High-intensity illumination induces the over-accumulation of carotenoids by modulates the expression of photosynthetic genes in Euglena sanguinea

High-intensity illumination induces the over-accumulation of carotenoids by modulates the expression of photosynthetic genes in Euglena sanguinea | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article High-intensity illumination induces the over-accumulation of carotenoids by modulates the expression of photosynthetic genes in Euglena sanguinea Ya Zheng, Shuran Wan, Xiaodie Jiang, Haoran Dai, Lili Xu, Qingmin You, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6542765/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 08 Sep, 2025 Read the published version in Photosynthesis Research → Version 1 posted 14 You are reading this latest preprint version Abstract Euglena sanguinea (Ehrenberg 1831) has recently gained prominence due to its bloom events and associated red tide phenomena. Despite this, research on E. sanguinea remains relatively sparse. In this study, we isolated and purified algal strains collected from the water of the Shanghai Botanical Garden, identifying them as E. sanguinea based on 16S and 23S rDNA sequence alignment. The cellular density and carotenoid content of E. sanguinea were observed to vary under different abiotic culture conditions, including varying temperatures, light intensities, potassium iodide, and sucrose. Notably, the most significant rapid accumulation of carotenoids in E. sanguinea was observed under continuous culture at a light intensity of 6000 lux. Furthermore, exposure to a strong intensity resulted in changes in the activity of the antioxidant enzymes and MDA content. Moreover, through de novo transcriptome sequencing and GO analysis of E. sanguinea cultured under different light intensities, we identified a total of 111 differentially expressed genes (DEGs), comprising 44 upregulated and 67 downregulated genes. The up-regulated DEGs included three genes encoding photosystem II protein D1, photosystem II protein K, and cytochrome b6/f complex subunit V. Therefore, we hypothesize that light stress regulates photosynthesis in E. sanguinea by altering the expression of these DEGs, thereby regulating the process of the photosynthetic electron transport and, consequently, the accumulation of photosynthetic pigments in vivo. These findings provide foundational reference data for investigating the photoprotective mechanisms in E. sanguinea and offer a theoretical basis for exploring carotenoid synthesis pathways in plants. Euglena sanguinea Illumination intensity Photosynthesis Carotenoid RNA-Seq Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Euglena is a genus of flagellated algae, characterized by the absence of a cell wall and the presence of a red eyespot. These organisms contain numerous chloroplasts, enabling photosynthesis, and are classified as single-celled eukaryotic organisms exhibiting both plant and animal characteristics (Gupta et al., 2021 ). Active substances accumulated within the algal cells, such as carotenoids, polysaccharides, and lipids, hold potential applications in energy development, healthcare, and agricultural production (Garson et al., 1989). Euglena sanguinea Ehrenberg 1831, the earliest documented green “Euglena” species, primarily inhabits lakes and ponds, with a widespread distribution across freshwater bodies in Africa, Asia, Europe, North America, South America, and Oceania (Kulczycka et al., 2018 ; Deli et al., 2014 ). As a common bloom-forming species in eutrophic waters, E. sanguinea is of increasing concern due to its propensity to form red blooms on the water surface, driven by its strong phototaxis and motility, leading to significant water pollution in recent years. It exhibits considerable resilience, adapting to various environmental fluctuations (Coleman et al., 1988 ). Current research, both domestically and internationally, primarily focuses on the morphological characteristics of E. sanguinea , optimization of culture conditions and the development and utilization of bioactive substances (Kings et al., 2017 ; Vuuren et al., 2021; Sivanesh et al., 2022 ). The pigments within phytoplankton can be categorized into three main groups: chlorophylls, carotenoids, and phycobilin. While chlorophyll-a is universally present in all phytoplankton, the distribution of other pigments varies across different phytoplankton species (Seoane et al., 2009 ). Based on their function in photosynthesis, these pigments can be classified into two broad categories: light-harvesting pigments (LHPs) and photoprotective pigments (PP). LHPs encompass a diverse range of pigments, with chlorophyll-a being a crucial light-harvesting pigment in algae. Other LHPs include fucoxanthin, chlorophyll-c, and peridinin, which serve as accessory pigments (Barlow et al., 2008 ). Photoprotective pigments primarily consist of carotenoids, with variations in their composition among different algal species. These include violaxanthin, diadinoxanthin, diatoxanthin, and zeaxanthin (Aiken et al., 2009 ). Carotenoids are a class of compounds composed of hydrocarbons and their oxygenated derivatives, all derived from a non-cyclic C40H56 structure. Approximately 600 different carotenoids have been isolated from nature, and the environmental conditions significantly influence the accumulation of carotenoids, particularly β-carotene, in microalgae (Oren et al., 2005). As accessory pigments, carotenoids primarily participate in light harvesting under low-light conditions and provide photoprotection under high-light conditions. They absorb portions of visible light, such as blue-violet light, and transfer the absorbed energy to the chloroplasts (Takaichi et al., 2011). Carotenoids can serve as precursors to vitamin A, an essential micronutrient and potent antioxidant in humans. Consequently, carotenoids extracted from algae can be utilized in the production of vitamin A (Yabuzaki et al., 2017; Takaichi et al., 2011). To date, numerous studies have investigated the impact of various environmental conditions, including temperature, nutrient availability, light intensity, and pH, on the pigment composition of microalgae (Schlüter et al., 2000 ; Du et al., 2024). Alterations in these abiotic factors can induce oxidative stress responses within algal cells, thereby modulating carotenoid synthesis. Temperature plays a critical role in algal growth and development, primarily influencing the enzymatic activity within the carotenoid biosynthetic pathway, which in turn controls the rate and expression levels of carotenoid synthesis. For instance, under nitrogen starvation acclimation, Haematococcus pluvialis exhibited a 1.4-fold increase in astaxanthin accumulation at 27°C compared to 20°C (Giannelli et al., 2015 ). Similarly, elevated temperatures have been shown to enhance lutein synthesis in Chlorella protothecoides (Shi et al., 2006 ). Furthermore, Gómez et al. (2005) reported a 3.5-fold increase in α-carotene accumulation in Dunaliella salina when cultured at a lower temperature of 15°C. Light intensity serves as the primary energy source for microalgal growth and is a critical factor in regulating biomass accumulation (Iasimone et al., 2018 ). Within an optimal range of light intensities, an increase in light intensity typically results in a decrease in the concentration of light-harvesting pigments per cell, while the concentration of photoprotective pigments increases, with variations spanning several-fold to over tenfold (Henriksen et al., 2002 ; Rodriguez et al., 2006 ). For instance, light intensity is one of the most effective conditions for inducing the growth of Euglena sanguinea and the accumulation of astaxanthin (Laza-Martínez et al., 2019 ). Del Campo et al ( 2000 ) observed a significant increase in the production of zeaxanthin in Muriellopsis sp. when light intensity was increased from 184 to 460 µmol m⁻² s⁻¹. Furthermore, light quality influences carotenoid accumulation in algae. The pre-irradiation with blue light enhanced photosynthetic activity and tolerance to high-light stress. These findings suggest that β-carotene accumulation is regulated by light intensity, potentially aiding E. gracilis in adapting to the light environment under diurnal conditions (Tanno et al., 2020 ). However, the responses and underlying mechanisms of Euglena sanguinea to various environmental factors remain largely unexplored. Consequently, our findings revealed that light intensity significantly impacted photosynthetic pigment accumulation in E. sanguinea . Under high-light stress (6000 lux), the growth rate and chlorophyll a content of algal cells decreased significantly, accompanied by a marked reduction in superoxide dismutase (SOD) and peroxidase (POD) activities. Conversely, carotenoid content, euglena polysaccharide levels, catalase (CAT) activity, and malondialdehyde (MDA) content increased significantly. Furthermore, transcriptomic analysis of E. sanguinea cultivated under different light intensities (1500 lux and 6000 lux) identified significant differential expression in 111 genes (DEGs). Gene Ontology (GO) and KEGG enrichment analyses of these DEGs revealed significant enrichment in chloroplast thylakoid membrane, plastid thylakoid membrane, photosynthetic membrane, and outer membrane of organelles. Additionally, the KEGG pathways with the highest enrichment were photosynthesis and ribosome pathways. 2. Materials and methods 2.1 Sample Collection, Isolation, and Cultivation The algal strain was obtained from the Shanghai Botanical Garden (31°15′N, 121°45′E). Samples were collected using a 25 # plankton net and stored at 4°C. The algal strain was isolated via the capillary method and subsequently transferred to a 24-well cell culture plate containing Poly A medium. Cultures were maintained in an incubator at 25°C under a 12 h:12 h light-dark cycle with an irradiance of 1500 lux. Growth was monitored using an inverted microscope, and cultures were regularly assessed for contamination. For experimental cultures, 50 mL of fresh medium was added to a 250 mL conical flask, and logarithmic-phase algal cultures were inoculated at a 10:1 ratio, resulting in an initial cell density of approximately 1.0×10⁴ cells mL − 1 . Three replicates were established. Daily, 1 mL of culture was sampled aseptically, fixed with 40 µL of formaldehyde, and thoroughly mixed. Cell counts were performed in triplicate using a hemocytometer under an optical microscope. This process was continued until the stationary phase was reached, and a growth curve was generated. 2.2 Algal Strain Identification 1 mL of algal solution were collected and centrifuged at 12,000 g for 3 minutes, discarding the supernatant. To the pellet, 200 µL of a 10% Chelex100 solution was added, followed by vortexing for 10 seconds and incubation in a 95°C water bath for 20 minutes. Subsequently, vortex the sample for 15 seconds, followed by centrifugation at 13,000 g for 2 minutes to isolate the supernatant. Following DNA extraction, PCR amplification was conducted utilizing primers specific to the 16S rDNA and 23S rDNA gene fragments. The primer sequences are as follows: cpSSU -F (5'-TTGATCCTGGCTCAGGATGAACGCT-3') and cpSSU -R (5'-CAAGGAGGTGATCCAGCCGCACC TT-3'); 23S -F (5'-ATAAGCTTCATTGTCRARAGG-3') and 23S -R (5'-TATGCTTTCAGCATTATCCAC-3'). The PCR amplification protocol was as follows: an initial denaturation step at 94°C for 5 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 50°C for 1 minute, and extension at 72°C for 2 minutes. A final extension step was performed at 72°C for 11 minutes. The resulting PCR products were purified and subsequently submitted to BGI for sequencing. The obtained sequences were then subjected to BLAST analysis against the GenBank database to identify homologous sequences of Euglena. Sequence alignment was performed using BioEdit software, and the terminal, unaligned bases were trimmed to ensure uniform sequence lengths. Phylogenetic analyses were conducted using the aligned sequences. Two distinct phylogenetic trees were constructed using different methodologies. A maximum likelihood tree was generated using PHYML software, with 1,000 bootstrap replicates. A Bayesian phylogenetic tree, incorporating both 16S and 23S gene sequences, was constructed using MrBayes ver. 3.1.2. The resulting phylogenetic trees were visualized and edited using Figtree 1.4.2. 