genes (eg Mock et al Nature 541:\n536-640).\nResponse: Indeed, diatoms like Fragilariopsis cylindrus also contains a large number of Lhcx proteins.\nF. cylindrus is also able to form blooms in the Antarctic (Otte et al. 2003, J. Phycol. 59: 301-\n306, https://doi.org/10.1111/jpy.13325). However, the light-responsive mechanisms of these diatom\nproteins have not been elucidated. We explored and demonstrated a mechanism in A. anophagefferens\ninvolving two E residues that respond to the proton gradient to induce NPQ. As for whether these LHC\nproteins with photoprotective functions have other roles still requires further investigation, which we\nadded a discussion in the final paragraph of the manuscript as below:\nLines 325-337: It is worth noting that the total of 11 AaLhc2E proteins (and 62 total LHC proteins) in A.\nanophagefferens far exceeds that of many other algae but is comparable to some lineages living in highly\nlight-variable environments. The intertidal brown alga Ectocarpus siliculosus and the polar ocean-\nadapted diatom Fragilariopsis cylindrus, both contain 11 Lhcx-like/Lhcx proteins36,37. The intertidal\nenvironment undergoes extreme light changes daily. The polar environment experiences complete light\nto complete dark annually, but F. cylindrus flourishes and forms blooms there38. Emiliania huxleyi, which\nthrives and forms large-scale blooms in habitats ranging from the equator to the subarctic, possesses even\nmore (17) LHCs with photoprotective functions39, although the apparent plasticity within species can\npotentially be attributed to genetic differentiation into distinct geographic populations. Our findings\nprovide novel insights into a molecular mechanism by which LHCs mediate photoprotection in A.\nanophagefferens, with implications in other variable light-adapted lineages.\n8. L155 “Moreover, the two glutamic residues were located very closely to each other (<3.0 Å apart) in\nall these 11 AaLHC proteins, indicating the potential that these two E residues serve as a cascade of\nproton flux.” Not sure what is meant by this?\nResponse: As documented in literature, acidic amino acid residues that are in close proximity are likely\nto induce changes in the overall protein structure upon protonation, thereby exerting specific functions.\nWe have made the necessary revisions in the manuscript as below:\nLines 157-160: Moreover, the two glutamic residues were located very closely to each other (<3.0 Å\napart) in all the 11 AaLhc2Es. The proximity potentially facilitates structural adjustments of the protein\nwhen they are protonated, as documented for LhcSR in the chlorophyte C. reinhardtii19.\n9. I find the schematic in Fig 3b a bit confusing. What are we supposed to learn from the LCHSR/PsbS\ndepictions underneath the Green algae/Mosses/vascular Plants?\nResponse: Thank you for your suggestion. Considering its relevance to the paper, we have removed Fig.\n3b and its description.\n10. “Light intensity was measured for 25 sampling sites at the water surface, 1 meter below the water\nsurface, and at the bottom. Light intensities were measured with a PAR sensor (Onset HOBO MX2202,\nUSA). “ As far as I know, this sensor measures relative light levels, how were these converted to the\nabsolute numbers shown? Additionally, light stress is often associated with abrupt increases, rather than\nconstant high light. Did the authors look at the temporal profile of light intensity?\nResponse: We multiplied the values obtained with the HOBO MX2202 (in units of lux) by 0.018 to\nconvert them to PAR units (μmol photons m−2 s−1) (Thimijan and Heins, 1983).\nWe did not measure the changes in local light intensity over time, but worked on the premise that light\nchanges day to night, and in shallow waters, mixing brings cells up and down.\nReference:\nThimijan, R. W. & Heins, R. D. Photometric, Radiometric, and Quantum Light Units of Measure: A\nReview of Procedures for Interconversion. HortScience 18, 818-822 (1983).\nhttps://doi.org/10.21273/HORTSCI.18.6.818\n11. The experimental section lacks detail about the experimental conditions for the various light\nenvironments and phytoPAM instrument settings.\nResponse: We described the light settings in each different experiment to increase the clarity of the\nexperimental conditions. We also added a description of the Phyto-PAM settings.