Physiological and Transcriptomic Responses of Sargassum hemiphyllum to Ocean Acidification and Nitrogen Enrichment

Physiological and Transcriptomic Responses of Sargassum hemiphyllum to Ocean Acidification and Nitrogen Enrichment | 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 Physiological and Transcriptomic Responses of Sargassum hemiphyllum to Ocean Acidification and Nitrogen Enrichment Jing Chen, Xiao Ke, Jinhui Wu, Yurong Wang, Honghao Liang, Jie Zheng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6499830/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 13 Nov, 2025 Read the published version in BMC Genomics → Version 1 posted 10 You are reading this latest preprint version Abstract Sargassum hemiphyllum is a major brown macroalga and has important ecological and economic significance. Ocean acidification and nitrogen enrichment are serious threats to marine ecosystems primarily by altering the physiology of organisms. However, the response of S. hemiphyllum to the combined effects of ocean acidification and elevated nitrogen levels remains unclear. This study conducted a 7-day dual-factor experiment to investigate the physiological and transcriptional responses of S. hemiphyllum under two CO2 levels (400 μatm and 1000 μatm) and two NO3⁻ levels (50 μmol/L and 300 μmol/L). The results showed that high CO2 and NO3- concentrations promoted the synthesis of photosynthetic pigments including qN and NPQ. Physiological results showed that high CO2 and the combined high NO3- and CO2 treatments enhanced growth rate and NO3- uptake rate, but NR activity was significantly decreased. Transcriptome analysis identified differentially expressed genes involved in oxidative phosphorylation, carbon metabolism, the TCA cycle, and nitrogen metabolic pathways. Notably, genes related to oxidative phosphorylation and TCA cycle were significantly up-regulated under high NO3- and dual-factor treatments, suggesting that carbohydrate metabolism and energy metabolism of S. hemiphyllum were significantly enhanced. The qRT-PCR analysis revealed that the expression levels of key genes involved in carbon fixation and nitrogen metabolism, including PFK, PRK, GAPDH, Rubisco, NR, and MDH, were significantly downregulated. These findings elucidate the molecular mechanisms by which S. hemiphyllum adapts to ocean acidification and nitrogen enrichment, offering valuable insights for understanding its capacity to withstand changing marine environments. Sargassum hemiphyllum acidification nitrate nitrogen physiology transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Sargassum hemiphyllum is a major brown macroalga widely distributed along the coasts of China, Korea, and Japan [1]. Its high diversity and biomass play a critical role in ocean ecosystems by forming natural seaweed beds that serve as spawning, nursery, and feeding grounds for many marine animals [2]. It is also an economically important seaweed, serving as raw material for phycocolloids and as feed for highly valued aquaculture species such as holothurians, sea urchins, abalones and winkles [3]. Therefore, S. hemiphyllum has significant economic and ecological value. However, its natural biomass has declined annually due to environmental pollution. The ocean absorbs excess carbon dioxide (CO 2 ) from the atmosphere, causing ocean acidification [4]. Various anthropogenic activities, such as excessive use of chemical fertilizers and industrial pollution discharges, causing an increase in nitrogen levels [5]. Ocean acidification and elevated nitrogen content in seawater are major threats to marine ecosystems by altering organism physiology and disrupting ecological interactions [6, 7]. Hence, this topic has attracted the interest of many researchers. How macroalgae respond to ocean acidification has drawn increasing attention in recent years. Carbon dioxide concentrating mechanisms (CCMs) enable macroalgae to absorb dissolved inorganic carbon (DIC) when CO 2 : O 2 ratio is high or CO 2 concentrations are insufficient to maximize the rate of photosynthesis [8]. Macroalgae lacking CCMs, or with CCMs of low DIC affinity, may benefit from ocean acidification as CO 2 limitations are alleviated [7, 9, 10]. When seawater CO 2 concentrations exceed those produced internally by CCMs, diffusive CO 2 can enter Rubisco [11-13]. This adaptation may downregulate CCMs and save energy, thereby enhancing growth rates and providing competitive advantages under ocean acidification [10]. However, The physiological mechanisms of macroalgae in response to ocean acidification still require further research [14]. Another major concern is how the concentration of inorganic nitrogen (N) affects the growth performance of macroalgae. Studies have shown that elevated external nitrogen (N) concentrations can promote the growth of seaweeds [15]. When algae absorb nitrate, it is first reduced to nitrite and subsequently to ammonium through the action of the enzymes nitrate reductase and nitrite reductase. The ammonium is then used to make amino acids through the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway, which is common in most algal species [16]. Previous studies indicated that the transcription and activity of enzymes involved in nitrate metabolism are regulated by various external factors, for example, cellular fluxes of carbon and nitrogen substrates [17]. Nitrogen is one of the essential nutrients that regulate biomass production and photosynthetic efficiency in marine ecosystems. S. hemiphyllum plays a vital role in carbon sequestration and nitrogen absorption, functioning as a biological filter to mitigate enrichment and help maintain the ecological balance of marine ecosystems [7, 18, 19]. However, relatively few studies reported the relationship between intracellular carbon and nitrogen metabolism in macroalgae . To elucidate the combined impacts of nitrogenous nutrients and ocean acidification on the growth of Sargassum , it is essential to examine how carbon and nitrogen influence the algal physiological mechanisms. The aim of this study is to investigate how the altered CO 2 concentrations and elevated nitrogen levels affect the physiology of Sargassum in a lab setting. In this study, we set two level of carbon (400 μatm and 1000 μatm) and two nitrogen supply levels (50 μmol L −1 and 300 μmol L −1 ). The thalli were used in the study to examine the physiological, biochemical and transcriptomic characteristics of carbon and nitrogen. Our results showed that the combined treatment of high CO 2 and nitrate concentrations enhanced the growth and photosynthetic efficiency of S. hemiphyllum . We hypothesize that S. hemiphyllum has a significant advantage in coping with ocean acidification and high nitrogen environments. The findings present that the carbon and nitrogen within S. hemiphyllum were reallocated under acidification and elevated nitrogen conditions, which not only helps to comprehend the regulatory mechanisms of seaweed growth, but also contributes to optimizing cultivation to promote rapid growth of the algae. Materials and methods Algal strains and culture conditions Sargassum hemiphyllum var. chinense was collected from Nanao Island, Shantou University, Guangdong Province, China (117.11°E, 23.48°N). The thalli were cultured in aseptic seawater and illuminated with 80 μmol photons m -2 s -1 under a 12 h:12 h light/dark photoperiod. Experimental cultures were set up in four groups: control (NO₃⁻-N: 50 μmol/L, CO₂: 400 μatm), high nitrate (NO₃⁻-N: 300 μmol/L, CO₂: 400 μatm), high CO₂ (NO₃⁻-N: 50 μmol/L, CO₂: 1000 μatm), and high carbon-nitrogen (NO₃⁻-N: 300 μmol/L, CO₂: 1000 μatm) (Supplementary Fig. S1). Each experimental group consisted of three biological replicates, and the seawater was replaced every two days to ensure a continuous nutrient supply. After seven days of culture, the algae were collected and stored at -80℃ for subsequent RNA extraction and other measurements. Chlorophyll fluorescence Chlorophyll fluorescence parameters were determined using the chlorophyll fluorescence imaging system (WALZ Image-PAM, Zealquest Scientific Technology China). The maximum quantum yield (Fv/Fm) of the photosystem Ⅱ (PSⅡ) in S. hemiphyllum (dark-adapted for 15 min) was measured using a saturation pulse (5000 μmol photons m -2 s -1 , 0.8 s). The relative electron transport rate (rETR) is the light-saturated photosynthetic electron transport rate, α is the electron transport efficiency, and E represents the photosynthetically active radiation [20]. The maximum quantum yield (Fv/Fm) was measured via a pulse amplitude-modulated (PAM) fluorometer (Imaging-PAM, Walz, Germany) [21]. The calculations of Y(II), qP, and NPQ [22] were performed as follows: Relative growth rates, the NO 3 - uptake rate, nitrate reductase (NR) activity, total nitrogen and carbon contents Relative growth rates (RGR) were represented as the percentage increase in daily fresh weight (FW) biomass of S. hemiphyllum , and were calculated using the following formula [23]: RGR (% d -1 ) = [(lnW t - lnW 0 ) / t] × 100 The NO 3 - uptake rate was determined by measuring the decrease in NO 3 - concentration in the cultured seawater over a certain time. The calculation formula is as follows: NO₃⁻ uptake rate The Nitrate reductase (NR) activity was measured using NR activity assay kit. NR catalyzes the reduction of nitrate to nitrite in the presence of NADH, measuring its absorption peak at 340 nm. Approximately 0.1 g of fresh algal was ground in 1 mL of extraction solution, then centrifuged at 4°C 4000 g for 10 min. The absorption value of the supernatant was determined by the spectrophotometry at a wavelength of 340 nm. Approximately 0.2 g of S. hemiphyllum was dried at 60℃ for 48 h, then finely ground into powder using a high-flux tissue grinder (Scientz-48, Ningbo Scientz Biotechnology, China). About 2 mg of the powder was used to measure cellular carbon (C) and nitrogen (N) content using an Elementar Vario Cube (Elementar, Germany). Antioxidant characteristics Briefly, 0.1 g thallus was ground into powder and mixed with 1 mL pre-cooled