2.3 Cell Density Determination Algal cultures in the exponential growth phase were exposed to various stress conditions, including different light intensities, temperatures, and elicitor treatments (sucrose and potassium iodide). These cultures were maintained under stress for five days, with daily measurements of cell density and carotenoid content. For each condition, a 100 mL aliquot of cells in the exponential growth phase was transferred to a 250 mL conical flask, with three replicates per treatment. Cultures were then subjected to the defined stress conditions. Light microscopy (LM) was performed using an Axio Imager A2 microscope (Carl Zeiss Inc., Hallbergmoos, Germany) equipped with a DP72 microscope camera (Olympus, Tokyo, Japan). Cell density was determined by microscopic counts using a hemocytometer on a 100 mL culture sample. 2.4 Determination of Chlorophyll and Carotenoid Content Photosynthetic pigment content, including chlorophyll a, chlorophyll b, and carotenoids, in Euglena sanguinea cells was quantified using a UV-Vis spectrophotometer following ethanol extraction. The experimental procedure was as follows: 5 mL of algal culture was transferred to 15 mL centrifuge tubes, with three biological replicates. The tubes were centrifuged at 4°C, 6,000 g for 10 min, and the supernatant was discarded. The pellet was resuspended in 5 mL of 95% ethanol, thoroughly mixed, and incubated in the dark at 4°C for 24 h to facilitate pigment extraction. Following extraction, samples were vortexed and centrifuged at 8,000 g for 15 min. The supernatant was collected, and absorbance was measured at 649 nm, 665 nm, and 470 nm using a quartz cuvette in a UV-Vis spectrophotometer. Chlorophyll a, chlorophyll b, and carotenoid contents were calculated using the following equations: Chlorophyll a (Chl a) = 13.95 × A665–6.88 × A649, Chlorophyll b (Chl b) = 24.96 × A649–7.32 × A665, Carotenoids (Car) = (1000 × A470–2.05 × Chlorophyll a -114.8 × Chlorophyll b) / 245. The units for Chl a, Chl b, and Car in the formulas are all mg L − 1 . 2.5 Determination of Euglena Polysaccharide Content Approximately 10 mg of freeze-dried algal powder was weighed and transferred into a capped 15 mL centrifuge tube. Subsequently, 4 mL of ethanol was added, and the mixture was vortexed for 10 s, repeated twice. Following a 1 h incubation at room temperature, the sample was centrifuged at 5000 g for 5 min, and the supernatant was discarded to obtain the crude Euglena polysaccharidae fraction. Protein removal was achieved by adding 1 mL of 1% SDS solution. The mixture was then transferred to a 1.5 mL EP tube and heated in an 85°C water bath for 30 min. After centrifugation at 8000 r/min for 3 min, the supernatant was removed, and 0.1% SDS solution was added. The sample was incubated at room temperature for 30 min, followed by centrifugation at 8000 r/min for 5 min, and the supernatant was discarded. This process was repeated until complete protein removal was achieved. The resulting pellet, designated Pm, was dried in a 50°C drying oven for 24 h. Subsequently, 1 mL of 0.5 mol/L NaOH was added. Polysaccharide quantification was performed using the phenol-sulfuric acid method, with glucose as the standard, and a polysaccharide standard curve was generated. For Pm quantification, 50 µL of the Pm solution was diluted to 1.0 mL with distilled water, and the Pm content was determined using the standard curve. 2.6 Determination of Antioxidant Enzyme Activity and MDA content The algal suspension (100 mL) underwent centrifugation at 8000 g for 10 minutes at 4°C. The resulting pellet was resuspended in 5 mL of phosphate-buffered saline (PBS) solution within a 15 mL centrifuge tube. This tube was maintained on ice and subsequently homogenized via a tissue grinder. The homogenate was then centrifuged at 10000 g for 20 minutes in a high-speed refrigerated centrifuge, and the supernatant was collected. Soluble protein content was quantified using the Coomassie Brilliant Blue method (Beyotime Biotechnology Kit), with a standard curve employed for accurate determination. Antioxidant enzyme activity was evaluated using Superoxide Dismutase (SOD) and Peroxidase (POD) assay kits (Solarbio), and measurements were obtained utilizing a microplate reader (Varioskan LUX). Cellular malondialdehyde (MDA) content was quantified using a micro MDA assay kit obtained from Solebaer Biotechnology Co., LTD (China). Enzymatic analysis was employed to determine MDA levels within the cells. The extraction protocol for MDA from algal cells was adapted from established methods for antioxidant enzyme extraction. 2.7 RNA Preparation and Transcriptome Data Analysis Total RNA was extracted from 100 mL of Euglena sanguinea cultures, cultivated under 1500 lux and 6000 lux illumination, with three biological replicates, using the Qubit RNA kit. RNA purity was assessed using a Nano Photometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was quantified using the Qubit® RNA Assay Kit in a Qubit®2.0 Fluorometer (Life Technologies, CA, USA). cDNA libraries were constructed using the VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina®. De novo assembly was performed using Trinity (v. 2.0.6) with default parameters. Given the absence of a complete genome sequence for any species within the E. sanguinea genus and the low homology observed between our transcriptome data and that of Euglena gracilis , a nonparametric analysis was employed. Differentially expressed genes (DEGs) were identified based on a mean fold change threshold of ≥ 2 or ≤ 0.5, and a P -value ≤ 0.05. Unigenes were annotated using nine public databases: NCBI nucleotide sequences (NT), NCBI non-redundant protein sequences (NR), Conserved Domain Database (CDD), Clusters of Orthologous Groups of proteins (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), Pfam, SwissProt, and TrEMBL. GO functional enrichment and KEGG pathway analyses were conducted using Goatools and KOBAS (Xie et al., 2011 ). 2.8 Data Analysis Triplicate biological replicates were employed, and the data are presented as means ± S.E.M. Prior to analysis, homogeneity of variance and normal distribution were assessed. Data analysis was performed using the Data Processing System (DPS Version 7.05). Multiple comparisons were conducted using one-way ANOVA followed by Tukey's test. Statistical significance was defined as * P < 0.05 and ** P < 0.01. Data visualization was performed using GraphPad Prism (version 8.01, San Diego, USA). 3. Results 3.1 Strain Collection and Identification of Euglena sanguinea Algal strains were isolated from water samples collected from the Shanghai Botanical Garden and cultured in Ploy A medium. Microscopic analysis of the isolates revealed cells with a green pigmentation, measuring 30–170 µm in length and 17–44 µm in diameter. The cells exhibited a flexible cell wall, high motility, and significant morphological plasticity. The nucleus was observed to be either medial or posterior in position, and a red eyespot was present. Based on these morphological features, the isolated algal strain was provisionally identified as Euglena sanguinea . Molecular identification was conducted via 16S rDNA and 23S rDNA sequence analysis. PCR amplification generated products of 1349 bp and 1538 bp, respectively. The obtained sequences (S13B6) were subjected to a homology search against sequences within the Euglena genus, and phylogenetic trees were constructed using Bayesian and maximum likelihood methodologies. The resulting topologies of the 16S rDNA and 23S rDNA trees exhibited congruence. Consequently, the support values from the maximum likelihood tree were annotated onto the Bayesian tree, as illustrated in Fig. 1 and Table S1 . The integrated morphological and phylogenetic analyses confirmed the identification of the isolated algal strains as E. sanguinea. 3.2 Influence of Varying Culture Conditions on Carotenoid Content Carotenoids, prevalent across diverse taxa including plants, fungi, bacteria, and algae, function as accessory pigments within photosynthetic pathways, primarily absorbing visible light, particularly in the blue-violet spectrum. This absorbed photonic energy is then channeled to the chloroplasts, thereby facilitating light harvesting and photoprotection in photosynthetic organisms (Takaichi et al., 2011). To elucidate the factors governing carotenoid biosynthesis in E. sanguinea , the microalgae were initially cultivated continuously for four days to achieve exponential growth. Subsequently, cultures were maintained under a range of light intensities (1500 lux, 4000 lux, 6000 lux, 8000 lux, and 10000 lux) for 4–5 days, with daily quantification of carotenoid content. Results indicated that varying light intensities significantly impacted carotenoid accumulation (Fig. 2 A). At 6000 lux, carotenoid accumulation exhibited the most substantial increase after five days (30.5%) relative to the 1500 lux control. Conversely, at 10000 lux, carotenoid accumulation was significantly suppressed. Consequently, a light intensity of 6000 lux was selected for subsequent experiments involving other factor inductions in E. sanguinea . Following this, under 6000 lux illumination, E. sanguinea was cultivated under varying conditions, including different temperatures (20°C, 25°C, 30°C, 35°C), potassium iodide concentrations (0.01 mM, 0.1 mM, 1.0 mM, 5.0 mM, 10.0 mM), and sucrose concentrations (0.1 mM, 1.0 mM, 10.0 mM, 50.0 mM). The results demonstrated that temperature also influenced carotenoid accumulation. The most significant carotenoid accumulation was observed at 20°C, with a 4% increase compared to the control group (Fig. 2 B). Furthermore, the addition of varying concentrations of potassium iodide and sucrose to the culture medium also affected carotenoid accumulation. Specifically, carotenoid accumulation was most pronounced when the culture medium contained 1 mM potassium iodide or 50 mM sucrose, with increases of 9% and 15% compared to the control group, respectively (Fig. 2 C, 2 D). Based on these findings, we conclude that light intensity is a significant factor influencing carotenoid accumulation in E. sanguinea , with optimal accumulation observed at 6000 lux. Furthermore, other environmental factors, including temperature, potassium iodide concentration, and sucrose concentration, also modulate carotenoid accumulation in this microalga. 