\nLines 395-400: In the experiment on A. anophagefferens, NPQ was induced by 10 min of 764 μmol\nphotons m−2 s−1, followed by a 10-min recovery phase in darkness to provide information on the\nrelaxation kinetics. After 5 min high light exposure, 1 mM and 20 mM NH Cl were added respectively.\nflux of 764 μmol photons m−2 s−1, followed by a 30-min recovery phase in the dark.\n12. “Recombinant protein of AaLhc27E and AaLhc27Q were refolded in ref. 52,53.” Rephrase, I guess\nthe protocol from these references was used?\nResponse: Thank you for catching the error. We have corrected it to: Recombinant protein of AaLHC27E\nand AaLHC27Q were refolded following the method described in ref. 58,59.\nReviewer #2 (Remarks to the Author):\nComments\nThe authors present new and original data on the function of LHC genes in A. anophagefferens, a boom\nforming brown tide alga. This species contains an unusual high number of LHC genes. The authors could\nidentify a couple of genes which have a similar function as LHCx genes in green algae or diatoms playing\na central role in LHC aggregation. The latter is of crucial importance in the formation of the non-\nphotochemical quenching under high light stress. An efficient NPQ is part of an effective photoprotection\nunder excessive light exposure. The authors show on the basis of gene expression upon light shift\nexperiments and sequence comparison with LHCx genes from other algal classes that at least ten of the\nAaLhc genes cluster within the LHCx group. A detailed analysis of the AaLhc27 genes shows the\npresence of two glutamic acid residues exposed to the lumen which is a uniform structure element of pH\ndependent protein aggregation in LHCs. Using FTIR spectroscopy of wildtype and modified\nheterologous proteins samples clearly prove the expected function of AaLhc27 . This part of the word is\nsound and merits publication.\nHowever, the author conclude from their experiments that the LHCx triggered NPQ formation explains\nthe ability of the species to form blooms by better growth performance along light gradients. I think that\nthe data do not allow to justify this conclusion, because of the following arguments\nResponse: We thank the reviewer for the positive evaluation of our work and the comment about our\nbloom connection conclusion. We have carefully considered this comment and toned down the statements.\n1. This conclusion is based on the hypothesis that solely protein aggregation alone determines NPQ\ncapacity and the latter solely determines growth. This is obviously wrong. The efficiency of NPQ\nformation is dependent on the amplitude and its kinetic. The latter is controlled by the activity of the de-\nepoxidase enzyme. If this enzyme operates with higher activity in A. anophagefferens, it would also\nexplain in parts the advantange in species competition. In Fig 4 the initial value of NPQ is much higher\nthan in Fig 2. Why? Also the control should show strong NPQ formation because the necessary\ncomponents are not inactivated. The data would also indicate that the presence of a changed AaLHC27E\ngene inactivates the native NPQ mechanism in the diatom.\nResponse: Thank you for your suggestions. We have added data on growth and pigments. Due to\ndifferences among species, A. anophagefferens (Fig. 2e) and T. pseudonana (Fig. 4c) exhibit different\nabilities to induce NPQ under initial light stress (20 s). We repeated the experiment shown in Fig. 4c and\nextended the detection time. The results indicate that the introduction of the protein AaLhc27Q did not\nalter the ability of T. pseudonana to induce NPQ (consistent with the control group), while the protein\nAaLhc27E enhanced the ability to induce NPQ, confirming that the glutamate (E) residue has the\nfunction of responding to proton gradients and inducing NPQ.\nFig. 4c | NPQ induction differences in mutants.\nFig. 4d | Changes in diadinoxanthin (Dd) and diatoxanthin (Dt) concentrations of T. pseudonana and its\ntwo mutants.\n2. The assumption that NPQ is the only component that determines growth is in contrast to the literature.