extraction buffer. We used the CAT assay, MDA and SOD kits (Nanjing Jian cheng Bioengineering Institute, Nanjing, China) for each enzyme assay. The CAT assay was used in the study according to the method of Góth [24]. The SOD assay was based on SOD ability to oxidise hydroxylamine by the xanthine-xanthine oxidase system [25]. One unit (U) of SOD activity was calculated as the inhibition by 50% of the oxidation of hydroxylamine without an enzyme source. In addition, the malondialdehyde (MDA) assay was previous method [26]. RNA extraction and cDNA library construction Total RNA was extracted from S. hemiphyllum using the RNAprep Pure Plant Kit (TIANGEN, China). The quality of RNA was assessed using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). The cDNA library was constructed with the mRNA library preparation kit (MGIEasy TM , MGI, Shenzhen, China), with mRNA enriched for the poly(A) tail using magnetic beads conjugated with Oligo(dT). The cDNA library was transformed into double-stranded cDNA using random N6 primers, followed by the addition of A-Tailing Mix and RNA Index Adapters for ligation. Finally, the double-stranded cDNA library was amplified by polymerase chain reaction (PCR) and sequenced using the BGISEQ-500 platform at the Wuhan Institute of Genomics. Transcriptome analysis Gene expression levels were represented by FPKM and calculated using RSEM software [27]. FPKM values for each sample were log-transformed as 𝑥 = log 2 (FPKM + 1). The normalized score (Z) was calculated using the formula: Z = (𝑥 - μ)/σ, μ: the mean of all sample FPKM values, σ: the standard deviation of all sample FPKM values, 𝑥: the log-transformed FPKM value. BLAST software was used to map genes to various databases (NT, NR, KOG, KEGG) for functional annotation. DEseq2 was detected different expression genes (DEGs). DEGs with |log 2 (fold change)| ≥ 1.5 and an adjusted p value ≤ 0.05 were considered significantly differentially expressed [28]. GO and KEGG enrichment analyses were conducted to study the function and metabolic pathway significantly enriched by DEGs, and the significant enrichment level was Q value ≤ 0.05 [29]. RNA extraction and RT-qPCR Approximately 0.15 g of S. hemiphyllum was collected and was homogenized by using liquid nitrogen and purified via RNAprep Pure Plant Kit with RNase-Free DNAase I to isolate total cellular RNA. The concentration and purity of the RNA were assessed using Nanodrop microspectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). After determination the RNA quality using agarose gel electrophoresis, complementary cDNA synthesis was carried out using Rever Tra Ace q PCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative real-time PCR (qRT-PCR) was performed with an Applied Biosystems 7300 Real Time PCR System and Roche Light Cycler 96 system, using TB Green® Premix Ex Taq™ II to measure the relative transcript levels of the genes associated with metabolic changes. The ribosomal protein L35 (Unigene10602_All) was used as the endogenous control. Reference Gene Quantitative Primer were RL35-RT-F and RL35-RT-R. Primers used in this study were designed on primer 6.0 and are shown in Supplementary Table S1. Cycling conditions were 10 min at 95°C with 40 cycles for the melting (30 s at 95°C), annealing (30 s at 60°C), and extension (30 s at 72°C). The genes of interest and the primers are listed in Supplementary Table S1. The data were quantitatively analyzed by 2 − △△ CT . Statistical analysis All data were expressed as the mean ± standard deviation (SD) (n = 3). Statistical analysis was performed using SPSS 25. Individual and interactive effects of nitrate and CO 2 were analyzed using two-way ANOVA with a confidential level of 95%. Significant differences between treatment groups were tested using independent t -tests p < 0.05 (Two-tailed distribution; Two-sample equal variance). Different letters on the top of the bars mark the statistically significant groups after t -tests. Results Chlorophyll fluorescence parameters The chlorophyll fluorescence parameters of S. hemiphyllum under different culture conditions are shown in the Table 1. Fv/Fm showed no significant change under different treatments. Photochemical quenching (qP) reflects the level of photosynthetic activity in algae. The results showed that high concentrations of CO 2 significantly increased qP of algae to 0.58 ± 0.05 a and 0.58 ± 0.005 a under +CO 2 and +CN conditions, respectively ( p < 0.05). qN and NPQ exhibited the same trend, with a significant increase under different treatment conditions (+N, +CO 2 , and +CN) compared to the control group. The study found that CO 2 had a main effect on the relative electron transport rate (rETR) of algae (Table 1). High concentrations of CO₂ significantly increased the relative electron transport rate (rETR) of the algae ( p < 0.05), with +CO₂ and +CN showing values of 19.55 ± 0.65 a and 21.05 ± 0.26 a , respectively. The value of α ranged from 0.18 to 0.21, with the highest α value observed in +CN group ( p < 0.05). CO 2 had a significant main effect on the light saturation point (Ik) of the algae (Table 1). High CO 2 concentrations significantly increased Ik, with values of 56.26 ± 2.46 a and 54.41 ± 1.23 a under +CO 2 and +CN conditions, respectively. Fluorescence parameters Control +N +CO 2 +CN Fv/Fm 0.7 0.01 a 0.7135 0.0008 a 0.7052 0.0005 a 0.6983 0.01 a qP 0.46 0.006 b 0.51 0.02 b 0.05 a 0.005 a Y(Ⅱ) 0.29 0.03 b 0.31 0.01 b 0.31 0.02 b 0.37 0.01 a qN 0.29 0.02 b 0.49 0.06 a 0.04 a 0.14 0.03 a NPQ 0.07 0.004 b 0.15 0.03 a 0.15 0.02 a 0.14 0.03 a rETR 15.78 1.54 b 16.46 0.61 b 0.65 a 21.05 0.26 a α 0.18 0.003 b 0.19 0.01 ab 0.19 0.01 b 0.21 0.004 a Ik 47.51 4.03 b 3.33 b 56.26 2.46 a 1.23 a Table 1. Effects of different culture conditions on chlorophyll-induced fluorescence parameters in S. hemiphyllum. Control group: 50 μmol/L NO 3 - -N，400 μatm CO 2 ; +N group: 300 μmol/L NO 3 - -N，400 μatm CO 2 ; +CO 2 group: 50 μmol/L NO 3 - -N, 1000 μatm CO 2 ; +CN group: 300 μmol/L NO 3 - -N, 1000 μatm CO 2 , significant differences ( p < 0.05, n ≥ 3). Physiological analysis of S. hemiphyllum The relative growth rate (RGR), NO 3 - uptake rate, nitrate reductase activity (NR), total carbon (C), total nitrogen (N) and C:N ratio were measured in different carbon and nitrogen treatments (Control, +N, +CO 2 , +CN), as shown in Fig. 1. The RGR of S. hemiphyllum were significantly promoted in +CO 2 and +CN groups ( p < 0.05, Fig. 1a). The highest RGR was found in +CN group (2.96 % g -1 FW d -1 ). The NO 3 - uptake rate was significantly increased in the +N, +CO 2 and +CN groups (Fig. 1b). However, the activity of NR was statistically significantly lower in the +N, +CO₂, and +CN groups compared to the control group (Fig. 1c). There were no significant differences in total carbon and nitrogen content between the different treatment groups (Fig. 1d-1e). In addition, the C:N ratio in the +CN group was the lowest about 22.49 (Fig. 1f). To understand the mechanisms responsible for the maintenance of the cellular state in the different environments, we investigated the enzymatic components of the antioxidant machinery (Supplementary Fig. S2). The CAT activity of S. hemiphyllum grown at +N and +CO 2 environments were higher than that of the algae grown at Control and +CN environments. The MDA activity of S. hemiphyllum was the lowest in +N group. No significant differences were observed in the SOD activities in different environments. Identification and validation of differentially expressed genes The reliability and consistency of the data were demonstrated by a strong correlation coefficient (R 2 > 0.909) of gene expression between biological replicates (Supplementary Fig. S3). Principal Component Analysis (PCA) was performed to compare transcriptome differences between groups (Supplementary Fig. S4). Comparative transcriptome analysis was performed to screen DEGs in three groups: Control vs +C, Control vs +N, and Control vs +CN. A total of 842 unigenes were differentially expressed in the Control vs +C group, with 364 upregulated and 478 downregulated. In the Control vs +N group, 1215 unigenes were differentially expressed, including 652 upregulated and 563 downregulated. Moreover, there were 1776 DEGs, which 795 were upregulated and 981 were downregulated in Control vs +CN group (Supplementary Fig. S5). Differential genes were screened with p value ≤ 0.05 and an absolute value of log 2 (FC) ≥ 1 as the criteria (Fig. 2a, b, c). Total 279 DEGs were identified in the Control vs +C group, with 119 significantly upregulated and 160 significantly downregulated (Fig. 2a). There were 111 genes significantly different, with 67 were upregulated and 44 were downregulated (Fig. 2b). Furthermore, 398 DEGs were identified in Control vs +CN group, including 176 upregulated and 222 downregulated genes (Fig. 2c). Enrichment analysis of differentially expressed genes To explore the regulatory roles of DEGs in different treatment conditions, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Based on the annotation results of Unigenes in the GO database, the DEGs were categorized by their GO functions across different comparison groups (Supplementary Fig. S6). The GO analysis revealed enrichment in three categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Simultaneously, GO enrichment analysis was performed on DEGs with a Q-value ≤ 0.05, and the top 20 GO terms were selected for plotting (Fig. 3a, b, c). As shown in Figure 3a, the significantly enriched GO functions of DEGs in the Control vs +C group primarily included amine oxidase activity, amine metabolic process and quinone binding. In the Control vs +N group, DEGs were primarily associated with oxidoreductase activity, heme binding and proton transmembrane transport (Fig. 3b). Under +CN conditions, the significantly enriched GO functions of DEGs were predominantly related to ornithine-oxo-acid transaminase activity, thiol-dependent deubiquitinase activity, phospholipid binding, and ornithine transaminase activity (Fig. 3c). Additionally, the KEGG pathway enrichment analysis of the DEGs