3.3 Effects of Light intensity on Photosynthetic Pigments and Paramylon To investigate the impact of varying light intensities on photosynthetic pigment synthesis in E. sanguinea , a five-day morphological observation was conducted under different light intensities. Although no significant alteration in algal morphological size was observed across the light treatments, the carotenoid content in the algal cells exhibited a marked increase with escalating light intensity within the range of 1500–6000 lux (as indicated by the orange-red cluster in Fig. 3 A). Furthermore, the rate of carotenoid accumulation decelerated when light intensity exceeded 8000 lux. Consequently, the data suggest that the most rapid carotenoid accumulation, culminating in a peak value on the fifth day, occurred under a light intensity of 6000 lux. Subsequently, we assessed the photosynthetic pigment variations in E. sanguinea on the fifth day under light intensities of 1500 lux and 6000 lux, respectively. The pigments quantified included chlorophyll a, chlorophyll b, and total carotenoids. The results indicated that, relative to the control group (1500 lux), high-light stress (6000 lux) induced a reduction in chlorophyll content, with a significant 20.5% decrease in chlorophyll a (Chla) content ( P < 0.05). Conversely, the content of carotenoids, acting as accessory photosynthetic pigments, significantly increased by 30.6% ( P < 0.05) under high-light intensity (Fig. 3 B). Moreover, continuous high-light stress for five days significantly promoted polysaccharide accumulation in E. sanguinea ( P < 0.05) (Fig. 3 C). These findings suggest that high light intensities can enhance the accumulation of polysaccharides and carotenoids, while simultaneously suppressing chlorophyll synthesis. 3.4 High-intensity light elicits oxidative stress response in E. sanguinea To investigate the induction of oxidative stress responses in E. sanguinea by photo-stress, soluble protein standard curves were generated under continuous four-day photo-stress conditions. Samples were normalized to the measured soluble protein content of the algal cells, and the levels of antioxidant stress response enzymes, including SOD, CAT, POD, and malondialdehyde (MDA), were quantified. The results demonstrated that the antioxidant enzyme activities of SOD and POD in E. sanguinea cultivated under 6000 lux light intensity were significantly downregulated compared to the control group (1500 lux) ( P < 0.01), with respective decreases of 48.2% and 52.9% (Figs. 4 A, 4 B). Conversely, CAT enzyme activity in the algae exhibited a significant increase of 63.7% ( P < 0.05) under high light intensity stress (Fig. 4 C). Furthermore, the MDA content in Euglena under 6000 lux stress was also significantly elevated by 56.2% compared to the control group ( P < 0.01) (Fig. 4 D). These findings suggest that high light intensity modulates the oxidative stress responses in E. sanguinea . 3.5 Light intensity-dependent modulation of gene expression profiles in E. sanguinea To investigate the impact of light intensity on gene expression, we conducted transcriptomic profiling of E. sanguinea under different light intensities (1500 lux and 6000 lux). Sequencing yielded a total of 32,153 identified genes. The number of expressed genes, total mapped reads, and unique matches for each sample are detailed in Table S2. High-intensity light stress induced differential expression in 111 genes (DEGs), comprising 44 up-regulated and 67 down-regulated genes (absolute log2 fold-change > 1, P < 0.05) (Fig. 5 A). The evolutionary genealogy of genes: non-supervised orthologous groups (EggNOG) classification of these DEGs revealed enrichment in the cytoskeleton, energy production and conversion, and cell cycle control, cell division, and chromosome partitioning categories (Fig. 5 B). To further elucidate the biological responses elicited by high-intensity light stress, we conducted GO (gene ontology) annotation (Figure S2) and GO enrichment analysis (Fig. 5 C) of differentially expressed genes (DEGs), categorized by their functional roles. The predominant GO terms encompassed chloroplast thylakoid membrane, plastid thylakoid membrane, photosynthetic membrane, and organelle outer membrane. Furthermore, based on cellular component (CC) analysis, the DEGs were primarily enriched in plastid, chloroplast, and organelle bounding membrane. Regarding biological process (BP), the DEGs were significantly enriched in photosynthesis. Molecular function (MF) analysis revealed enrichment of DEGs in electron transfer activity, rRNA binding, copper ion binding, and ATP-dependent activity. Additionally, KEGG pathway analysis was performed on these DEGs. The two most highly enriched KEGG pathways were photosynthesis and Ribosome (Fig. 5 D). Notably, upregulated DEGs included three genes encoding photosystem II protein D1 ( Euglena gracilis ), photosystem II protein K ( Eutreptiella eupharyngea ), and cytochrome b6/f complex subunit V ( Euglena deses ). Consequently, we hypothesize that light stress modulates photosynthesis in Euglena species by modulating the expression of these DEGs, thereby regulating photosynthetic membrane function and, consequently, influencing the in vivo accumulation of photosynthetic pigments. 4. Discussion The complex chloroplast morphology of Euglena sanguinea presents challenges for taxonomic identification (Laza-Martinez et al., 2019). Over the course of more than two centuries of research, twelve novel algal groups were initially considered posited as analogous to E. sanguinea . However, identification based solely on morphological observations and physiological-biochemical indicators, using traditional taxonomic methods, proved difficult. Currently, descriptions and analyses of E. sanguinea and related species integrate morphological and molecular biological approaches, which has reduced the number of similar groups to four ( E. sanguinea , E. sociabilis , E. splendensand and E. laciniata ). Furthermore, new phenotypic characteristics and updated diagnostic descriptions have been established, including the presence of fusiform muciferous sacs, numerous chloroplasts, different types of paramylon bodies, and the presence of paramylon granules near the cell's stigma (Karnkowska-Ishikawa et al., 2013 ). Nevertheless, taxonomic verification of this species remains challenging for researchers lacking extensive experience. Therefore, accurate identification of E. sanguinea based on molecular biology is essential. The prevailing methodology employed currently employs its nSSU rDNA as a molecular barcode (Kulczycka et al., 2018 ). This study employed a dual identification approach, combining morphological analysis with homology data comparison of 16S rDNA and 23S rDNA base sequences, confirming the algal strain collected from the Shanghai Botanical Garden's river as E. sanguinea . The growth and the accumulation of bioactive substances in microalgae are significantly influenced by environmental factors, including temperature, salinity, light, and pH (Ogbonna et al., 2000). Research indicates that light conditions play a crucial role in the growth and development of microalgae, with light intensity, photoperiod, and light quality exerting the most significant effects (Takada et al., 2012 ). In this study, we initially investigated the impact of four abiotic factors (light, temperature, potassium iodide, and glucose) on the growth and physiological biochemistry of E. sanguinea . We observed that the cell density of E. sanguinea was modulated to varying degrees by these abiotic factors. Notably, light intensity and temperature exhibited the most pronounced effects on the microalgae (Fig S1 ). Previous studies have demonstrated that under stress conditions such as high light, high salinity, or nutrient limitation, microalgae can produce excess lipids or carotenoids (Lamers et al., 2010 ). Carotenoids, acting as photoprotective pigments, absorb excess energy, thereby preventing damage to cellular membranes and providing photoprotection (Sun et al., 2018 ). We observed a decline in chlorophyll a content within E. sanguinea concurrent with escalating light intensity. Furthermore, carotenoid accumulation exhibited a marked increase, particularly under cultivation at 6000 lux. However, at a light intensity of 10000 lux, carotenoid accumulation was significantly inhibited. This observation aligns with findings in Haematococcus pluvialis , where high-intensity light induces the accumulation of astaxanthin and canthaxanthin within cytoplasmic vesicles, accompanied by a color change from green to red (Lemoine et al., 2010; Cezare-Gomes et al., 2019 ). Furthermore, in both higher plants and algae, the accumulation of triacylglycerols is frequently coupled with the synthesis of carotenoids. Under high light conditions, carotenoids are transported to lipid bodies, forming abundant carotenoid-containing lipid bodies outside the chloroplasts, thus preventing the overstimulation of chloroplasts by intense light and ensuring efficient photosynthesis (Ferrell and Sariskyreed, 2010 ). Lamers et al. ( 2010 ) investigated the changes in β-carotene and fatty acid content in Dunaliella salina under photoinduction, observing that the accumulation of carotenoids was accompanied by a decrease in fatty acid unsaturation. The plants generate a large amount of reactive oxygen species (ROS) under low temperature, high salinity, or nitrogen deficiency conditions. ROS, acting as signaling molecules, can initiate the organism's defense system by promoting the synthesis of cellular carotenoids, thereby enabling the organism to withstand oxidative stress damage (Alscher et al., 1997 ). We further observed that exposure to a light intensity of 6000 lux resulted in a reduction in the activity of the antioxidant enzymes SOD and POD within the algae, while CAT activity and MDA content increased. These antioxidant enzymes play a crucial role in the organism's antioxidant defense system by working synergistically to scavenge free radicals. Specifically, POD and CAT are responsible for the decomposition of hydrogen peroxide in the cytoplasm and peroxisomes, respectively, thereby preventing oxidative damage caused by its accumulation (Abo-Shady et al., 2023 ). Li et al. ( 2008 ) found that under high-light stress, the cellular morphology of Haematococcus pluvialis transitioned from green motile cells to brown cells, with the highest activities of SOD and CAT observed. However, due to prolonged photooxidative stress and high levels of peroxidation, the cells ultimately turned red, accompanied by the accumulation of astaxanthin. Furthermore, the polysaccharide content of E. sanguinea significantly increased under 6000 lux illumination. Given the antioxidant properties of Euglena polysaccharides, it is hypothesized that the rapid accumulation of carotenoids in E. sanguinea under light stress may be regulated by ROS. These responses are essential for maintaining normal photosynthetic function in microalgae. To investigate the regulatory effects and mechanisms of light on E. sanguinea growth, we conducted a transcriptomic analysis of cells cultivated under 1500 lux and 6000 lux illumination. A total of 111 differentially expressed genes (DEGs) were identified, comprising 44 upregulated and 67 downregulated genes. These DEGs were subjected to GO and KEGG enrichment analyses. The results indicate that light intensity primarily influences the photosynthetic processes and membrane systems of E. sanguinea , specifically including the chloroplast thylakoid membrane, plastid thylakoid membrane, photosynthetic membrane, and organelle outer membrane. The chloroplast thylakoid membrane is considered the primary site for carotenoid synthesis. Photosynthetic proteins responsible for carrying out photosynthesis are located on the thylakoid membrane structure within the cell. Various environmental stresses render the thylakoid membrane particularly susceptible to damage, especially under high-illumination conditions (Collins et al., 2011 ). The accumulation of diverse carotenoids in photosynthetic organisms is believed to serve multiple physiological functions, with the most critical being the facilitation of stress tolerance. A prime example is the photoprotective function executed by the xanthophyll cycle (Bassi and Caffarri, 2000 ; Jahns et al., 2009 ; Lin et al., 2002 ). The levels of