\nThe growth experiment in Fig 4 clearly shows that at the beginning of the experiment at low cell density,\nwhen the cells are exposed to higher light than in the later growth stages due to self shading, no growth\ndifference is observed. The data do not give any hint to the operation by ROS (no data given and therefore,\nthe idea is only speculation) but clearly indicate that not photoprotection by light acclimation is different\nbetween both cell lines. During light acclimation also the dark reaction capacities are increased leading\nto a higher CO2 fixation capacity under saturating light. This is exactly the condition when the cells are\nexposed to 200 µE/m² sec. However, these parameters are not analysed in the study.\nResponse: Thank you for your suggestion. We added pigment analyses as well as growth experiments\nunder different light intensities. As shown in Fig. 4e (copied below), AaLhx27E-expressing Thalassiosira\npseudonana exhibited superior growth under high light irradiation.\nFig. 4e | Growth differences in mutants and wild type under different light intensities.\n3. The authors show in figure 1 the uniform vertical distribution of . A. anophagefferens and conclude\nfrom this observation that this species species specifically equally performes in all light climates leading\nto population dominance. This observation can also explained by optimized nutrient uptake in different\nlayers by different ecotypes or by optimized C-allocation in the cells of A. anophagefferens compared to\ncompeting species. Therefore, the number of LHC genes and the specific functions of LHC aggregation\nis only one component which may explain bloom formation.\nResponse: We agree with your perspective that A. anophagefferens's adaptation to low-light, high-\norganic-matter, and high-trace-element concentrations in estuaries collectively contributes to its\ncompetitive advantage. We have revised the last paragraph to reflect that the role of LHCs is a light-\nresponse-related cause for the occurrence of brown tides.\n4. Minor points\nFig 1 What is the depth of the sea flor?\nResponse: The information on seafloor depth is included in Fig. 1a, and more detailed information has\nbeen supplemented in Supplementary Table 7.\nFig. 1a | Map of sampling stations.\nSupplementary Table 7. The latitude and longitude of this study area.\nLongitude Latitude Station Depth (m) Data collection\n119°37'55″E 39°54'07″N S1 7.1 phytoplankton sampling\n119°39'42″E 39°52'55″N S2 7.7 phytoplankton sampling\n119°41'14″E 39°51'44″N S3 7.9 phytoplankton sampling\n119°42'46″E 39°50'21″N S4 8.2 phytoplankton sampling\n119°44'18″E 39°49'10″N S5 9.5 phytoplankton sampling\n119°32'39″E 39°49'32″N S6 7.5 phytoplankton sampling\n119°34'41″E 39°48'43″N S7 8.2 phytoplankton sampling\n119°36'45″E 39°47'52″N S8 8.8 phytoplankton sampling\n119°38'50″E 39°47'03″N S9 9.3 phytoplankton sampling\n119°40'50″E 39°46'14″N S10 10.5 phytoplankton sampling\n119°28'51″E 39°48'06″N S11 6.4 phytoplankton sampling\n119°30'53″E 39°47'13″N S12 7.7 phytoplankton sampling\n119°32'55″E 39°46'18″N S13 9.0 phytoplankton sampling\nLongitude Latitude Station Depth (m) Data collection\n119°34'57″E 39°45'25″N S14 10.3 phytoplankton sampling\n119°37'00″E 39°44'30″N S15 11.3 phytoplankton sampling\n119°26'11″E 39°46'13″N S16 6.3 phytoplankton sampling\n119°28'11″E 39°45'23″N S17 7.7 phytoplankton sampling\n119°30'13″E 39°44'31″N S18 9.1 phytoplankton sampling\n119°32'15″E 39°43'39″N S19 10.8 phytoplankton sampling\n119°34'15″E 39°42'48″N S20 12.4 phytoplankton sampling\n119°52'17″E 39°58'51″N S21 10.8 light measurement\n119°53'25″E 39°57'35″N S22 11.8 light measurement\n119°56'06″E 39°54'34″N S23 17.7 light measurement\n119°59'32″E 39°50'40″N S24 19.9 light measurement\n119°44'41″E 39°56'50″N S25 5.1 light measurement\n119°45'09″E 39°56'20″N S26 7.2 light measurement\n119°46'16″E 39°55'04″N S27 10.7 light measurement\n119°49'30″E 39°51'23″N S28 15.6 light measurement\n119°52'48″E 39°47'31″N S29 18.7 light measurement\n119°56'03″E 39°43'53″N S30 21.1 light measurement\n119°37'11″E 39°54'19″N S31 6.8 light measurement\n119°37'46″E 39°53'43″N S32 7.4 light measurement\n119°39'30″E 39°51'57″N S33 8.3 light measurement\n119°42'59″E 39°48'25″N S34 12.3 light measurement\n119°47'03″E 39°44'16″N S35 16.8 light measurement\n119°50'48″E 39°40'30″N S36 19.0 light measurement\n119°31'55″E 39°48'34″N S37 9.1 light