was presented in (Fig. 3d, e, f). The top 30 KEGG pathways enrichment analysis demonstrated that the DEGs were mainly enriched in tyrosine metabolism, phenylalanine metabolism, and carotenoid biosynthesis in the Control vs +C (Fig. 3d). The KEGG classification results indicated that the DEGs were primarily categorized into oxidative phosphorylation, carbon metabolism, and glycolysis/gluconeogenesis in the Control vs +N group (Fig. 3e). In the Control vs +CN group, most DEGs were involved in nucleocytoplasmic transport, fatty acid degradation, and oxidative phosphorylation (Fig. 3f). Differentially expressed genes associated with oxidative phosphorylation Through comparative transcriptome analysis, a considerable number of DEGs involved in oxidative phosphorylation were identified in the +C vs Control, +N vs Control and +CN vs Control (Fig. 4). Genes encoding NADH dehydrogenase, including Ndufs2, NdhB, Ndufa12 and Ndufb9, were all significantly upregulated in the +N vs Control. All genes, except NdhB, showed significant upregulation in the +C vs Control and +CN vs Control. The succinate dehydrogenase complex (SDHC) gene was remarkably upregulated in both the +N vs Control and +CN vs Control. Compared to the control group, the treatment groups (+C, +N, +CN) exhibited a notable decrease in the expression of cytochrome bc1 complex ( QCR7 ) and several cytochrome c oxidase genes ( COX6B, COX11, COX15, COX17 and CYC ). ATP synthase genes (ATP1 and ATP15) were significantly upregulated under all treatments (+C, +N, and +CN). ATP2 showed a significant decrease under carbon treatment. Analysis of the DEGs related to the photosynthesis and carbon metabolic pathway The expression levels of genes related to photosynthesis and carbon metabolic pathway are shown in Fig. 5. It was observed that most genes of Calvin cycle ( PGK, GAPDH, Aldolase, FBPase, tktA/B, PRK ) were up-regulated in +N vs Control (Fig. 5a, b). Rubisco and rpiA were down-regulated in all treatment groups. A significant increase in the expression of genes related to light-harvesting complex antenna proteins (Lhca1, Lhca5 and Lhca2) were observed in +N vs Control and +C vs Control, whereas these genes did not show significant changes in +CN vs Control group. In the photosynthesis pathway, the expression levels of photosynthesis-related genes ( PsaA, α, β, a ,) showed significant decrease in treated groups. Six EMP genes ( PFK , ALDO , GAPDH, P GK, gpmA and ENO ) and eight TCA genes ( pdhB, pdhC, PC, CS, ACLY, DLST, DLD and MDH ) were upregulated in +N vs Control group (Fig. 5c, d). The expression levels of genes related to the TCA cycle were increased in the +N vs Control and +CN vs Control groups, while these genes were downregulated in +C vs Control group (Fig. 5d). Analysis of the DEGs related to the Nitrogen metabolic pathway Nitrogen metabolism, as one of the fundamental processes in the life activities of seaweed, plays a crucial role in its growth and development. The transcriptome data of this study found that the genes encoding nitrate reductase (NR) and nitrite reductase (NiR) were significantly downregulated under +N vs Control, +C vs Control, and +CN vs Control (Fig. 6). The genes encoding glutamine synthetase (GS) and glutamate oxaloacetate transaminase (GOGAT) were significantly upregulated (Fig. 6). Transcription expression of genes related to carbon fixation and nitrogen utilization To further explore the relationship between DEGs and carbon-nitrogen metabolic pathways, the expression of carbon and nitrogen related genes 6-Phosphofructokinase (PFK), Phosphoribulose kinase (PRK), Glyceraldehyde-3-phosphate dehydrogenase (GRPDH), Ribulose diphosphate carboxylase (Rubisco), nitrate reductase (NR), malate dehydrogenase (MDH) were detected by qRT-PCR (Fig. 7). The expression level of PFK, PRK and GAPDH were similar trend, with the highest in +N group and the lowest in +CN treatments (Fig. 7a, b, c). For the Rubisco gene, there was no significance between +CO 2 and +CN groups, with the lowest in +N group (Fig. 7d). In addition, the highest expression of NR and MDH was found in Control and +N groups respectively (Fig. 7e, f). Discussion Ocean acidification and nutrient loading have raised global concern as they disrupt algal physiology and ecological interactions, jeopardizing the survival of marine organisms. S. hemiphyllum is widespread in coasts the most acidified and nitrogen nutrient zone. Here, we cultured S. hemiphyllum in different carbon and nitrogen concentrations and analyzed its physiological and transcriptomic characteristics. Understanding algae responses to carbon and nitrogen dynamics is critical for exploring their physiological adaptations to ocean acidification and nitrogen enrichment. CO 2 and nitrogen are critical factors affecting nutrient uptake efficiency, photosynthetic rate, respiration, and protein and amino acid metabolism. These factors have a significant impact on the growth rate of macroalgae [15, 30-32]. For example, the growth, photosynthesis, and nutrient uptake of S. japonica were significantly enhanced under elevated CO 2 concentrations [33]. Excess nutrient availability significantly promoted the growth and physiological performance of S. japonica [34] . In this study, Fv/Fm, Y(Ⅱ) and α had no significant differences in the different treatments. This phenomenon was also observed in Gracilariopsis lemaneiformis, where photosynthetic parameters were insignificant change in different levels of CO 2 and temperature [35]. In this study, both qN and NPQ were significantly higher in the experimental groups (+C, +N and +CN) compared to the control group. In addition, the rETR, Ik and qP were obviously enhanced between the +CO 2 and +CN groups. Similar results were reported that increased CO 2 benefits the photosynthetic production of Porphyra haitanensis [36] . The RGR of algae serves as a direct indicator for evaluating how environmental factors influence algal growth. For example, G. lemaneiformis exhibited a significantly lower RGR under low CO₂ levels compared to high CO₂ conditions [37]. Previous studies have also reported increased RGR in G. lemaneiformis and Gracilaria sp . under elevated CO₂ conditions [38, 39]. We observed consistent trend that the RGR increased in high-carbon treatments (+C and +CN groups). The algae efficiently utilized the inorganic carbon (HCO 3 − ) driving photosynthetic carbon concentration mechanisms (CCMs). Under high CO 2 conditions, CCMs enhance carbon utilization for photosynthesis, promoting growth and increasing RGR in Sargassum . In the present study, the enrichment of CO 2 enhanced the nitrogen absorption rate in different algae, for example the green algae Ulva Lactuca , the red algae Gracilaria sp . and the brown algae Hizikila fusiforme [40-42]. These results are similar with our previous study that high CO 2 concentrations stimulate the rate of NO 3 − absorption. NR is a substrate-inducing enzyme and is positively correlated with the increase in intracellular NO 3 − concentration [39]. However, our data showed that NRase activity were significantly decreased in different treatment environments, and both transcriptomic and quantitative results showed the same trends. When a relatively high concentration of nitrogen the algae obtains sufficient nutrients for physiological metabolism, and the CAT and SOD activities of the algal cells increase [37]. However high nitrogen did not increase their activity in our study (Supplementary Fig. S2). In previous studies, transcriptome analysis was used in algae molecular response to varying carbon or nitrogen environments [43, 44]. In this study, transcriptomic analysis was conducted to investigate the responses of Sargassum to different C and N treatments. The analysis of DEGs highlighted the oxidative phosphorylation, photosynthesis and carbon fixation, the TCA cycle and nitrogen metabolic pathways. The oxidative phosphorylation in cells is the main energy generation to sustain vital life activities. There are four main membrane-bound complexes-complex I [45], complex II [46], complex III [47, 48], and complex IV [49] participate in mitochondrial oxidative phosphorylation. Transcriptome analysis revealed that both the +N and +CN groups significantly upregulated genes related to NADH dehydrogenase, succinate dehydrogenase complex, cytochrome c complex and ATPase. The expression of V-type proton ATPase was increased when Chlorococcum littorale was exposed to high CO 2 levels [50]. We also found that the expression of ATPases (ATP1 and ATP15) was significantly improved in the +C treated group (Fig. 5). The Calvin cycle is an important process of carbon fixation in photosynthesis, where chloroplasts convert CO 2 into carbohydrates [51]. Ribulose bisphosphate carboxylase oxygenase (Rubisco) is an important enzyme in the Calvin cycle and plays a crucial role in photosynthesis carbon fixation [52]. Previous studies have yielded mixed results on the effects of CO 2 on Rubisco in algae [53-57]. McCarthy et al observed an increase in Rubisco content under elevated CO 2 concentrations in Thalassiosira pseudonana and Emiliania huxleyi [54], while other studies found that Rubisco content either decreases or remains unchanged [55, 56]. In our experiments, Rubisco activity and gene expression decreased at high CO 2 conditions (Fig. 5 and Fig. 7). This result seemingly implies that the regulation of the carbon concentrating mechanism (CCM) does not maintain a saturating CO 2 concentration at the site of Rubisco fixation. In addition, other genes related to the Calvin cycle ( PGK, GAPDH, Aldolase, FBPase, tktA/B, PRK ) were significantly upregulated. It suggests that more carbon is fixed through the Calvin cycle to sustain rapid cell growth under high CO 2 concentration. Enhanced carbon fixation raises the demand for ATP and NADPH. Light-harvesting complex antenna proteins (Lhca1, Lhca2 and Lhca5) were upregulated in the +C, +N and +CN groups. The photon energy harvested by LHCs in the thylakoid membrane of chloroplast flows through to the electron transport chain for NADPH reduction, while the