carotenoids are influenced by numerous external factors; generally, photosynthetic organisms accumulate substantial amounts of carotenoids under adverse conditions. For instance, E. sanguinea accumulates astaxanthin and β-carotene under nutrient deficiency, high-illumination, high-temperature, or high-salinity conditions (Tanno et al., 2020 ). He et al. ( 2018 ) demonstrated, through transcriptomic analysis, that high-illumination conditions in Haematococcus pluvialis induced the promotion of carotenoid biosynthesis, with altered expression of genes associated with photosynthesis-antenna proteins and carbon fixation pathways. Therefore, we hypothesize that light stress primarily impacts carotenoid accumulation within the cell by modulating the expression of photosynthetic proteins on the thylakoid membrane of E. sanguinea . Notably, the up-regulated DEGs including three genes encoding photosystem II protein D1 ( Euglena agilis ), photosystem II protein K ( Eutreptiella eupharyngea ) and cytochrome b6/f complex subunit V ( Euglena deses ). Photosystem I (PSI) and Photosystem II (PSII) are light-harvesting protein complexes embedded within the thylakoid membranes. PSI, being a larger complex, is predominantly located in the stroma lamellae, whereas PSII complexes are primarily concentrated in the stacked grana thylakoid membrane regions (Umena et al., 2011 ). To mitigate irreversible photodamage to PSII under high light conditions, plants have evolved a sophisticated repair mechanism. This includes the disassembly of PSII, proteolytic degradation of the damaged D1 reaction center protein, and the co-translational insertion of newly synthesized D1 protein into partially disassembled PSII core complexes (Theis and Schroda, 2016 ). The D1 protein, encoded by the chloroplast gene psbA , is highly conserved across higher plants and forms the fundamental scaffold of the PSII reaction center, in conjunction with the D2 protein. The D1 protein not only provides binding sites for various cofactors but also maintains the conformational stability of the PSII reaction center and is particularly sensitive to various stress conditions (Zhang et al., 2000 ). Consequently, high light intensity can induce the upregulation of D1 expression in E. sanguinea , potentially representing a crucial mechanism for enhancing photosynthetic efficiency (Adir et al., 2003 ; Aro et al., 2005 ; Mulo et al., 2009 ). The cytochrome b6f (Cytb6f) complex, a pivotal component and rate-limiting step in photosynthetic electron transport, serves as a critical redox-sensing hub (Kurisu et al., 2003 ; Cramer et al., 2005 ). Consequently, Cytb6f represents a promising target for genetic engineering aimed at enhancing photosynthetic efficiency (Tikhonov et al., 2014; Malone et al., 2021 ). Therefore, high light intensity induces the overexpression of the cytochrome b6f complex in E. sanguinea , further validating that Cytb6f expression serves as a critical indicator for assessing photosynthetic efficiency. In conclusion, this study provides preliminary insights into the physiological and biochemical responses of E. sanguinea under various culture conditions and elucidates transcriptional changes in algal cells under light stress. These findings provide foundational data for investigating the mechanisms by which light regulates the growth and photosynthetic pigment synthesis in E. sanguinea , with the potential to inform solutions for E. sanguinea -related "red tide" events, large-scale cultivation, and research into carotenoid synthesis mechanisms. Declarations Funding Fundings for this work was provided by Natural Science Foundation of Shanghai (No. 23ZR1446800), and National Natural Science Foundation of China (NO. 32270383). Author Contribution Y. Z and W.T. P wrote the main manuscript text; S.R.W and X.D. J prepared figures 1; H.R.D, Q.M.Y and L.L.X prepared figures 2-5; Q.X.W presented conceptualization. All authors reviewed the manuscript. Acknowledgement We thank Majorbio company for its transcriptome data analysis in this study. Fundings for this work was provided by Natural Science Foundation of Shanghai (No. 23ZR1446800), and National Natural Science Foundation of China (NO. 32270383). 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Light microscopy was used to observe the morphology of \u003cem\u003eE. sanguinea\u003c/em\u003e, and a concatenated phylogenetic tree was constructed based on 16S and 23S rRNA gene sequences. Branching node values represent maximum likelihood bootstrap support/Bayesian posterior probabilities. Support values below 500/0.5 are not displayed, indicated by \"-\".\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6542765/v1/01507c4c6a2ca17900fd9c66.jpeg"},{"id":83075805,"identity":"e62b81cf-93ba-4de1-b7e7-203f29903165","added_by":"auto","created_at":"2025-05-19 18:07:09","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":470297,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCarotenoid content analysis of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e Euglena sanguinea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under four culture conditions. \u003c/strong\u003eThe experimental design incorporated variations in light intensity (\u003cstrong\u003eA\u003c/strong\u003e), temperature (\u003cstrong\u003eB\u003c/strong\u003e), potassium iodide concentration (\u003cstrong\u003eC\u003c/strong\u003e), and sucrose concentration (\u003cstrong\u003eD\u003c/strong\u003e). The algal cultures in conditions \u003cstrong\u003eB\u003c/strong\u003e, \u003cstrong\u003eC\u003c/strong\u003e, and \u003cstrong\u003eD\u003c/strong\u003e were maintained at 6000 lux, with the exception of the control group, which was exposed to 1500 lux. Data are presented as means ± standard deviations (n=3).\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6542765/v1/56f35f3ba3eb0daf01ff0dbf.jpg"},{"id":83075806,"identity":"6cea3828-56b4-458d-8160-51e94122b1b4","added_by":"auto","created_at":"2025-05-19 18:07:09","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":238140,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe impact of varying light intensities on photosynthetic pigments and paramylon. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Microscopic analysis of \u003cem\u003eE. sanguinea\u003c/em\u003e under five distinct light intensity conditions. Scale bar, 20 μm; (\u003cstrong\u003eB\u003c/strong\u003e) Comparative analysis of photosynthetic pigment concentrations in algal cultures on day five under differing light conditions. Chla, chlorophyll a; Chlb, chlorophyll b; Caro, carotenoids; (\u003cstrong\u003eC\u003c/strong\u003e) Comparison of Paramylon (Pm) content in algal cultures on day five under 1500 lux and 6000 lux illumination. *: \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6542765/v1/d73177c9149e408569d0c065.jpg"},{"id":83075577,"identity":"ed7221a3-e1dc-486a-bb8f-ea9dd317d214","added_by":"auto","created_at":"2025-05-19 17:59:09","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOxidative stress responses of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. sanguinea\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einduced by high-intensity light. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Differences in SOD activity in algae cultured under two light intensities (1500 lux and 6000 lux); (\u003cstrong\u003eB\u003c/strong\u003e) POD activity; (\u003cstrong\u003eC\u003c/strong\u003e) CAT activity; (\u003cstrong\u003eD\u003c/strong\u003e) MDA content. *, indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05; **, indicates \u003cem\u003eP\u003c/em\u003e\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6542765/v1/c9cc98c37b38fafd2c4bfee2.png"},{"id":83076460,"identity":"b3ed4a94-2301-4566-ae51-6a91a9978ed9","added_by":"auto","created_at":"2025-05-19 18:15:09","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscript alterations were assessed using RNA-seq in algae cultured under different light intensities.\u003c/strong\u003e (\u003cstrong\u003eA\u003c/strong\u003e) A volcano plot illustrates differentially expressed genes (DEGs) identified through a comparison of control algae (cultured at 1500 lux) and those exposed to 6000 lux. (\u003cstrong\u003eB\u003c/strong\u003e) The EggNOG (evolutionary genealogy of genes: Non-supervised Orthologous Groups) classification provides insights into the functional categorization of genes between the two algal groups. (\u003cstrong\u003eC\u003c/strong\u003e) Gene Ontology (GO) enrichment analysis was performed on DEGs in the 6000 lux group relative to the 1500 lux group, elucidating enriched biological processes. (\u003cstrong\u003eD\u003c/strong\u003e) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was conducted to identify pathways enriched in algae cultured at 6500 lux compared to the control group, providing a pathway-level understanding of the observed transcriptional responses.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6542765/v1/2e9b3cdcb04cd14ad94f3140.jpeg"},{"id":91358898,"identity":"cc1911db-831d-4279-8ab9-888730b64b69","added_by":"auto","created_at":"2025-09-15 16:00:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2239194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6542765/v1/6b8fb0c7-0bee-4e1d-b7dd-e4ae90731ae9.pdf"},{"id":83075599,"identity":"b3629fda-c14d-4ab6-ad0e-d18488f202ce","added_by":"auto","created_at":"2025-05-19 17:59:10","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":12392154,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6542765/v1/3c4feaf77af5e20e9b3cf0e1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"High-intensity illumination induces the over-accumulation of carotenoids by modulates the expression of photosynthetic genes in Euglena sanguinea","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEuglena is a genus of flagellated algae, characterized by the absence of a cell wall and the presence of a red eyespot. These organisms contain numerous chloroplasts, enabling photosynthesis, and are classified as single-celled eukaryotic organisms exhibiting both plant and animal characteristics (Gupta et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Active substances accumulated within the algal cells, such as carotenoids, polysaccharides, and lipids, hold potential applications in energy development, healthcare, and agricultural production (Garson et al., 1989). \u003cem\u003eEuglena sanguinea\u003c/em\u003e Ehrenberg 1831, the earliest documented green \u0026ldquo;Euglena\u0026rdquo; species, primarily inhabits lakes and ponds, with a widespread distribution across freshwater bodies in Africa, Asia, Europe, North America, South America, and Oceania (Kulczycka et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Deli et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). As a common bloom-forming species in eutrophic waters, \u003cem\u003eE. sanguinea\u003c/em\u003e is of increasing concern due to its propensity to form red blooms on the water surface, driven by its strong phototaxis and motility, leading to significant water pollution in recent years. It exhibits considerable resilience, adapting to various environmental fluctuations (Coleman et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e1988\u003c/span\u003e). Current research, both domestically and internationally, primarily focuses on the morphological characteristics of \u003cem\u003eE. sanguinea\u003c/em\u003e, optimization of culture conditions and the development and utilization of bioactive substances (Kings et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Vuuren et al., 2021; Sivanesh et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe pigments within phytoplankton can be categorized into three main groups: chlorophylls, carotenoids, and phycobilin. While chlorophyll-a is universally present in all phytoplankton, the distribution of other pigments varies across different phytoplankton species (Seoane et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Based on their function in photosynthesis, these pigments can be classified into two broad categories: light-harvesting pigments (LHPs) and photoprotective pigments (PP). LHPs encompass a diverse range of pigments, with chlorophyll-a being a crucial light-harvesting pigment in algae. Other LHPs include fucoxanthin, chlorophyll-c, and peridinin, which serve as accessory pigments (Barlow et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Photoprotective pigments primarily consist of carotenoids, with variations in their composition among different algal species. These include violaxanthin, diadinoxanthin, diatoxanthin, and zeaxanthin (Aiken et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Carotenoids are a class of compounds composed of hydrocarbons and their oxygenated derivatives, all derived from a non-cyclic C40H56 structure. Approximately 600 different carotenoids have been isolated from nature, and the environmental conditions significantly influence the accumulation of carotenoids, particularly β-carotene, in microalgae (Oren et al., 2005). As accessory pigments, carotenoids primarily participate in light harvesting under low-light conditions and provide photoprotection under high-light conditions. They absorb portions of visible light, such as blue-violet light, and transfer the absorbed energy to the chloroplasts (Takaichi et al., 2011). Carotenoids can serve as precursors to vitamin A, an essential micronutrient and potent antioxidant in humans. Consequently, carotenoids extracted from algae can be utilized in the production of vitamin A (Yabuzaki et al., 2017; Takaichi et al., 2011).\u003c/p\u003e \u003cp\u003eTo date, numerous studies have investigated the impact of various environmental conditions, including temperature, nutrient availability, light intensity, and pH, on the pigment composition of microalgae (Schl\u0026uuml;ter et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Du et al., 2024). Alterations in these abiotic factors can induce oxidative stress responses within algal cells, thereby modulating carotenoid synthesis. Temperature plays a critical role in algal growth and development, primarily influencing the enzymatic activity within the carotenoid biosynthetic pathway, which in turn controls the rate and expression levels of carotenoid synthesis. For instance, under nitrogen starvation acclimation, \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e exhibited a 1.4-fold increase in astaxanthin accumulation at 27\u0026deg;C compared to 20\u0026deg;C (Giannelli et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, elevated temperatures have been shown to enhance lutein synthesis in \u003cem\u003eChlorella protothecoides\u003c/em\u003e (Shi et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Furthermore, G\u0026oacute;mez et al. (2005) reported a 3.5-fold increase in α-carotene accumulation in \u003cem\u003eDunaliella salina\u003c/em\u003e when cultured at a lower temperature of 15\u0026deg;C.\u003c/p\u003e \u003cp\u003eLight intensity serves as the primary energy source for microalgal growth and is a critical factor in regulating biomass accumulation (Iasimone et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Within an optimal range of light intensities, an increase in light intensity typically results in a decrease in the concentration of light-harvesting pigments per cell, while the concentration of photoprotective pigments increases, with variations spanning several-fold to over tenfold (Henriksen et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Rodriguez et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). For instance, light intensity is one of the most effective conditions for inducing the growth of \u003cem\u003eEuglena sanguinea\u003c/em\u003e and the accumulation of astaxanthin (Laza-Mart\u0026iacute;nez et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Del Campo et al (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2000\u003c/span\u003e) observed a significant increase in the production of zeaxanthin in \u003cem\u003eMuriellopsis sp.\u003c/em\u003e when light intensity was increased from 184 to 460 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1;. Furthermore, light quality influences carotenoid accumulation in algae. The pre-irradiation with blue light enhanced photosynthetic activity and tolerance to high-light stress. These findings suggest that β-carotene accumulation is regulated by light intensity, potentially aiding \u003cem\u003eE. gracilis\u003c/em\u003e in adapting to the light environment under diurnal conditions (Tanno et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHowever, the responses and underlying mechanisms of \u003cem\u003eEuglena sanguinea\u003c/em\u003e to various environmental factors remain largely unexplored. Consequently, our findings revealed that light intensity significantly impacted photosynthetic pigment accumulation in \u003cem\u003eE. sanguinea\u003c/em\u003e. Under high-light stress (6000 lux), the growth rate and chlorophyll a content of algal cells decreased significantly, accompanied by a marked reduction in superoxide dismutase (SOD) and peroxidase (POD) activities. Conversely, carotenoid content, euglena polysaccharide levels, catalase (CAT) activity, and malondialdehyde (MDA) content increased significantly. Furthermore, transcriptomic analysis of \u003cem\u003eE. sanguinea\u003c/em\u003e cultivated under different light intensities (1500 lux and 6000 lux) identified significant differential expression in 111 genes (DEGs). Gene Ontology (GO) and KEGG enrichment analyses of these DEGs revealed significant enrichment in chloroplast thylakoid membrane, plastid thylakoid membrane, photosynthetic membrane, and outer membrane of organelles. Additionally, the KEGG pathways with the highest enrichment were photosynthesis and ribosome pathways.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Sample Collection, Isolation, and Cultivation\u003c/h2\u003e \u003cp\u003eThe algal strain was obtained from the Shanghai Botanical Garden (31\u0026deg;15\u0026prime;N, 121\u0026deg;45\u0026prime;E). Samples were collected using a 25\u003csup\u003e#\u003c/sup\u003e plankton net and stored at 4\u0026deg;C. The algal strain was isolated via the capillary method and subsequently transferred to a 24-well cell culture plate containing Poly A medium. Cultures were maintained in an incubator at 25\u0026deg;C under a 12 h:12 h light-dark cycle with an irradiance of 1500 lux. Growth was monitored using an inverted microscope, and cultures were regularly assessed for contamination.\u003c/p\u003e \u003cp\u003eFor experimental cultures, 50 mL of fresh medium was added to a 250 mL conical flask, and logarithmic-phase algal cultures were inoculated at a 10:1 ratio, resulting in an initial cell density of approximately 1.0\u0026times;10⁴ cells mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Three replicates were established. Daily, 1 mL of culture was sampled aseptically, fixed with 40 \u0026micro;L of formaldehyde, and thoroughly mixed. Cell counts were performed in triplicate using a hemocytometer under an optical microscope. This process was continued until the stationary phase was reached, and a growth curve was generated.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Algal Strain Identification\u003c/h2\u003e \u003cp\u003e1 mL of algal solution were collected and centrifuged at 12,000 \u003cem\u003eg\u003c/em\u003e for 3 minutes, discarding the supernatant. To the pellet, 200 \u0026micro;L of a 10% Chelex100 solution was added, followed by vortexing for 10 seconds and incubation in a 95\u0026deg;C water bath for 20 minutes. Subsequently, vortex the sample for 15 seconds, followed by centrifugation at 13,000 \u003cem\u003eg\u003c/em\u003e for 2 minutes to isolate the supernatant. Following DNA extraction, PCR amplification was conducted utilizing primers specific to the 16S rDNA and 23S rDNA gene fragments. The primer sequences are as follows: \u003cem\u003ecpSSU\u003c/em\u003e-F (5'-TTGATCCTGGCTCAGGATGAACGCT-3') and \u003cem\u003ecpSSU\u003c/em\u003e-R (5'-CAAGGAGGTGATCCAGCCGCACC\u003c/p\u003e \u003cp\u003eTT-3'); \u003cem\u003e23S\u003c/em\u003e-F (5'-ATAAGCTTCATTGTCRARAGG-3') and \u003cem\u003e23S\u003c/em\u003e-R (5'-TATGCTTTCAGCATTATCCAC-3'). The PCR amplification protocol was as follows: an initial denaturation step at 94\u0026deg;C for 5 minutes, followed by 30 cycles of denaturation at 94\u0026deg;C for 30 seconds, annealing at 50\u0026deg;C for 1 minute, and extension at 72\u0026deg;C for 2 minutes. A final extension step was performed at 72\u0026deg;C for 11 minutes. The resulting PCR products were purified and subsequently submitted to BGI for sequencing. The obtained sequences were then subjected to BLAST analysis against the GenBank database to identify homologous sequences of Euglena. Sequence alignment was performed using BioEdit software, and the terminal, unaligned bases were trimmed to ensure uniform sequence lengths. Phylogenetic analyses were conducted using the aligned sequences. Two distinct phylogenetic trees were constructed using different methodologies. A maximum likelihood tree was generated using PHYML software, with 1,000 bootstrap replicates. A Bayesian phylogenetic tree, incorporating both 16S and 23S gene sequences, was constructed using MrBayes ver. 3.1.2. The resulting phylogenetic trees were visualized and edited using Figtree 1.4.2.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Cell Density Determination\u003c/h2\u003e \u003cp\u003eAlgal cultures in the exponential growth phase were exposed to various stress conditions, including different light intensities, temperatures, and elicitor treatments (sucrose and potassium iodide). These cultures were maintained under stress for five days, with daily measurements of cell density and carotenoid content. For each condition, a 100 mL aliquot of cells in the exponential growth phase was transferred to a 250 mL conical flask, with three replicates per treatment. Cultures were then subjected to the defined stress conditions. Light microscopy (LM) was performed using an Axio Imager A2 microscope (Carl Zeiss Inc., Hallbergmoos, Germany) equipped with a DP72 microscope camera (Olympus, Tokyo, Japan). Cell density was determined by microscopic counts using a hemocytometer on a 100 mL culture sample.