measurement\n119°32'52″E 39°47'50″N S38 9.3 light measurement\n119°35'12″E 39°46'03″N S39 10.3 light measurement\n119°38'46″E 39°43'17″N S40 14.9 light measurement\n119°42'38″E 39°40'18″N S41 17.3 light measurement\n119°46'51″E 39°37'16″N S42 18.9 light measurement\n119°23'56″E 39°45'50″N S43 3.1 light measurement\n119°24'37″E 39°45'24″N S44 5.6 light measurement\n119°27'13″E 39°43'45″N S45 8.9 light measurement\n119°31'49″E 39°40'49″N S46 11.2 light measurement\n119°35'59″E 39°38'10″N S47 13.4 light measurement\n119°41'30″E 39°34'47″N S48 16.8 light measurement\n119°21'15″E 39°41'33″N S49 7.0 light measurement\n119°23'54″E 39°39'54″N S50 7.7 light measurement\n119°27'36″E 39°37'33″N S51 9.1 light measurement\n119°32'45″E 39°34'19″N S52 13 light measurement\n119°18'33″E 39°36'23″N S53 3.3 light measurement\n119°21'09″E 39°34'56″N S54 8.9 light measurement\n119°16'17″E 39°30'24″N S55 2.4 light measurement\n5. Fig 4 Please explain why a competitive growth experiment between the WT diatom and A.\nanophagefferens has not been performed?\nResponse: Thank you for your suggestion. We have supplemented the growth experiments of wild-type\nT. pseudonana and its two mutant strains under different light intensities (50, 200, and 800 μmol photons\nm−2 s−1), showing superior growth of the AaLhc27E-expressing strain. This study was focused on\nmolecular mechanisms of photoprotection in A. anophagefferens, and therefore we did not conduct the\ncompetition experiments on this and diatoms. We plan to use WT and AaLhc27E-expressing diatoms\nalong with A. anophagefferens for competition experiments in future studies.\n6. In conclusion\nThe paper must be revised with re-review with including the state of the art of light acclimation and\nrevision of the conclusion.\nResponse: Thank you for your suggestions. We have revised the results that lacked direct evidence, added\ndescriptions of pigments conversion, and rephrased the conclusion.\nReviewer #3 (Remarks to the Author):\nThis study investigates the critical role of the glutamic acid (E) residues located in the plastid luminal\nand C-terminal domains of the light-harvesting complex (LHC) protein gene AaLhc27 from Aureococcus\nanophagefferens, in its capacity to adapt to rapid light responses. To evaluate the function of these\nglutamic acid residues, the authors mutated the E102 and E210 residues to glutamine (Q102 and Q210),\nrespectively, in the AaLhc27 protein. The results showed that proton gradient sensitivity was completely\nlost in the mutants, as determined through Fourier transform infrared (FTIR) spectroscopy.\nTo further investigate this effect, the authors performed constant pH molecular dynamics (CpHMD)\nsimulations. Using AlphaFold, they predicted the 3D structure of the protein and modelled at two distinct\npH values (pH 4.5 and pH 7.0) for both the E and Q variants. The authors then conducted a 100 ns\nCpHMD simulation using the Gromacs software. Their analysis revealed significant changes in the root\nmean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), and\nsolvent-accessible surface area (SASA) for the glutamate (E) variant at different pH levels. In contrast,\nthese changes were minimal in the glutamine (Q) mutants, suggesting that the glutamic acid residues\nplay an important role in structural adaptations to pH variations.\nGiven that the observed differences in the computed properties were relatively small and very basic, I\nwould suggest extending the simulation length into the microsecond timescale. Longer simulations\nwould allow for a more comprehensive characterization of the conformational dynamics, providing a\nclearer understanding of how the protein adapts over longer timescales.\nAdditionally, I recommend that the authors consider using Markov State Modeling (MSM) and time-\nlagged independent component analysis (tICA) as complementary tools for analyzing the conformational\ndynamics of the protein. These approaches would be particularly valuable for capturing slow, pH-induced\nstructural transitions, as well as identifying key conformational states and transitions. MSM could help\nto model the state space and transition dynamics of the protein, while tICA could facilitate the\nidentification of slow collective motions that are crucial for understanding the pH-dependent structural\nchanges in AaLhc27. These analyses could provide deeper insights into the role of the glutamic acid\nresidues and enhance the overall understanding of how AaLhc27 adapts to rapid light conditions.