proton gradient generated across the membrane drives ATP synthesis [58]. Lowering of linear electron transport could be a mechanism to retain more reductant (NADPH) than energy in the form of ATP [59]. The glycolysis is a universal cytosolic pathway for the degradation of hexoses to generate pyruvate [60]. It is first oxidized by the mitochondrial pyruvate dehydrogenase complex (PDC) to form acetyl-CoA, which enters the TCA cycle to generate reductant for ATP production [61, 62]. In the +N vs control group, most genes in glycolysis and the TCA cycle showed an upward-regulation trend. The concerted upregulation of these genes could accelerate the TCA cycle, leading to the generation of more NADH and GTP/ATP. This suggests that more metabolic energy and intermediates are generated through accelerated glycolysis and the TCA cycle, supporting robust cell growth under high NO 3 - concentration. The nitrogen metabolism of macroalgae is a complex and intricate physiological process that involves the uptake of nitrogen sources through cellular absorption systems. Nitrogen enters the cells via transporter proteins on the cell membrane, providing raw materials for subsequent nitrogen assimilation processes. Nitrate is one of the important inorganic nitrogen sources for macroalgae. Under the action of nitrate reductase (NR) and nitrite reductase (NiR), nitrate is first reduced to nitrite and then further reduced to ammonium. Assimilation of NH 4 + , either derived from direct uptake or from reduction of NO 2 - , occurs via a series of reactions involving the enzymes glutamine (Gln) synthetase (GS) and glutamate (Glu) synthase (GOGAT) [63, 64]. In transcriptome data, NR and NiR were decreased in +N, +C and +CN treatments than control, respectively. Meanwhile, the expression level of NR showed the same results that expression in the three different conditions were significantly lower than in the control. Compared to the control group, the GS and GOGAT were significantly increased in different treatment groups. These results indicated that exogenous carbon and nitrogen did not promote the expression of NR and NiR, the accumulation of NH 4 + within the cells, which promoted the expression of GS and GOGAT (Fig. 7). Conclusion The continuous emission of CO 2 and enrichment of nitrogen nutrients are causing irreversible changes in oceans, significantly impacting marine ecosystems. Currently, little is known about how algae adapt to ocean acidification and varying nitrogen content. Our study demonstrates that ocean acidification and nitrogen content synergistically influence the development and growth of S. hemiphyllum. The combination of physiological and transcriptomic approaches, we have systematically demonstrated that photosynthesis, the antioxidant system, energy metabolism and carbohydrate synthesis were involved in responses of the S. hemiphyllum in different treatment conditions (+N, +CO 2 and +CN). This study is the first to investigate the transcriptional response of S. hemiphyllum under high CO 2 and high NO 3 - concentrations using RNA-Seq. It provides a theoretical and experimental basis for understanding macroalgae adaptation to ocean acidification and nitrogen enrichment. Therefore, subsequent research will focus on analyzing changes in algal metabolites and metabolic pathways to explore the regulatory mechanisms of algae responses to environmental changes. Abbreviations CCMs Carbon dioxide concentrating mechanisms DIC dissolved inorganic carbon RGR Relative growth rates rETR relative electron transport rate PCA Principal Component Analysis DEGs Differentially expressed genes GO Gene Ontology KEGG Kyoto Encyclopedia of Genes and Genomes Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Data availability The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center (Nucleic Acids Res 2024), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA024761) that are publicly accessible at https://bigd.big.ac.cn/gsa/browse/CRA024761. Competing Interest The authors declare no competing interests. Funding This work was supported by the National Key R&D Program of China (2023YFD2400100); Research on breeding technology of candidate species for Guangdong modern marine ranching (2024-MRB-00-001); Supported by China Agriculture Research System of MOF and MARA (CARS-50); Science and Technology Plan Projects of Guangdong Province (No. 2021B1212050025); the Program for University Innovat ion Team of Guangdong Province (2022KCXTD008); and the STU Scientific Research Initiation Grant (NTF23030T). Author contributions J.C. designed the experiments and H.D. supervised the overall project. X.K. collected and curated the data. X.K. and H.L. conducted the experiments. J.Z. and J.W. and Y.W. prepared figures1-4. J.C. performed the formal analysis and wrote the original draft of the manuscript. T.L. critically reviewed the manuscript for intellectual content. All authors reviewed the manuscript. Authors’ information Jing Chen: [email protected] Xiao Ke: [email protected] Jinhui Wu: [email protected] Yurong Wang: [email protected] Honghao Liang: [email protected] Jie Zheng: [email protected] Tangcheng Li: [email protected] Hong Du: [email protected] Acknowledgements The author thanks the lab members for assistance. This study was carried out with the support of Guangdong Provincial Key Laboratory of Marine Disaster Prediction and Prevention, Shantou University. References Yu Z, Hu C, Sun H, Li H, Peng P. Pond culture of seaweed Sargassum hemiphyllum in southern China. Chinese Journal of Oceanology and Limnology. 2013, 31(2):300-305. Rivera M, Scrosati R. Population dynamics of Sargassum lapazeanum (Fucales, Phaeophyta) from the Gulf of California, Mexico. Phycologia. 2006, 45(2):178-189. Han T, Shi R, Qi Z, Huang H. The overgrowth of epiphytic Ulva prolifera during seedling cultivation of Sargassum hemiphyllum can be mitigated by regulating nitrogen availability. 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Supplementary Files SupplementaryFig.S1.pdf SupplementaryFig.S2.pdf SupplementaryFig.S3.pdf SupplementaryFig.S4.pdf SupplementaryFig.S5.pdf SupplementaryFig.S6.pdf Cite Share Download PDF Status: Published Journal Publication published 13 Nov, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 14 Jul, 2025 Reviews received at journal 14 Jul, 2025 Reviewers agreed at journal 16 Jun, 2025 Reviews received at journal 11 Jun, 2025 Reviewers agreed at journal 11 May, 2025 Reviewers invited by journal 09 May, 2025 Editor invited by journal 23 Apr, 2025 Editor assigned by journal 22 Apr, 2025 Submission checks completed at journal 22 Apr, 2025 First submitted to journal 21 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6499830","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454811575,"identity":"5a4861ba-e406-43c7-99a4-c24cd661c8d6","order_by":0,"name":"Jing Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYDACCRA2YGDgh3CZSdAi2cBMihYQMDhArBb+2c3HHlgU3LHbfP78MQmGCuvEBvazB/BbcudYuoGEwbPkbTeS2SQYzqQnNvDkJeDVYiCRYyYhYXA42ewGM5sEY9vhxAYJHgMCWvK/gbUY9x8GavlHlJYcNpAWOwMGoMMYG4jQInEjDeywBIkbycYWCcfSjdt4cvBr4Z+R/Exa4s9he/7+gw9vfKixlu1nP4NfCwgwA+MmsQHESgBiNoLqgYDxAwODPTEKR8EoGAWjYIQCAAwuPlekH4k9AAAAAElFTkSuQmCC","orcid":"","institution":"University of Shantou","correspondingAuthor":true,"prefix":"","firstName":"Jing","middleName":"","lastName":"Chen","suffix":""},{"id":454811576,"identity":"58ff2ed6-2aa1-4db2-bcc9-9172c3df49e1","order_by":1,"name":"Xiao Ke","email":"","orcid":"","institution":"University of Shantou","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Ke","suffix":""},{"id":454811577,"identity":"01981765-183f-43ed-abfc-8def1a386de2","order_by":2,"name":"Jinhui Wu","email":"","orcid":"","institution":"Agro-Tech Extension Center of Guangdong Province","correspondingAuthor":false,"prefix":"","firstName":"Jinhui","middleName":"","lastName":"Wu","suffix":""},{"id":454811578,"identity":"60b4592d-4bd6-431a-afb8-9688e6dc0c0a","order_by":3,"name":"Yurong Wang","email":"","orcid":"","institution":"University of Shantou","correspondingAuthor":false,"prefix":"","firstName":"Yurong","middleName":"","lastName":"Wang","suffix":""},{"id":454811579,"identity":"3c35c345-cadf-4823-af2c-6ac2595e1c7f","order_by":4,"name":"Honghao Liang","email":"","orcid":"","institution":"University of Shantou","correspondingAuthor":false,"prefix":"","firstName":"Honghao","middleName":"","lastName":"Liang","suffix":""},{"id":454811580,"identity":"9593bf23-0f02-412e-852e-d6c72b1874e8","order_by":5,"name":"Jie Zheng","email":"","orcid":"","institution":"University of Shantou","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zheng","suffix":""},{"id":454811581,"identity":"ed765e3d-f9f3-41a1-a44e-c7cc23ee38ab","order_by":6,"name":"Tangcheng Li","email":"","orcid":"","institution":"University of Shantou","correspondingAuthor":false,"prefix":"","firstName":"Tangcheng","middleName":"","lastName":"Li","suffix":""},{"id":454811582,"identity":"6c4f939e-e1b4-4fa7-8ae4-8b5ae4f7e24f","order_by":7,"name":"Hong Du","email":"","orcid":"","institution":"University of Shantou","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Du","suffix":""}],"badges":[],"createdAt":"2025-04-22 03:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6499830/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6499830/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-12157-w","type":"published","date":"2025-11-13T15:57:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82655590,"identity":"ed5c3c78-0754-4b05-ab13-d8b38e0adb52","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":207102,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of physiological parameters of \u003cem\u003eS. hemiphyllum\u003c/em\u003e under different treatment conditions. (a) Relative growth rate (RGR); (b) NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e uptake rate of \u003cem\u003eS. hemiphyllum\u003c/em\u003e under Control (normal group), +N (high nitrate group), +CO\u003csub\u003e2\u003c/sub\u003e (high CO\u003csub\u003e2\u003c/sub\u003e group), +CN (high carbon and nitrate group); (c) Nitrate reductase activity (NR) in Control, +N, +CO\u003csub\u003e2 \u003c/sub\u003eand +CN; (d) Total carbon content as % dry weight; (e) Total nitrogen content as % dry weight ; (f) Total carbon to nitrogen ratio (C:N). Bar plot heights represent mean values, with error bars indicating SD (\u003cem\u003en \u003c/em\u003e= 3). Different lowercase letters indicate significant differences (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/f2ac1be21ed02e0e073be784.png"},{"id":82655597,"identity":"07815cf4-a857-4e44-915d-8ac2327e2709","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":269159,"visible":true,"origin":"","legend":"\u003cp\u003eRNA-seq data expression of \u003cem\u003eS. hemiphyllum\u003c/em\u003e in different treatment conditions. (a) The volcanic map of DEGs in Control vs +C group; (b) Volcano plot in Control vs +N group; (c) Volcano plot in Control vs +CN group. The abscissa showed the log2 fold change values and the ordinate represented the -log10 (\u003cem\u003ep\u003c/em\u003e-value), red dots indicated the significantly up-regulated genes, blue dots indicated the significantly down-regulated genes, and gray indicated no significant differences.