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Determination of Chlorophyll and Carotenoid Content\u003c/h2\u003e \u003cp\u003ePhotosynthetic pigment content, including chlorophyll a, chlorophyll b, and carotenoids, in \u003cem\u003eEuglena sanguinea\u003c/em\u003e cells was quantified using a UV-Vis spectrophotometer following ethanol extraction. The experimental procedure was as follows: 5 mL of algal culture was transferred to 15 mL centrifuge tubes, with three biological replicates. The tubes were centrifuged at 4\u0026deg;C, 6,000 \u003cem\u003eg\u003c/em\u003e for 10 min, and the supernatant was discarded. The pellet was resuspended in 5 mL of 95% ethanol, thoroughly mixed, and incubated in the dark at 4\u0026deg;C for 24 h to facilitate pigment extraction. Following extraction, samples were vortexed and centrifuged at 8,000 \u003cem\u003eg\u003c/em\u003e for 15 min. The supernatant was collected, and absorbance was measured at 649 nm, 665 nm, and 470 nm using a quartz cuvette in a UV-Vis spectrophotometer. Chlorophyll a, chlorophyll b, and carotenoid contents were calculated using the following equations: Chlorophyll a (Chl a)\u0026thinsp;=\u0026thinsp;13.95 \u0026times; A665\u0026ndash;6.88 \u0026times; A649, Chlorophyll b (Chl b)\u0026thinsp;=\u0026thinsp;24.96 \u0026times; A649\u0026ndash;7.32 \u0026times; A665, Carotenoids (Car) = (1000 \u0026times; A470\u0026ndash;2.05 \u0026times; Chlorophyll a -114.8 \u0026times; Chlorophyll b) / 245. The units for Chl a, Chl b, and Car in the formulas are all mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e2.5 Determination of\u003c/em\u003e Euglena \u003cem\u003ePolysaccharide Content\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eApproximately 10 mg of freeze-dried algal powder was weighed and transferred into a capped 15 mL centrifuge tube. Subsequently, 4 mL of ethanol was added, and the mixture was vortexed for 10 s, repeated twice. Following a 1 h incubation at room temperature, the sample was centrifuged at 5000 \u003cem\u003eg\u003c/em\u003e for 5 min, and the supernatant was discarded to obtain the crude Euglena polysaccharidae fraction. Protein removal was achieved by adding 1 mL of 1% SDS solution. The mixture was then transferred to a 1.5 mL EP tube and heated in an 85\u0026deg;C water bath for 30 min. After centrifugation at 8000 r/min for 3 min, the supernatant was removed, and 0.1% SDS solution was added. The sample was incubated at room temperature for 30 min, followed by centrifugation at 8000 r/min for 5 min, and the supernatant was discarded. This process was repeated until complete protein removal was achieved. The resulting pellet, designated Pm, was dried in a 50\u0026deg;C drying oven for 24 h. Subsequently, 1 mL of 0.5 mol/L NaOH was added. Polysaccharide quantification was performed using the phenol-sulfuric acid method, with glucose as the standard, and a polysaccharide standard curve was generated. For Pm quantification, 50 \u0026micro;L of the Pm solution was diluted to 1.0 mL with distilled water, and the Pm content was determined using the standard curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Determination of Antioxidant Enzyme Activity and MDA content\u003c/h2\u003e \u003cp\u003eThe algal suspension (100 mL) underwent centrifugation at 8000 g for 10 minutes at 4\u0026deg;C. The resulting pellet was resuspended in 5 mL of phosphate-buffered saline (PBS) solution within a 15 mL centrifuge tube. This tube was maintained on ice and subsequently homogenized via a tissue grinder. The homogenate was then centrifuged at 10000 \u003cem\u003eg\u003c/em\u003e for 20 minutes in a high-speed refrigerated centrifuge, and the supernatant was collected. Soluble protein content was quantified using the Coomassie Brilliant Blue method (Beyotime Biotechnology Kit), with a standard curve employed for accurate determination. Antioxidant enzyme activity was evaluated using Superoxide Dismutase (SOD) and Peroxidase (POD) assay kits (Solarbio), and measurements were obtained utilizing a microplate reader (Varioskan LUX).\u003c/p\u003e \u003cp\u003eCellular malondialdehyde (MDA) content was quantified using a micro MDA assay kit obtained from Solebaer Biotechnology Co., LTD (China). Enzymatic analysis was employed to determine MDA levels within the cells. The extraction protocol for MDA from algal cells was adapted from established methods for antioxidant enzyme extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 RNA Preparation and Transcriptome Data Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted from 100 mL of \u003cem\u003eEuglena sanguinea\u003c/em\u003e cultures, cultivated under 1500 lux and 6000 lux illumination, with three biological replicates, using the Qubit RNA kit. RNA purity was assessed using a Nano Photometer\u0026reg; spectrophotometer (IMPLEN, CA, USA). RNA concentration was quantified using the Qubit\u0026reg; RNA Assay Kit in a Qubit\u0026reg;2.0 Fluorometer (Life Technologies, CA, USA). cDNA libraries were constructed using the VAHTSTM mRNA-seq V2 Library Prep Kit for Illumina\u0026reg;. De novo assembly was performed using Trinity (v. 2.0.6) with default parameters. Given the absence of a complete genome sequence for any species within the \u003cem\u003eE. sanguinea\u003c/em\u003e genus and the low homology observed between our transcriptome data and that of \u003cem\u003eEuglena gracilis\u003c/em\u003e, a nonparametric analysis was employed.\u003c/p\u003e \u003cp\u003eDifferentially expressed genes (DEGs) were identified based on a mean fold change threshold of \u0026ge;\u0026thinsp;2 or \u0026le;\u0026thinsp;0.5, and a \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05. Unigenes were annotated using nine public databases: NCBI nucleotide sequences (NT), NCBI non-redundant protein sequences (NR), Conserved Domain Database (CDD), Clusters of Orthologous Groups of proteins (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), Pfam, SwissProt, and TrEMBL. GO functional enrichment and KEGG pathway analyses were conducted using Goatools and KOBAS (Xie et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Data Analysis\u003c/h2\u003e \u003cp\u003eTriplicate biological replicates were employed, and the data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Prior to analysis, homogeneity of variance and normal distribution were assessed. Data analysis was performed using the Data Processing System (DPS Version 7.05). Multiple comparisons were conducted using one-way ANOVA followed by Tukey's test. Statistical significance was defined as *\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and **\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01. Data visualization was performed using GraphPad Prism (version 8.01, San Diego, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Strain Collection and Identification of Euglena sanguinea\u003c/h2\u003e \u003cp\u003eAlgal strains were isolated from water samples collected from the Shanghai Botanical Garden and cultured in Ploy A medium. Microscopic analysis of the isolates revealed cells with a green pigmentation, measuring 30\u0026ndash;170 \u0026micro;m in length and 17\u0026ndash;44 \u0026micro;m in diameter. The cells exhibited a flexible cell wall, high motility, and significant morphological plasticity. The nucleus was observed to be either medial or posterior in position, and a red eyespot was present. Based on these morphological features, the isolated algal strain was provisionally identified as \u003cem\u003eEuglena sanguinea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eMolecular identification was conducted via 16S rDNA and 23S rDNA sequence analysis. PCR amplification generated products of 1349 bp and 1538 bp, respectively. The obtained sequences (S13B6) were subjected to a homology search against sequences within the Euglena genus, and phylogenetic trees were constructed using Bayesian and maximum likelihood methodologies. The resulting topologies of the 16S rDNA and 23S rDNA trees exhibited congruence. Consequently, the support values from the maximum likelihood tree were annotated onto the Bayesian tree, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The integrated morphological and phylogenetic analyses confirmed the identification of the isolated algal strains as \u003cem\u003eE. sanguinea.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Influence of Varying Culture Conditions on Carotenoid Content\u003c/h2\u003e \u003cp\u003eCarotenoids, prevalent across diverse taxa including plants, fungi, bacteria, and algae, function as accessory pigments within photosynthetic pathways, primarily absorbing visible light, particularly in the blue-violet spectrum. This absorbed photonic energy is then channeled to the chloroplasts, thereby facilitating light harvesting and photoprotection in photosynthetic organisms (Takaichi et al., 2011). To elucidate the factors governing carotenoid biosynthesis in \u003cem\u003eE. sanguinea\u003c/em\u003e, the microalgae were initially cultivated continuously for four days to achieve exponential growth. Subsequently, cultures were maintained under a range of light intensities (1500 lux, 4000 lux, 6000 lux, 8000 lux, and 10000 lux) for 4\u0026ndash;5 days, with daily quantification of carotenoid content. Results indicated that varying light intensities significantly impacted carotenoid accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). At 6000 lux, carotenoid accumulation exhibited the most substantial increase after five days (30.5%) relative to the 1500 lux control. Conversely, at 10000 lux, carotenoid accumulation was significantly suppressed. Consequently, a light intensity of 6000 lux was selected for subsequent experiments involving other factor inductions in \u003cem\u003eE. sanguinea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eFollowing this, under 6000 lux illumination, \u003cem\u003eE. sanguinea was\u003c/em\u003e cultivated under varying conditions, including different temperatures (20\u0026deg;C, 25\u0026deg;C, 30\u0026deg;C, 35\u0026deg;C), potassium iodide concentrations (0.01 mM, 0.1 mM, 1.0 mM, 5.0 mM, 10.0 mM), and sucrose concentrations (0.1 mM, 1.0 mM, 10.0 mM, 50.0 mM). The results demonstrated that temperature also influenced carotenoid accumulation. The most significant carotenoid accumulation was observed at 20\u0026deg;C, with a 4% increase compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, the addition of varying concentrations of potassium iodide and sucrose to the culture medium also affected carotenoid accumulation. Specifically, carotenoid accumulation was most pronounced when the culture medium contained 1 mM potassium iodide or 50 mM sucrose, with increases of 9% and 15% compared to the control group, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Based on these findings, we conclude that light intensity is a significant factor influencing carotenoid accumulation in \u003cem\u003eE. sanguinea\u003c/em\u003e, with optimal accumulation observed at 6000 lux. Furthermore, other environmental factors, including temperature, potassium iodide concentration, and sucrose concentration, also modulate carotenoid accumulation in this microalga.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Effects of Light intensity on Photosynthetic Pigments and Paramylon\u003c/h2\u003e \u003cp\u003eTo investigate the impact of varying light intensities on photosynthetic pigment synthesis in \u003cem\u003eE. sanguinea\u003c/em\u003e, a five-day morphological observation was conducted under different light intensities. Although no significant alteration in algal morphological size was observed across the light treatments, the carotenoid content in the algal cells exhibited a marked increase with escalating light intensity within the range of 1500\u0026ndash;6000 lux (as indicated by the orange-red cluster in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Furthermore, the rate of carotenoid accumulation decelerated when light intensity exceeded 8000 lux. Consequently, the data suggest that the most rapid carotenoid accumulation, culminating in a peak value on the fifth day, occurred under a light intensity of 6000 lux.