\nResponse: Thank you for your valuable advice. According to your suggestion, we extended the molecular\ndynamics simulation time to 1000 ns to enhance the accuracy of our results. Also, we added Markov\nState Modeling (MSM) and time-lagged independent component analysis (tICA) for protein\nconformation analysis. These changes were very beneficial to our study. Here are the additional results\nfrom this section:\nLines 303-318: Through 1000 ns of constant pH molecular dynamics (CpHMD) simulations, the dynamic\nstability changed little from a neutral environment to an acidic environment (Supplementary Fig. 4 and\nSupplementary Tables 3-6). To further observe the major motion states of the four systems\n(AaLHC27E_pH_4.5, AaLHC27E_pH_7, AaLHC27Q_pH_4.5, and AaLHC27Q_pH_7), the slowest\nmotions were captured using time-lagged independent component analysis (tICA), and Markov state\nmodels (MSMs) were built to examine the differences in state transitions across the systems. The MSM\nmodels showed that each state transition varied in time and conformation across the four systems.\nSpecifically, in the AaLHC27E system, both neutral and acidic conditions led to the formation of three\nmetastable states, while the AaLHC27Q system formed four metastable states under both conditions.\nThe most significant structural difference among the four systems was in the lumenal side (e.g. residues\n118-130). Under both acidic and neutral conditions, the AaLHC27Q system exhibited a clear helix\nsecondary structure. In contrast, the AaLHC27E system exhibited a partial loop structure along with the\nhelix structure under both conditions (Supplementary Fig. 5-6).\nSupplementary Fig. 4 | Constant pH molecular dynamics simulations (CpHMD) simulations of\nAaLhc27. The root mean square deviation (a), root mean square fluctuation (b), radius of gyration (c),\ntotal solvent accessible surface area (d), hydrophobic solvent accessible surface area (e), and hydrophilic\nsolvent accessible surface area (f) of AaLHC27E and its mutant AaLHC27Q at acidic pH and neutral pH.\nSupplementary Fig. 5 | Principal component analysis (PCA) and time-lagged independent\ncomponent analysis (tICA) of AaLhc27.\nSupplementary Fig. 6 | Markov state models (MSM) to study the AaLhc27.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: Thank you for your co-review of our manuscript. We truly value the insights and efforts\ncontributed by all reviewers involved. We have carefully considered the feedback provided and have\nmade the necessary revisions to enhance the quality of our work. Thank you once again for your time\nand valuable input."}, {"round": 2, "reviewer_comments": {"reviewer_2": "The authors have carefully improved the ms and have added new data as requested by the reviewers. However, after reading the rebuttal and the revised ms I have still reservations to publish the paper because of following reasons.\n1. I have mentioned in my review that the NPQ mechanism alone can not explain the strong bloom formation of Aureococcus anophagefferens. The authors have accepted this argument and have modified the conclusion at the end of the chapter “results and discussion.”. However, in the abstract (which is normally the most read part of a paper) they state again:\n“This photoprotection mechanism, along with low light adaptation, enables this alga to thrive throughout water columns with spatially contrasting and temporally fluctuating irradiance, thereby promoting bloom formation.” I mentioned that only a competition experiment can show that A. anophagefferens can outcompete other diatoms. In the rebuttal the authors reply:\n“This study was focused on molecular mechanisms of photoprotection in A. anophagefferens, and therefore we did not conduct the\ncompetition experiments on this and diatom”. If so, then the conclusion with respect to bloom formation is outside the focus of this study.\n2. The new additional data are not convincing.