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/1e60a8ab0ff79050177c4a04.png"},{"id":82655594,"identity":"550e3b47-9171-48ad-bf66-7c5e4bb0eadb","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":986593,"visible":true,"origin":"","legend":"\u003cp\u003eEnriched GO terms and KEGG pathway of DEGs. (a) Enriched GO terms for Control vs +C; (b) Enriched GO terms for Control vs +N; (c) Enriched GO terms for Control vs +CN. The X-axis represents the enrichment ratio, the Y-axis represents the GO Term. (d) Enriched KEGG pathways for Control vs +C; (e) Enriched KEGG pathways for Control vs +N; (f) Enriched KEGG pathways for Control vs +CN. The KEGG pathway names are listed along the Y-axis. The top 30 KEGG pathways enriched with DEGs are shown, with the Y-axis representing KEGG pathway descriptions and the X-axis representing gene ratio. The bubble size indicates the number of DEGs, and the colors ranging from red to blue represent the significance of enrichment.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/05c91a64b6b2d819484f05a7.png"},{"id":82655591,"identity":"52ed0845-c642-4a7d-8c6d-159d52eb4e4b","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":696581,"visible":true,"origin":"","legend":"\u003cp\u003eThe proposed oxidative phosphorylation pathway and the expression of associated genes in \u003cem\u003eS. hemiphyllum\u003c/em\u003e. The heatmap of the relative expression of DEGs in the +C vs Control, +N vs Control and +CN vs Control.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/c3fa7fa393c8c58342c0c85e.png"},{"id":82655599,"identity":"7d377b96-f0ed-4b14-acc1-6145645eed8d","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":904158,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed genes (DEGs) involved in the photosynthesis and carbon metabolic pathway in \u003cem\u003eS. hemiphyllum \u003c/em\u003efollowing control, the +C, +N and +CN treatments. (a) Schematic overview of photosynthesis and carbon fixation; (b) Heatmap of relative expression of DEGs; (c) Scheme of EMP and the TCA cycle; (d) Heatmap of relative expression of DEGs in the +C vs Control, +N vs Control and +CN vs Control groups.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/1c5e94483af001104f4dafbb.png"},{"id":82655608,"identity":"16a3e39b-aa0e-425a-9bca-b648de56da4d","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":139122,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed genes (DEGs) involved in the nitrogen metabolic pathwayof \u003cem\u003eS. hemiphyllum\u003c/em\u003e in +C vs Control, +N vs Control and +CN vs Control\u003cstrong\u003e \u003c/strong\u003egroups.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/8015acd9ad905d413d6b5c43.png"},{"id":82656072,"identity":"f2ec7f98-e06e-4ee3-970c-ef6d2418faf1","added_by":"auto","created_at":"2025-05-13 18:48:27","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":186402,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR expression profile of marker genes \u003cem\u003ePFK, PRK, GAPDH, Rubisco, NR\u003c/em\u003e and \u003cem\u003eMDH\u003c/em\u003e in \u003cem\u003eS. hemiphyllum\u003c/em\u003e following control, the +N, +CO\u003csub\u003e2\u003c/sub\u003e and +CN treatments. (a) Expression changes of \u003cem\u003ePFK\u003c/em\u003e; (b) Expression changes of \u003cem\u003ePRK\u003c/em\u003e; (c) Expression changes of \u003cem\u003eGAPDH\u003c/em\u003e; (d) Expression changes of \u003cem\u003eRubisco\u003c/em\u003e; (e) Expression changes of \u003cem\u003eNR\u003c/em\u003e; (f)Expression changes of \u003cem\u003eMDH\u003c/em\u003e. Data are representative of three independent experiments. Different lowercase letters indicate significant differences (\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/31b93acb4a2128987f76e4b7.png"},{"id":96104991,"identity":"07a27e78-1051-45e5-9d1f-9c8b6f6c1cf7","added_by":"auto","created_at":"2025-11-17 16:06:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4007769,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/9d13ce5b-5121-4af6-a708-7c7eed34af3c.pdf"},{"id":82656069,"identity":"43d66cdc-b933-41ec-9bf8-ca67b93aa012","added_by":"auto","created_at":"2025-05-13 18:48:27","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3709153,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/353d0bee67fae0ae6ed82261.pdf"},{"id":82656067,"identity":"7be3d3f9-bdd1-4be7-8ec2-134b96ddd0f0","added_by":"auto","created_at":"2025-05-13 18:48:27","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":407186,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/37f6ac4380acecd3a87fc5e6.pdf"},{"id":82655596,"identity":"8cc7d738-ac8d-4045-b1ed-83c03624bea2","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"pdf","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":441236,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/e4adb695560998d05c6239ed.pdf"},{"id":82655611,"identity":"b479a034-c20c-4ee0-bea1-8063b2c6d818","added_by":"auto","created_at":"2025-05-13 18:40:28","extension":"pdf","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":421407,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/791657e4f697c77f0c7fd0be.pdf"},{"id":82655613,"identity":"730be539-b302-4721-ab00-4d4436448aa5","added_by":"auto","created_at":"2025-05-13 18:40:28","extension":"pdf","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":395345,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/e06a103f269f2129beee353b.pdf"},{"id":82655606,"identity":"19b68ec3-e20c-483e-99c0-16d1a83bbf7f","added_by":"auto","created_at":"2025-05-13 18:40:27","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":543180,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFig.S6.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6499830/v1/af2546e820738a7f0be5248f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003ePhysiological and Transcriptomic Responses of \u003cem\u003eSargassum hemiphyllum\u003c/em\u003e to Ocean Acidification and Nitrogen Enrichment\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eSargassum hemiphyllum\u003c/em\u003e is a major brown macroalga widely distributed along the coasts of China, Korea, and Japan [1]. Its high diversity and biomass play a critical role in ocean ecosystems by forming natural seaweed beds that serve as spawning, nursery, and feeding grounds for many marine animals [2]. It is also an economically important seaweed, serving as raw material for phycocolloids and as feed for highly valued aquaculture species such as holothurians, sea urchins, abalones and winkles [3]. Therefore, \u003cem\u003eS. hemiphyllum\u003c/em\u003e has significant economic and ecological value. However, its natural biomass has declined annually due to environmental pollution. The ocean absorbs excess carbon dioxide (CO\u003csub\u003e2\u003c/sub\u003e) from the atmosphere, causing ocean acidification [4]. Various anthropogenic activities, such as excessive use of chemical fertilizers and industrial pollution discharges, causing an increase in nitrogen levels [5]. Ocean acidification and elevated nitrogen content in seawater are major threats to marine ecosystems by altering organism physiology and disrupting ecological interactions [6, 7]. Hence, this topic has attracted the interest of many researchers.\u003c/p\u003e\n\u003cp\u003eHow macroalgae respond to ocean acidification has drawn increasing attention in recent years. Carbon dioxide concentrating mechanisms (CCMs) enable macroalgae to absorb dissolved inorganic carbon (DIC) when CO\u003csub\u003e2\u003c/sub\u003e : O\u003csub\u003e2\u003c/sub\u003e ratio is high or CO\u003csub\u003e2\u003c/sub\u003e concentrations are insufficient to maximize the rate of photosynthesis [8]. Macroalgae lacking CCMs, or with CCMs of low DIC affinity, may benefit from ocean acidification as CO\u003csub\u003e2 \u003c/sub\u003elimitations are alleviated [7, 9, 10]. When seawater CO\u003csub\u003e2\u003c/sub\u003e concentrations exceed those produced internally by CCMs, diffusive CO\u003csub\u003e2\u003c/sub\u003e can enter Rubisco [11-13]. This adaptation may downregulate CCMs and save energy, thereby enhancing growth rates and providing competitive advantages under ocean acidification [10]. However, The physiological mechanisms of macroalgae in response to ocean acidification still require further research [14].