\u003c/p\u003e \u003cp\u003eSubsequently, we assessed the photosynthetic pigment variations in \u003cem\u003eE. sanguinea\u003c/em\u003e on the fifth day under light intensities of 1500 lux and 6000 lux, respectively. The pigments quantified included chlorophyll a, chlorophyll b, and total carotenoids. The results indicated that, relative to the control group (1500 lux), high-light stress (6000 lux) induced a reduction in chlorophyll content, with a significant 20.5% decrease in chlorophyll a (Chla) content (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, the content of carotenoids, acting as accessory photosynthetic pigments, significantly increased by 30.6% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) under high-light intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Moreover, continuous high-light stress for five days significantly promoted polysaccharide accumulation in \u003cem\u003eE. sanguinea\u003c/em\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These findings suggest that high light intensities can enhance the accumulation of polysaccharides and carotenoids, while simultaneously suppressing chlorophyll synthesis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.4 High-intensity light elicits oxidative stress response in E. sanguinea\u003c/h2\u003e \u003cp\u003eTo investigate the induction of oxidative stress responses in \u003cem\u003eE. sanguinea\u003c/em\u003e by photo-stress, soluble protein standard curves were generated under continuous four-day photo-stress conditions. Samples were normalized to the measured soluble protein content of the algal cells, and the levels of antioxidant stress response enzymes, including SOD, CAT, POD, and malondialdehyde (MDA), were quantified. The results demonstrated that the antioxidant enzyme activities of SOD and POD in \u003cem\u003eE. sanguinea\u003c/em\u003e cultivated under 6000 lux light intensity were significantly downregulated compared to the control group (1500 lux) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with respective decreases of 48.2% and 52.9% (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Conversely, CAT enzyme activity in the algae exhibited a significant increase of 63.7% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) under high light intensity stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, the MDA content in Euglena under 6000 lux stress was also significantly elevated by 56.2% compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings suggest that high light intensity modulates the oxidative stress responses in \u003cem\u003eE. sanguinea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Light intensity-dependent modulation of gene expression profiles in E. sanguinea\u003c/h2\u003e \u003cp\u003eTo investigate the impact of light intensity on gene expression, we conducted transcriptomic profiling of \u003cem\u003eE. sanguinea\u003c/em\u003e under different light intensities (1500 lux and 6000 lux). Sequencing yielded a total of 32,153 identified genes. The number of expressed genes, total mapped reads, and unique matches for each sample are detailed in Table S2. High-intensity light stress induced differential expression in 111 genes (DEGs), comprising 44 up-regulated and 67 down-regulated genes (absolute log2 fold-change\u0026thinsp;\u0026gt;\u0026thinsp;1, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The evolutionary genealogy of genes: non-supervised orthologous groups (EggNOG) classification of these DEGs revealed enrichment in the cytoskeleton, energy production and conversion, and cell cycle control, cell division, and chromosome partitioning categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eTo further elucidate the biological responses elicited by high-intensity light stress, we conducted GO (gene ontology) annotation (Figure S2) and GO enrichment analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) of differentially expressed genes (DEGs), categorized by their functional roles. The predominant GO terms encompassed chloroplast thylakoid membrane, plastid thylakoid membrane, photosynthetic membrane, and organelle outer membrane. Furthermore, based on cellular component (CC) analysis, the DEGs were primarily enriched in plastid, chloroplast, and organelle bounding membrane. Regarding biological process (BP), the DEGs were significantly enriched in photosynthesis. Molecular function (MF) analysis revealed enrichment of DEGs in electron transfer activity, rRNA binding, copper ion binding, and ATP-dependent activity. Additionally, KEGG pathway analysis was performed on these DEGs. The two most highly enriched KEGG pathways were photosynthesis and Ribosome (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Notably, upregulated DEGs included three genes encoding photosystem II protein D1 (\u003cem\u003eEuglena gracilis\u003c/em\u003e), photosystem II protein K (\u003cem\u003eEutreptiella eupharyngea\u003c/em\u003e), and cytochrome b6/f complex subunit V (\u003cem\u003eEuglena deses\u003c/em\u003e). Consequently, we hypothesize that light stress modulates photosynthesis in Euglena species by modulating the expression of these DEGs, thereby regulating photosynthetic membrane function and, consequently, influencing the in vivo accumulation of photosynthetic pigments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe complex chloroplast morphology of \u003cem\u003eEuglena sanguinea\u003c/em\u003e presents challenges for taxonomic identification (Laza-Martinez et al., 2019). Over the course of more than two centuries of research, twelve novel algal groups were initially considered posited as analogous to \u003cem\u003eE. sanguinea\u003c/em\u003e. However, identification based solely on morphological observations and physiological-biochemical indicators, using traditional taxonomic methods, proved difficult. Currently, descriptions and analyses of \u003cem\u003eE. sanguinea\u003c/em\u003e and related species integrate morphological and molecular biological approaches, which has reduced the number of similar groups to four (\u003cem\u003eE. sanguinea\u003c/em\u003e, \u003cem\u003eE. sociabilis\u003c/em\u003e, \u003cem\u003eE. splendensand\u003c/em\u003e and \u003cem\u003eE. laciniata\u003c/em\u003e). Furthermore, new phenotypic characteristics and updated diagnostic descriptions have been established, including the presence of fusiform muciferous sacs, numerous chloroplasts, different types of paramylon bodies, and the presence of paramylon granules near the cell's stigma (Karnkowska-Ishikawa et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Nevertheless, taxonomic verification of this species remains challenging for researchers lacking extensive experience. Therefore, accurate identification of \u003cem\u003eE. sanguinea\u003c/em\u003e based on molecular biology is essential. The prevailing methodology employed currently employs its nSSU rDNA as a molecular barcode (Kulczycka et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This study employed a dual identification approach, combining morphological analysis with homology data comparison of 16S rDNA and 23S rDNA base sequences, confirming the algal strain collected from the Shanghai Botanical Garden's river as \u003cem\u003eE. sanguinea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe growth and the accumulation of bioactive substances in microalgae are significantly influenced by environmental factors, including temperature, salinity, light, and pH (Ogbonna et al., 2000). Research indicates that light conditions play a crucial role in the growth and development of microalgae, with light intensity, photoperiod, and light quality exerting the most significant effects (Takada et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In this study, we initially investigated the impact of four abiotic factors (light, temperature, potassium iodide, and glucose) on the growth and physiological biochemistry of \u003cem\u003eE. sanguinea\u003c/em\u003e. We observed that the cell density of \u003cem\u003eE. sanguinea\u003c/em\u003e was modulated to varying degrees by these abiotic factors. Notably, light intensity and temperature exhibited the most pronounced effects on the microalgae (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Previous studies have demonstrated that under stress conditions such as high light, high salinity, or nutrient limitation, microalgae can produce excess lipids or carotenoids (Lamers et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Carotenoids, acting as photoprotective pigments, absorb excess energy, thereby preventing damage to cellular membranes and providing photoprotection (Sun et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). We observed a decline in chlorophyll a content within \u003cem\u003eE. sanguinea\u003c/em\u003e concurrent with escalating light intensity. Furthermore, carotenoid accumulation exhibited a marked increase, particularly under cultivation at 6000 lux. However, at a light intensity of 10000 lux, carotenoid accumulation was significantly inhibited. This observation aligns with findings in \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e, where high-intensity light induces the accumulation of astaxanthin and canthaxanthin within cytoplasmic vesicles, accompanied by a color change from green to red (Lemoine et al., 2010; Cezare-Gomes et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, in both higher plants and algae, the accumulation of triacylglycerols is frequently coupled with the synthesis of carotenoids. Under high light conditions, carotenoids are transported to lipid bodies, forming abundant carotenoid-containing lipid bodies outside the chloroplasts, thus preventing the overstimulation of chloroplasts by intense light and ensuring efficient photosynthesis (Ferrell and Sariskyreed, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Lamers et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) investigated the changes in β-carotene and fatty acid content in \u003cem\u003eDunaliella salina\u003c/em\u003e under photoinduction, observing that the accumulation of carotenoids was accompanied by a decrease in fatty acid unsaturation. The plants generate a large amount of reactive oxygen species (ROS) under low temperature, high salinity, or nitrogen deficiency conditions. ROS, acting as signaling molecules, can initiate the organism's defense system by promoting the synthesis of cellular carotenoids, thereby enabling the organism to withstand oxidative stress damage (Alscher et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1997\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWe further observed that exposure to a light intensity of 6000 lux resulted in a reduction in the activity of the antioxidant enzymes SOD and POD within the algae, while CAT activity and MDA content increased. These antioxidant enzymes play a crucial role in the organism's antioxidant defense system by working synergistically to scavenge free radicals. Specifically, POD and CAT are responsible for the decomposition of hydrogen peroxide in the cytoplasm and peroxisomes, respectively, thereby preventing oxidative damage caused by its accumulation (Abo-Shady et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Li et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) found that under high-light stress, the cellular morphology of \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e transitioned from green motile cells to brown cells, with the highest activities of SOD and CAT observed. However, due to prolonged photooxidative stress and high levels of peroxidation, the cells ultimately turned red, accompanied by the accumulation of astaxanthin. Furthermore, the polysaccharide content of \u003cem\u003eE. sanguinea\u003c/em\u003e significantly increased under 6000 lux illumination. Given the antioxidant properties of \u003cem\u003eEuglena\u003c/em\u003e polysaccharides, it is hypothesized that the rapid accumulation of carotenoids in \u003cem\u003eE. sanguinea\u003c/em\u003e under light stress may be regulated by ROS. These responses are essential for maintaining normal photosynthetic function in microalgae.