\n- Xanthophyll cycling:\n- The authors report that the deepoxidation status (DPS) after high light treatment is in the range of 0.1. This is in contrast to literature, where always value of 0.8 or higher have been reported by many authors independently (see literature below and refs therein). It is hard for me to believe that such a low DPS can really activate a strong NPQ by the mechanism of xanthophylls cycling. If the data are true, the authors should have discussed this strange value and think about of an Xanthophyll cycle independent mechanism of NPQ. Also the concentrations of Dt + Dx are very low compared to literature. This again asks for explanation and can not be published as it is.\n- The growth curves\n3. The authors argument is that the growth experiments show that the TpAalhc 27e grows better in high light, which is taken as argument that the modification of the Lhc is responsible for improved photoprotection. This is true, however, the growth differences between the different genotypes are quite small and the growth curves show non-typical behavior. Normally, the growth rate increases up to a saturating light intensity, especially when the cultures are diluted and light is not reduced by self shading (see e.g. paper below).In contrast to this general rule the cultures at low light show the highest growth rates indicated by the number of cells after 10 days cultivation. Also the slope during the exponential phase is higher or equal to higher light intensities. The reduction in growth rate by a factor of about three at 800 µE/m² sec compared to 50 µE/m² sec is a clear indication that all the cultures are severely sensitive to photoinhibition. I do not understand why the WT cultures of Tp show such a high sensitivity in growth in response to high light. Please refer to literature (see below).\nIn summary, the additional data increase the inconsistency of the paper and therefore, the paper is not ready for publication\nThe authors should take notice for literature:\n- on xanthophylls cycling e.g. Gruneva et al. BBA 2007\n- Shifts in growth light optima among diatom species support their succession during the spring bloom in the Arctic - Croteau\n- 2022 - Journal of Ecology", "reviewer_3": "The manuscript has been improved considerably by reacting to the reviewer questions and comments. Especially the part on the MD simulations and Markov state modelling has been revised extensively and now contributes significantly to the interpretation of the experimental findings. Thus, I recommended publication of this revised version.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "REVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThe authors have carefully improved the ms and have added new data as requested by the\nreviewers. However, after reading the rebuttal and the revised ms I have still reservations to\npublish the paper because of following reasons.\n1. I have mentioned in my review that the NPQ mechanism alone can not explain the strong\nbloom formation of Aureococcus anophagefferens. The authors have accepted this argument\nand have modified the conclusion at the end of the chapter “results and discussion.”. However,\nin the abstract (which is normally the most read part of a paper) they state again:\n“This photoprotection mechanism, along with low light adaptation, enables this alga to thrive\nthroughout water columns with spatially contrasting and temporally fluctuating irradiance,\nthereby promoting bloom formation.” I mentioned that only a competition experiment can show\nthat A. anophagefferens can outcompete other diatoms. In the rebuttal the authors reply: “This\nstudy was focused on molecular mechanisms of photoprotection in A. anophagefferens, and\ntherefore we did not conduct the\ncompetition experiments on this and diatom”. If so, then the conclusion with respect to bloom\nformation is outside the focus of this study.\nThank you for your suggestion. We agree with your point and revised the final section of the\nmanuscript in our last revision. Regrettably, we forgot to revise the abstract accordingly earlier.\nThis time we have revised the abstract as follows:\nL28: This photoprotection mechanism, along with low light adaptation, enables this alga to\nthrive throughout water columns with spatially contrasting and temporally fluctuating\nirradiance, thereby contributing to bloom formation.