\u003c/p\u003e\n\u003cp\u003eAnother major concern is how the concentration of inorganic nitrogen (N) affects the growth performance of macroalgae. Studies have shown that elevated external nitrogen (N) concentrations can promote the growth of seaweeds [15]. When algae absorb nitrate, it is first reduced to nitrite and subsequently to ammonium through the action of the enzymes nitrate reductase and nitrite reductase. The ammonium is then used to make amino acids through the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway, which is common in most algal species [16]. Previous studies indicated that the transcription and activity of enzymes involved in nitrate metabolism are regulated by various external factors, for example, cellular fluxes of carbon and nitrogen substrates [17]. Nitrogen is one of the essential nutrients that regulate biomass production and photosynthetic efficiency in marine ecosystems. \u003c/p\u003e\n\u003cp\u003e\u003cem\u003eS. hemiphyllum \u003c/em\u003eplays a vital role in carbon sequestration and nitrogen absorption, functioning as a biological filter to mitigate enrichment and help maintain the ecological balance of marine ecosystems [7, 18, 19]. However, relatively few studies reported the relationship between intracellular carbon and nitrogen metabolism in macroalgae\u003cem\u003e. \u003c/em\u003eTo elucidate the combined impacts of nitrogenous nutrients and ocean acidification on the growth of \u003cem\u003eSargassum\u003c/em\u003e, it is essential to examine how carbon and nitrogen influence the algal physiological mechanisms. \u003c/p\u003e\n\u003cp\u003eThe aim of this study is to investigate how the altered CO\u003csub\u003e2 \u003c/sub\u003econcentrations and elevated nitrogen levels affect the physiology of \u003cem\u003eSargassum\u003c/em\u003e in a lab setting. In this study, we set two level of carbon (400 \u0026mu;atm and 1000 \u0026mu;atm) and two nitrogen supply levels (50 \u0026mu;mol L\u003csup\u003e\u0026minus;1 \u003c/sup\u003eand 300 \u0026mu;mol L\u003csup\u003e\u0026minus;1\u003c/sup\u003e). The thalli were used in the study to examine the physiological, biochemical and transcriptomic characteristics of carbon and nitrogen. Our results showed that the combined treatment of high CO\u003csub\u003e2 \u003c/sub\u003eand\u003csub\u003e \u003c/sub\u003enitrate concentrations enhanced the growth and photosynthetic efficiency of \u003cem\u003eS. hemiphyllum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eWe hypothesize that \u003cem\u003eS. hemiphyllum \u003c/em\u003ehas a significant advantage in coping with ocean acidification and high nitrogen environments. The findings present that the carbon and nitrogen within \u003cem\u003eS. hemiphyllum\u003c/em\u003e were reallocated under acidification and elevated nitrogen conditions, which not only helps to comprehend the regulatory mechanisms of seaweed growth, but also contributes to optimizing cultivation to promote rapid growth of the algae.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAlgal strains and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSargassum hemiphyllum\u003c/em\u003e var. \u003cem\u003echinense\u003c/em\u003e was collected from Nanao Island, Shantou University, Guangdong Province, China (117.11\u0026deg;E, 23.48\u0026deg;N). The thalli were cultured in aseptic seawater and illuminated with 80 \u0026mu;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e under a 12 h:12 h light/dark photoperiod. Experimental cultures were set up in four groups: control (NO₃⁻-N: 50 \u0026mu;mol/L, CO₂: 400 \u0026mu;atm), high nitrate (NO₃⁻-N: 300 \u0026mu;mol/L, CO₂: 400 \u0026mu;atm), high CO₂ (NO₃⁻-N: 50 \u0026mu;mol/L, CO₂: 1000 \u0026mu;atm), and high carbon-nitrogen (NO₃⁻-N: 300 \u0026mu;mol/L, CO₂: 1000 \u0026mu;atm) (Supplementary Fig. S1). Each experimental group consisted of three biological replicates, and the seawater was replaced every two days to ensure a continuous nutrient supply. After seven days of culture, the algae were collected and stored at -80℃ for subsequent RNA extraction and other measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChlorophyll fluorescence\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChlorophyll fluorescence parameters were determined using the chlorophyll fluorescence imaging system (WALZ Image-PAM, Zealquest Scientific Technology China). The maximum quantum yield (Fv/Fm) of the photosystem Ⅱ (PSⅡ) in \u003cem\u003eS. hemiphyllum\u003c/em\u003e (dark-adapted for 15 min) was measured using a saturation pulse (5000 \u0026mu;mol photons m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, 0.8 s). The relative electron transport rate (rETR) is the light-saturated photosynthetic electron transport rate, \u0026alpha; is the electron transport efficiency, and E represents the photosynthetically active radiation [20]. The maximum quantum yield (Fv/Fm) was measured via a pulse amplitude-modulated (PAM) fluorometer (Imaging-PAM, Walz, Germany) [21]. The calculations of Y(II), qP, and NPQ [22] were performed as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelative growth rates, the NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e uptake rate, nitrate reductase (NR) activity, total nitrogen and carbon contents\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative growth rates (RGR) were represented as the percentage increase in daily fresh weight (FW) biomass of\u003cem\u003e S. hemiphyllum\u003c/em\u003e, and were calculated using the following formula [23]:\u003c/p\u003e\n\u003cp\u003eRGR (% d\u003csup\u003e-1\u003c/sup\u003e) = [(lnW\u003csub\u003et \u003c/sub\u003e- lnW\u003csub\u003e0\u003c/sub\u003e) / t] \u0026times; 100\u003c/p\u003e\n\u003cp\u003eThe NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e- \u003c/sup\u003euptake rate was determined by measuring the decrease in NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e concentration in the cultured seawater over a certain time. The calculation formula is as follows: NO₃⁻ uptake rate \u003cimg src=\"data:image/png;base64,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\"\u003eThe Nitrate reductase (NR) activity was measured using NR activity assay kit. NR catalyzes the reduction of nitrate to nitrite in the presence of NADH, measuring its absorption peak at 340 nm. Approximately 0.1 g of fresh algal was ground in 1 mL of extraction solution, then centrifuged at 4\u0026deg;C 4000 g for 10 min. The absorption value of the supernatant was determined by the spectrophotometry at a wavelength of 340 nm. Approximately 0.2 g of \u003cem\u003eS. hemiphyllum\u003c/em\u003e was dried at 60℃ for 48 h, then finely ground into powder using a high-flux tissue grinder (Scientz-48, Ningbo Scientz Biotechnology, China). About 2 mg of the powder was used to measure cellular carbon (C) and nitrogen (N) content using an Elementar Vario Cube (Elementar, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAntioxidant characteristics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBriefly, 0.1 g thallus was ground into powder and mixed with 1 mL pre-cooled extraction buffer. We used the CAT assay, MDA and SOD kits (Nanjing Jian cheng Bioengineering Institute, Nanjing, China) for each enzyme assay. The CAT assay was used in the study according to the method of G\u0026oacute;th [24]. The SOD assay was based on SOD ability to oxidise hydroxylamine by the xanthine-xanthine oxidase system [25]. One unit (U) of SOD activity was calculated as the inhibition by 50% of the oxidation of hydroxylamine without an enzyme source. In addition, the malondialdehyde (MDA) assay was previous method [26].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and cDNA library construction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from \u003cem\u003eS. hemiphyllum\u003c/em\u003e using the RNAprep Pure Plant Kit (TIANGEN, China). The quality of RNA was assessed using NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, MA, USA). The cDNA library was constructed with the mRNA library preparation kit (MGIEasy\u003csup\u003eTM\u003c/sup\u003e, MGI, Shenzhen, China), with mRNA enriched for the poly(A) tail using magnetic beads conjugated with Oligo(dT). The cDNA library was transformed into double-stranded cDNA using random N6 primers, followed by the addition of A-Tailing Mix and RNA Index Adapters for ligation. Finally, the double-stranded cDNA library was amplified by polymerase chain reaction (PCR) and sequenced using the BGISEQ-500 platform at the Wuhan Institute of Genomics.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene expression levels were represented by FPKM and calculated using RSEM software [27]. FPKM values for each sample were log-transformed as 𝑥 = log\u003csub\u003e2 \u003c/sub\u003e(FPKM + 1). The normalized score (Z) was calculated using the formula: Z = (𝑥 - \u0026mu;)/\u0026sigma;, \u0026mu;: the mean of all sample FPKM values, \u0026sigma;: the standard deviation of all sample FPKM values, 𝑥: the log-transformed FPKM value. BLAST software was used to map genes to various databases (NT, NR, KOG, KEGG) for functional annotation. DEseq2 was detected different expression genes (DEGs). DEGs with |log\u003csub\u003e2\u003c/sub\u003e (fold change)| \u0026ge; 1.5 and an adjusted \u003cem\u003ep \u003c/em\u003evalue \u0026le; 0.05 were considered significantly differentially expressed [28]. GO and KEGG enrichment analyses were conducted to study the function and metabolic pathway significantly enriched by DEGs, and the significant enrichment level was Q value \u0026le; 0.05 [29].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA extraction and \u003c/strong\u003e\u003cstrong\u003eRT-qPCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApproximately 0.15 g of \u003cem\u003eS. hemiphyllum\u003c/em\u003e was collected and was homogenized by using liquid nitrogen and purified via RNAprep Pure Plant Kit with RNase-Free DNAase I to isolate total cellular RNA. The concentration and purity of the RNA were assessed using Nanodrop microspectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). After determination the RNA quality using agarose gel electrophoresis, complementary cDNA synthesis was carried out using Rever Tra Ace q PCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative real-time PCR (qRT-PCR) was performed with an Applied Biosystems 7300 Real Time PCR System and Roche Light Cycler 96 system, using TB Green\u0026reg; Premix Ex Taq\u0026trade; II to measure the relative transcript levels of the genes associated with metabolic changes. The ribosomal protein L35 (Unigene10602_All) was used as the endogenous control. Reference Gene Quantitative Primer were RL35-RT-F and RL35-RT-R. Primers used in this study were designed on primer 6.0 and are shown in Supplementary Table S1. Cycling conditions were 10 min at 95\u0026deg;C with 40 cycles for the melting (30 s at 95\u0026deg;C), annealing (30 s at 60\u0026deg;C), and extension (30 s at 72\u0026deg;C). The genes of interest and the primers are listed in Supplementary Table S1. The data were quantitatively analyzed by 2\u003csup\u003e\u0026minus;\u003c/sup\u003e\u003csup\u003e△△\u003c/sup\u003e\u003csup\u003eCT\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data were expressed as the mean \u0026plusmn; standard deviation (SD) (n = 3). Statistical analysis was performed using SPSS 25. Individual and interactive effects of nitrate and CO\u003csub\u003e2\u003c/sub\u003e were analyzed using two-way ANOVA with a confidential level of 95%. Significant differences between treatment groups were tested using independent \u003cem\u003et\u003c/em\u003e-tests \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 (Two-tailed distribution; Two-sample equal variance). Different letters on the top of the bars mark the statistically significant groups after \u003cem\u003et\u003c/em\u003e-tests.