\u003c/p\u003e \u003cp\u003eTo investigate the regulatory effects and mechanisms of light on \u003cem\u003eE. sanguinea\u003c/em\u003e growth, we conducted a transcriptomic analysis of cells cultivated under 1500 lux and 6000 lux illumination. A total of 111 differentially expressed genes (DEGs) were identified, comprising 44 upregulated and 67 downregulated genes. These DEGs were subjected to GO and KEGG enrichment analyses. The results indicate that light intensity primarily influences the photosynthetic processes and membrane systems of \u003cem\u003eE. sanguinea\u003c/em\u003e, specifically including the chloroplast thylakoid membrane, plastid thylakoid membrane, photosynthetic membrane, and organelle outer membrane.\u003c/p\u003e \u003cp\u003eThe chloroplast thylakoid membrane is considered the primary site for carotenoid synthesis. Photosynthetic proteins responsible for carrying out photosynthesis are located on the thylakoid membrane structure within the cell. Various environmental stresses render the thylakoid membrane particularly susceptible to damage, especially under high-illumination conditions (Collins et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The accumulation of diverse carotenoids in photosynthetic organisms is believed to serve multiple physiological functions, with the most critical being the facilitation of stress tolerance. A prime example is the photoprotective function executed by the xanthophyll cycle (Bassi and Caffarri, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Jahns et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The levels of carotenoids are influenced by numerous external factors; generally, photosynthetic organisms accumulate substantial amounts of carotenoids under adverse conditions. For instance, \u003cem\u003eE. sanguinea\u003c/em\u003e accumulates astaxanthin and β-carotene under nutrient deficiency, high-illumination, high-temperature, or high-salinity conditions (Tanno et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). He et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) demonstrated, through transcriptomic analysis, that high-illumination conditions in \u003cem\u003eHaematococcus pluvialis\u003c/em\u003e induced the promotion of carotenoid biosynthesis, with altered expression of genes associated with photosynthesis-antenna proteins and carbon fixation pathways. Therefore, we hypothesize that light stress primarily impacts carotenoid accumulation within the cell by modulating the expression of photosynthetic proteins on the thylakoid membrane of \u003cem\u003eE. sanguinea\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNotably, the up-regulated DEGs including three genes encoding photosystem II protein D1 (\u003cem\u003eEuglena agilis\u003c/em\u003e), photosystem II protein K (\u003cem\u003eEutreptiella eupharyngea\u003c/em\u003e) and cytochrome b6/f complex subunit V (\u003cem\u003eEuglena deses\u003c/em\u003e). Photosystem I (PSI) and Photosystem II (PSII) are light-harvesting protein complexes embedded within the thylakoid membranes. PSI, being a larger complex, is predominantly located in the stroma lamellae, whereas PSII complexes are primarily concentrated in the stacked grana thylakoid membrane regions (Umena et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). To mitigate irreversible photodamage to PSII under high light conditions, plants have evolved a sophisticated repair mechanism. This includes the disassembly of PSII, proteolytic degradation of the damaged D1 reaction center protein, and the co-translational insertion of newly synthesized D1 protein into partially disassembled PSII core complexes (Theis and Schroda, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The D1 protein, encoded by the chloroplast gene \u003cem\u003epsbA\u003c/em\u003e, is highly conserved across higher plants and forms the fundamental scaffold of the PSII reaction center, in conjunction with the D2 protein. The D1 protein not only provides binding sites for various cofactors but also maintains the conformational stability of the PSII reaction center and is particularly sensitive to various stress conditions (Zhang et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Consequently, high light intensity can induce the upregulation of D1 expression in \u003cem\u003eE. sanguinea\u003c/em\u003e, potentially representing a crucial mechanism for enhancing photosynthetic efficiency (Adir et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Aro et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Mulo et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe cytochrome b6f (Cytb6f) complex, a pivotal component and rate-limiting step in photosynthetic electron transport, serves as a critical redox-sensing hub (Kurisu et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Cramer et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Consequently, Cytb6f represents a promising target for genetic engineering aimed at enhancing photosynthetic efficiency (Tikhonov et al., 2014; Malone et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, high light intensity induces the overexpression of the cytochrome b6f complex in \u003cem\u003eE. sanguinea\u003c/em\u003e, further validating that \u003cem\u003eCytb6f\u003c/em\u003e expression serves as a critical indicator for assessing photosynthetic efficiency.\u003c/p\u003e \u003cp\u003eIn conclusion, this study provides preliminary insights into the physiological and biochemical responses of \u003cem\u003eE. sanguinea\u003c/em\u003e under various culture conditions and elucidates transcriptional changes in algal cells under light stress. These findings provide foundational data for investigating the mechanisms by which light regulates the growth and photosynthetic pigment synthesis in \u003cem\u003eE. sanguinea\u003c/em\u003e, with the potential to inform solutions for \u003cem\u003eE. sanguinea\u003c/em\u003e-related \"red tide\" events, large-scale cultivation, and research into carotenoid synthesis mechanisms.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eFundings for this work was provided by Natural Science Foundation of Shanghai (No. 23ZR1446800), and National Natural Science Foundation of China (NO. 32270383).\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eY. Z and W.T. P wrote the main manuscript text; S.R.W and X.D. J prepared figures 1; H.R.D, Q.M.Y and L.L.X prepared figures 2-5; Q.X.W presented conceptualization. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank Majorbio company for its transcriptome data analysis in this study. Fundings for this work was provided by Natural Science Foundation of Shanghai (No. 23ZR1446800), and National Natural Science Foundation of China (NO. 32270383).\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe raw sequence data are available from the National Center for Biotechnology Information Short Read Archive database (http://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA1254926. All other data are available in this text and Supplementary Files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAdir, N., Zer, H., Shochat, S., Ohad, I., 2003. Photoinhibition-a historical perspective. Photosynthesis research. 76, 343\u0026ndash;370. https://doi.org/10.1023/ A:1024969518145.\u003c/li\u003e\n \u003cli\u003eAro, E. 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The Plant cell. 12, 1769\u0026ndash;1782. https://doi.org/10.1105/tpc.12.9.1769.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Euglena sanguinea, Illumination intensity, Photosynthesis, Carotenoid, RNA-Seq","lastPublishedDoi":"10.21203/rs.3.rs-6542765/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6542765/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eEuglena sanguinea\u003c/em\u003e (Ehrenberg 1831) has recently gained prominence due to its bloom events and associated red tide phenomena. Despite this, research on \u003cem\u003eE. sanguinea\u003c/em\u003e remains relatively sparse. In this study, we isolated and purified algal strains collected from the water of the Shanghai Botanical Garden, identifying them as \u003cem\u003eE. sanguinea\u003c/em\u003e based on 16S and 23S rDNA sequence alignment. The cellular density and carotenoid content of \u003cem\u003eE. sanguinea\u003c/em\u003e were observed to vary under different abiotic culture conditions, including varying temperatures, light intensities, potassium iodide, and sucrose. Notably, the most significant rapid accumulation of carotenoids in \u003cem\u003eE. sanguinea\u003c/em\u003e was observed under continuous culture at a light intensity of 6000 lux. Furthermore, exposure to a strong intensity resulted in changes in the activity of the antioxidant enzymes and MDA content. Moreover, through de novo transcriptome sequencing and GO analysis of \u003cem\u003eE. sanguinea\u003c/em\u003e cultured under different light intensities, we identified a total of 111 differentially expressed genes (DEGs), comprising 44 upregulated and 67 downregulated genes. The up-regulated DEGs included three genes encoding photosystem II protein D1, photosystem II protein K, and cytochrome b6/f complex subunit V. Therefore, we hypothesize that light stress regulates photosynthesis in \u003cem\u003eE. sanguinea\u003c/em\u003e by altering the expression of these DEGs, thereby regulating the process of the photosynthetic electron transport and, consequently, the accumulation of photosynthetic pigments in vivo. These findings provide foundational reference data for investigating the photoprotective mechanisms in \u003cem\u003eE. sanguinea\u003c/em\u003e and offer a theoretical basis for exploring carotenoid synthesis pathways in plants.\u003c/p\u003e","manuscriptTitle":"High-intensity illumination induces the over-accumulation of carotenoids by modulates the expression of photosynthetic genes in Euglena sanguinea","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-19 17:59:05","doi":"10.21203/rs.3.rs-6542765/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-18T16:12:17+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-15T21:06:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-09T06:26:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191423631431775557190993297651204378676","date":"2025-06-01T09:18:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332441595618630982999908131579133699880","date":"2025-06-01T07:26:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86542089029259836455305057550430967498","date":"2025-05-31T15:06:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-29T14:34:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-05-27T01:55:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"45655972801482420434577733349005383328","date":"2025-05-20T13:31:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130418396706011759180655471186236204986","date":"2025-05-16T22:33:40+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-15T13:18:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-15T05:46:27+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-13T05:20:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photosynthesis Research","date":"2025-04-28T00:36:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"e74bbc0a-0776-4bc5-88f2-df2babea35f2","owner":[],"postedDate":"May 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T15:57:56+00:00","versionOfRecord":{"articleIdentity":"rs-6542765","link":"https://doi.org/10.1007/s11120-025-01168-z","journal":{"identity":"photosynthesis-research","isVorOnly":false,"title":"Photosynthesis Research"},"publishedOn":"2025-09-08 15:56:53","publishedOnDateReadable":"September 8th, 2025"},"versionCreatedAt":"2025-05-19 17:59:05","video":"","vorDoi":"10.1007/s11120-025-01168-z","vorDoiUrl":"https://doi.org/10.1007/s11120-025-01168-z","workflowStages":[]},"version":"v1","identity":"rs-6542765","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6542765","identity":"rs-6542765","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}