\n2. The new additional data are not convincing.\n- Xanthophyll cycling:\n- The authors report that the deepoxidation status (DPS) after high light treatment is in the range\nof 0.1. This is in contrast to literature, where always value of 0.8 or higher have been reported\nby many authors independently (see literature below and refs therein). It is hard for me to\nbelieve that such a low DPS can really activate a strong NPQ by the mechanism of xanthophylls\ncycling. If the data are true, the authors should have discussed this strange value and think about\nof an Xanthophyll cycle independent mechanism of NPQ. Also the concentrations of Dt + Dx\nare very low compared to literature. This again asks for explanation and can not be published\nas it is.\nThank you for your valuable suggestion. We repeated the experiment multiple times during this\nround of revision. In these new experiments, we observed higher DPS values than our earlier\nresults, but they are consistently lower than the levels seen in Phaeodactylum tricornutum. After\ncarefully reviewing the paper you provided and other relevant literature, we hypothesize that\ndistinct Ndh isoforms contribute to this difference. The putative non-transmembrane Ndh2 in\nThalassiosira pseudonana is unlikely to contribute to proton-gradient formation and therefore\nmay provide no additional stimulation of DDE activity. Moreover, Ndh2 is not expected to\nrelease detectable protons on the stromal side, which could leave the stromal microenvironment\npermissive for Dt epoxidation. At present, these ideas remain speculative for T. pseudonana\nand lack solid evidence. We are therefore keen to pursue follow-up work, focusing on the\nfunctional roles of the key protein Ndh in this process.\nWe have revised Figure 4d and updated the manuscript as follows:\nL256: Compared to the wild-type and Tp_AaLhc27Q, the DPS of Tp_AaLhc27E increased\nmore rapidly upon light induction (Fig. 4d), indicating that the xanthophyll cycle is involved in\nthe AaLHC27E-based NPQ regulation. Notably, the centric diatom T. pseudonana exhibit\nlower DPS values than the pennate diatom P. tricornutum32. Apart from the influence of growth\nconditions and light history33, this difference may result from the distinct topologies of\nNADPH-dehydrogenases (type-1 and type-2)32. The suppression of DPS elevation upon\nDCMU addition (Fig. 4d) suggests that T. pseudonana likely possesses a non-membrane-\nspanning type-2 NADPH dehydrogenase, which is probably not involved in generating the\nlumenal proton gradient that facilitates DDE-mediated de-epoxidation. Whether this enzyme\nreleases protons on the stromal side and, together with abundant NADPH, promotes the\nepoxidation of Dt also warrants further investigation.\nReference:\nnonphotochemical quenching (NPQ) among microalgae: Implications for growth strategy and\necology. Journal of Phycology 56, 245-263 (2020).\nhttps://doi.org/https://doi.org/10.1111/jpy.12944\nactivity and of non-photochemical fluorescence quenching by two alternative electron\nflows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana.\nBiochimica et Biophysica Acta (BBA) - Bioenergetics 1787, 929-938 (2009).\nhttps://doi.org/https://doi.org/10.1016/j.bbabio.2009.02.004\n- The growth curves\n3. The authors argument is that the growth experiments show that the TpAalhc 27e grows better\nin high light, which is taken as argument that the modification of the Lhc is responsible for\nimproved photoprotection. This is true, however, the growth differences between the different\ngenotypes are quite small and the growth curves show non-typical behavior. Normally, the\ngrowth rate increases up to a saturating light intensity, especially when the cultures are diluted\nand light is not reduced by self shading (see e.g. paper below). In contrast to this general rule\nthe cultures at low light show the highest growth rates indicated by the number of cells after 10\ndays cultivation. Also the slope during the exponential phase is higher or equal to higher light\nintensities. The reduction in growth rate by a factor of about three at 800 µE/m² sec compared\nto 50 µE/m² sec is a clear indication that all the cultures are severely sensitive to photoinhibition.