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eChlorophyll fluorescence parameters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe chlorophyll fluorescence parameters of \u003cem\u003eS. hemiphyllum\u003c/em\u003e under different culture conditions are shown in the Table 1. Fv/Fm showed no significant change under different treatments. Photochemical quenching (qP) reflects the level of photosynthetic activity in algae. The results showed that high concentrations of CO\u003csub\u003e2\u003c/sub\u003e significantly increased qP of algae to 0.58 \u0026plusmn; 0.05\u003csup\u003ea\u003c/sup\u003e and 0.58 \u0026plusmn; 0.005\u003csup\u003ea\u003c/sup\u003e under +CO\u003csub\u003e2\u003c/sub\u003e and +CN conditions, respectively (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05). qN and NPQ exhibited the same trend, with a significant increase under different treatment conditions (+N, +CO\u003csub\u003e2\u003c/sub\u003e, and +CN) compared to the control group. The study found that CO\u003csub\u003e2\u003c/sub\u003e had a main effect on the relative electron transport rate (rETR) of algae (Table 1). High concentrations of CO₂ significantly increased the relative electron transport rate (rETR) of the algae (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05), with +CO₂ and +CN showing values of 19.55 \u0026plusmn; 0.65\u003csup\u003ea\u003c/sup\u003e and 21.05 \u0026plusmn; 0.26\u003csup\u003ea\u003c/sup\u003e, respectively. The value of \u0026alpha; ranged from 0.18 to 0.21, with the highest \u0026alpha; value observed in +CN group (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05). CO\u003csub\u003e2\u003c/sub\u003e had a significant main effect on the light saturation point (Ik) of the algae (Table 1). High CO\u003csub\u003e2\u003c/sub\u003e concentrations significantly increased Ik, with values of 56.26 \u0026plusmn; 2.46\u003csup\u003ea\u003c/sup\u003e and 54.41 \u0026plusmn; 1.23\u003csup\u003ea\u003c/sup\u003e under +CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand +CN conditions, respectively.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"571\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFluorescence parameters\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+N\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+CO\u003csub\u003e2\u003c/sub\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e+CN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFv/Fm\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.7\u003c/strong\u003e \u003cstrong\u003e0.01\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.7135\u003c/strong\u003e \u003cstrong\u003e0.0008\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.7052\u003c/strong\u003e \u003cstrong\u003e0.0005\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.6983\u003c/strong\u003e \u003cstrong\u003e0.01\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eqP\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.46\u003c/strong\u003e \u003cstrong\u003e0.006\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.51\u003c/strong\u003e \u003cstrong\u003e0.02\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003e0.05\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003e0.005\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eY(Ⅱ)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.29\u003c/strong\u003e \u003cstrong\u003e0.03\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.31\u003c/strong\u003e \u003cstrong\u003e0.01\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.31\u003c/strong\u003e \u003cstrong\u003e0.02\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.37\u003c/strong\u003e \u003cstrong\u003e0.01\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eqN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.29\u003c/strong\u003e \u003cstrong\u003e0.02\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.49\u003c/strong\u003e \u003cstrong\u003e0.06\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003e0.04\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.14\u003c/strong\u003e \u003cstrong\u003e0.03\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNPQ\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.07\u003c/strong\u003e \u003cstrong\u003e0.004\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.15\u003c/strong\u003e \u003cstrong\u003e0.03\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.15\u003c/strong\u003e \u003cstrong\u003e0.02\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.14\u003c/strong\u003e \u003cstrong\u003e0.03\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003erETR\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e15.78\u003c/strong\u003e \u003cstrong\u003e1.54\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e16.46\u003c/strong\u003e \u003cstrong\u003e0.61\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003e0.65\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e21.05\u003c/strong\u003e \u003cstrong\u003e0.26\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.18\u003c/strong\u003e \u003cstrong\u003e0.003\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.19\u003c/strong\u003e \u003cstrong\u003e0.01\u003csup\u003eab\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.19\u003c/strong\u003e \u003cstrong\u003e0.01\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e0.21\u003c/strong\u003e \u003cstrong\u003e0.004\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003eIk\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 18.2137%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e47.51\u003c/strong\u003e \u003cstrong\u003e4.03\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003e3.33\u003csup\u003eb\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 22.0665%;\"\u003e\n \u003cp\u003e\u003cstrong\u003e56.26\u003c/strong\u003e \u003cstrong\u003e2.46\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 19.4396%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003cstrong\u003e1.23\u003csup\u003ea\u003c/sup\u003e\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;Table 1. Effects of different culture conditions on chlorophyll-induced fluorescence parameters in \u003cem\u003eS. hemiphyllum.\u003c/em\u003e Control group: 50 \u0026mu;mol/L NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N，400 \u0026mu;atm CO\u003csub\u003e2\u003c/sub\u003e; +N group: 300 \u0026mu;mol/L NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N，400 \u0026mu;atm CO\u003csub\u003e2\u003c/sub\u003e; +CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003egroup: 50 \u0026mu;mol/L NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, 1000 \u0026mu;atm CO\u003csub\u003e2\u003c/sub\u003e; +CN group: 300 \u0026mu;mol/L NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e-N, 1000 \u0026mu;atm CO\u003csub\u003e2\u003c/sub\u003e, significant differences (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, \u003cem\u003en\u003c/em\u003e \u0026ge; 3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiological analysis of \u003cem\u003eS. hemiphyllum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe relative growth rate (RGR), NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e uptake rate,\u0026nbsp;nitrate reductase activity (NR),\u0026nbsp;total carbon (C), total nitrogen (N) and C:N\u0026nbsp;ratio were measured in different carbon and nitrogen treatments (Control, +N, +CO\u003csub\u003e2\u003c/sub\u003e, +CN), as shown in Fig. 1. The RGR of \u003cem\u003eS. hemiphyllum\u003c/em\u003e were significantly promoted in +CO\u003csub\u003e2\u003c/sub\u003e and +CN groups (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.05, Fig. 1a). The highest RGR was found in +CN group (2.96 % g\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eFW d\u003csup\u003e-1\u003c/sup\u003e). The NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e uptake rate was significantly increased in the +N, +CO\u003csub\u003e2\u003c/sub\u003e and +CN groups (Fig. 1b). However, the activity of NR was statistically significantly lower in the +N, +CO₂, and +CN groups compared to the control group (Fig. 1c). There were no significant differences in total carbon and nitrogen content between the different treatment groups (Fig. 1d-1e). In addition, the C:N ratio in the +CN group was the lowest about 22.49 (Fig. 1f).