\nI do not understand why the WT cultures of Tp show such a high sensitivity in growth in\nresponse to high light. Please refer to literature (see below).\nThank you for your insight. Following your advice and the references you provided, we ran\nnew experiments using semi-continuous cultures, instead of batch cultures we used initially,\nunder different light intensities, thereby maintaining the cultures at near-maximum specific\ngrowth rates for prolonged period. Based on the new growth rate measurements, we have\nrevised Figure 4e and updated the manuscript as follows:\nL267: Furthermore, our growth measurements indicated that the promoted high light adaptation\nby AaLHC27E enabled the AaLhc27E-expressing T. pseudonana to grow better under higher\nlight conditions (200 and 800 µmol photons m-2 s-1) compared to the AaLhc27Q-expressing\nstrain and the wild type strain (Fig. 4e). Given that T. pseudonana was transformed with only\none AaLhc2E, whereas A. anophagefferens possesses eleven AaLhc2Es, this suggests that the\nmultiple AaLhc2Es in A. anophagefferens may provide greater advantages in dissipating excess\nenergy and growing well under intense and fluctuating light conditions.\nIn summary, the additional data increase the inconsistency of the paper and therefore, the paper\nis not ready for publication\nThe authors should take notice for literature:\n- on xanthophylls cycling e.g. Gruneva et al. BBA 2007\n- Shifts in growth light optima among diatom species support their succession during the spring\nbloom in the Arctic - Croteau - 2022 - Journal of Ecology\nThank you once again for your valuable suggestions and the references, which have\nsignificantly improved our manuscript.\nReviewer #2 (Remarks to the Author):\nThe manuscript has been improved considerably by reacting to the reviewer questions and\ncomments. Especially the part on the MD simulations and Markov state modelling has been\nrevised extensively and now contributes significantly to the interpretation of the experimental\nfindings. Thus, I recommended publication of this revised version.\nThank you for your review and positive feedback.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This\nis part of the Nature Communications initiative to facilitate training in peer review and to\nprovide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for your review and positive feedback."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I think the reviewers have sufficiently addressed the outstanding comments 2 to 4, but their response to the first comment still maintains the untested link with bloom formation, with only a single word change. I don't think that there has been any clear evidence presented to establish this link and therefore any statement to suggest so without further experimentation is highly speculative. If the authors indeed 'agree with this statement', they should do better in adjusting the text accordingly."}, "author_response": "We have to respectfully disagree with this persistent opinion of this reviewer, which seems to deny\nthe important role of photoadaptation in bloom formation. Numerous studies have linked blooms to\nphotoadaptation, although other factors can be important too1-3, which we embrace in our\nmanuscript. We believe our current manuscript already makes it clear that other factors such as\nnutrients are also important while demonstrating that Aurecococcus anophagefferens'\nphotoadaptation mechanism provides an explanation of this species' ability to growth well in\nvariable light conditions contributing to its bloom.\nWith that said, we have decided to further tone this linkage down by changing the concluding\nsentence to \"This photoprotection mechanism, along with low light adaptation, enables this alga to\nthrive throughout water columns with spatially contrasting and temporally fluctuating irradiance,\nwith implications in bloom formation.\"\nReference:\necogenomics. Proceedings of the National Academy of Sciences 108, 4352 (2011).\nhttps://doi.org/10.1073/pnas.1016106108\nPhotosynthesis and Growth of Phaeocystis antarctica and Fragilariopsis cylindrus under\nDifferent Irradiance Levels. Integrative and Comparative Biology 50, 950-966 (2010).\nhttps://doi.org/10.1093/icb/icq021\n541, 536-540 (2017). https://doi.org/10.1038/nature20806"}]

Decision letter