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo understand the mechanisms responsible for the maintenance of the cellular state in the different environments, we investigated the enzymatic components of the antioxidant machinery (Supplementary Fig. S2). The CAT activity of \u003cem\u003eS. hemiphyllum\u003c/em\u003e grown at +N and +CO\u003csub\u003e2\u003c/sub\u003e environments were higher than that of the algae grown at Control and +CN environments. The MDA activity of \u003cem\u003eS. hemiphyllum\u003c/em\u003e was the lowest in +N group. No significant differences were observed in the SOD activities in different environments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification and validation of differentially expressed genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe reliability and consistency of the data were demonstrated by a strong correlation coefficient (R\u003csup\u003e2\u003c/sup\u003e \u0026gt; 0.909) of gene expression between biological replicates (Supplementary Fig. S3). Principal Component Analysis (PCA) was performed to compare transcriptome differences between groups (Supplementary Fig. S4). Comparative transcriptome analysis was performed to screen DEGs in three groups: Control vs +C, Control vs +N, and Control vs +CN. A total of 842 unigenes were differentially expressed in the Control vs +C group, with 364 upregulated and 478 downregulated. In the Control vs +N group, 1215 unigenes were differentially expressed, including 652 upregulated and 563 downregulated. Moreover, there were 1776 DEGs, which 795 were upregulated and 981 were downregulated in Control vs +CN group (Supplementary Fig. S5). Differential genes were screened with\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003evalue \u0026le; 0.05 and an absolute value of log\u003csub\u003e2\u003c/sub\u003e (FC) \u0026ge; 1 as the criteria (Fig. 2a, b, c). Total 279 DEGs were identified in the Control vs +C group, with 119 significantly upregulated and 160 significantly downregulated (Fig. 2a). There were 111 genes significantly different, with 67 were upregulated and 44 were downregulated (Fig. 2b). Furthermore, 398 DEGs were identified in Control vs +CN group, including 176 upregulated and 222 downregulated genes (Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnrichment analysis of differentially expressed genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the regulatory roles of DEGs in different treatment conditions, Gene Ontology (GO) annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed. Based on the annotation results of Unigenes in the GO database, the DEGs were categorized by their GO functions across different comparison groups (Supplementary Fig. S6). The GO analysis revealed enrichment in three categories: Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Simultaneously, GO enrichment analysis was performed on DEGs with a Q-value \u0026le; 0.05, and the top 20 GO terms were selected for plotting (Fig. 3a, b, c). As shown in Figure 3a, the significantly enriched GO functions of DEGs in the Control vs +C group primarily included amine oxidase activity, amine metabolic process and quinone binding. In the Control vs +N group, DEGs were primarily associated with oxidoreductase activity, heme binding and proton transmembrane transport (Fig. 3b). Under +CN conditions, the significantly enriched GO functions of DEGs were predominantly related to ornithine-oxo-acid transaminase activity, thiol-dependent deubiquitinase activity, phospholipid binding, and ornithine transaminase activity (Fig. 3c). Additionally, the KEGG pathway enrichment analysis of the DEGs was presented in (Fig. 3d, e, f). The top 30 KEGG pathways enrichment analysis demonstrated that the DEGs were mainly enriched in tyrosine metabolism, phenylalanine metabolism, and carotenoid biosynthesis in the Control vs +C (Fig. 3d). The KEGG classification results indicated that the DEGs were primarily categorized into oxidative phosphorylation, carbon metabolism, and glycolysis/gluconeogenesis in the Control vs +N group (Fig. 3e). In the Control vs +CN group, most DEGs were involved in nucleocytoplasmic transport, fatty acid degradation, and oxidative phosphorylation (Fig. 3f).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferentially expressed genes associated with oxidative phosphorylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough comparative transcriptome analysis, a considerable number of DEGs involved in oxidative phosphorylation were identified\u0026nbsp;in the +C vs Control, +N vs Control and +CN vs Control (Fig. 4). Genes encoding NADH dehydrogenase, including Ndufs2, NdhB, Ndufa12 and Ndufb9, were all significantly upregulated in the +N vs Control. All genes, except NdhB, showed significant upregulation in the +C vs Control and +CN vs Control. The succinate dehydrogenase complex (SDHC) gene was remarkably upregulated in both the +N vs Control and +CN vs Control.\u0026nbsp;Compared to the control group, the treatment groups (+C, +N, +CN) exhibited a notable decrease in the expression of cytochrome bc1 complex (\u003cem\u003eQCR7\u003c/em\u003e) and several cytochrome c oxidase genes (\u003cem\u003eCOX6B, COX11, COX15, COX17\u003c/em\u003e and \u003cem\u003eCYC\u003c/em\u003e). ATP synthase genes (ATP1 and ATP15) were significantly upregulated under all treatments (+C, +N, and +CN). ATP2 \u0026nbsp;showed a significant decrease under carbon treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the DEGs related to the\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;photosynthesis and carbon metabolic pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of genes related to photosynthesis and carbon metabolic pathway are shown in Fig. 5.\u0026nbsp;It was observed that most genes of Calvin cycle (\u003cem\u003ePGK, GAPDH, Aldolase, FBPase, tktA/B, PRK\u003c/em\u003e) were up-regulated in +N vs Control (Fig. 5a, b). Rubisco and rpiA were down-regulated in all treatment groups. A significant increase in the expression of genes related to light-harvesting complex antenna proteins (Lhca1, Lhca5 and Lhca2) were observed in +N vs Control and +C vs Control, whereas these genes did not show significant changes in +CN vs Control group. In the photosynthesis pathway, the expression levels of photosynthesis-related genes (\u003cem\u003ePsaA, \u0026alpha;, \u0026beta;, a\u003c/em\u003e,) showed significant decrease in treated groups. Six\u0026nbsp;EMP genes (\u003cem\u003ePFK\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003cem\u003eALDO\u003c/em\u003e\u003cem\u003e,\u0026nbsp;\u003c/em\u003e\u003cem\u003eGAPDH,\u003c/em\u003e\u003cem\u003e\u0026nbsp;P\u003c/em\u003e\u003cem\u003eGK, gpmA\u003c/em\u003e and \u003cem\u003eENO\u003c/em\u003e) and\u0026nbsp;eight TCA\u0026nbsp;genes\u0026nbsp;(\u003cem\u003epdhB, pdhC, PC, CS, ACLY, DLST, DLD\u0026nbsp;\u003c/em\u003eand \u003cem\u003eMDH\u003c/em\u003e) were upregulated in +N vs Control group (Fig. 5c, d). The expression levels of genes related to the TCA cycle were increased in the +N vs Control and +CN vs Control groups, while these genes were downregulated in +C vs Control group (Fig. 5d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of the DEGs related to the Nitrogen metabolic pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNitrogen metabolism, as one of the fundamental processes in the life activities of seaweed, plays a crucial role in its growth and development. The transcriptome data of this study found that the genes encoding nitrate reductase (NR) and nitrite reductase (NiR) were significantly downregulated under +N vs Control, +C vs Control, and +CN vs Control (Fig. 6). The genes encoding glutamine synthetase (GS) and glutamate oxaloacetate transaminase (GOGAT) were significantly upregulated (Fig. 6).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscription expression of genes related to carbon fixation and nitrogen utilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further explore the relationship between DEGs and carbon-nitrogen metabolic pathways, the expression of carbon and nitrogen related genes 6-Phosphofructokinase (PFK), Phosphoribulose kinase (PRK), Glyceraldehyde-3-phosphate dehydrogenase (GRPDH), Ribulose diphosphate carboxylase (Rubisco), nitrate reductase (NR), malate dehydrogenase (MDH) were detected by qRT-PCR (Fig. 7). The expression level of \u003cem\u003ePFK, PRK\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;GAPDH\u003c/em\u003e were similar trend, with the highest in +N group and the lowest in +CN treatments (Fig. 7a, b, c). For the \u003cem\u003eRubisco\u003c/em\u003e gene, there was no significance between +CO\u003csub\u003e2\u003c/sub\u003e and +CN groups, with the lowest in +N group (Fig. 7d). In addition, the highest expression of \u003cem\u003eNR\u003c/em\u003e and \u003cem\u003eMDH\u003c/em\u003e was found in Control and +N groups respectively (Fig. 7e, f).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOcean acidification and nutrient loading have raised global concern as they disrupt algal physiology and ecological interactions, jeopardizing the survival of marine organisms. \u003cem\u003eS. hemiphyllum\u003c/em\u003e is widespread in coasts the most acidified and nitrogen nutrient zone. Here, we cultured\u003cem\u003e S. hemiphyllum\u003c/em\u003e in different carbon and nitrogen concentrations and analyzed its physiological and transcriptomic characteristics. Understanding algae responses to carbon and nitrogen dynamics is critical for exploring their physiological adaptations to ocean acidification and nitrogen enrichment.\u003c/p\u003e\n\u003cp\u003eCO\u003csub\u003e2\u003c/sub\u003e and nitrogen are critical factors affecting nutrient uptake efficiency, photosynthetic rate, respiration, and protein and amino acid metabolism. These factors have a significant impact on the growth rate of macroalgae [15, 30-32]. For example, the growth, photosynthesis, and nutrient uptake of \u003cem\u003eS. japonica \u003c/em\u003ewere significantly enhanced under elevated CO\u003csub\u003e2\u003c/sub\u003e\u003cem\u003e \u003c/em\u003econcentrations [33]. Excess nutrient availability significantly promoted the growth and physiological performance of\u003cem\u003e S. japonica \u003c/em\u003e[34]\u003cem\u003e.\u003c/em\u003e In this study, Fv/Fm, Y(Ⅱ) and \u0026alpha; had no significant differences in the different treatments. This phenomenon was also observed in \u003cem\u003eGracilariopsis lemaneiformis,\u003c/em\u003e where photosynthetic parameters were insignificant change in different levels of CO\u003csub\u003e2\u003c/sub\u003e and temperature\u003csub\u003e \u003c/sub\u003e[35]. In this study, both qN and NPQ were significantly higher in the experimental groups (+C, +N and +CN) compared to the control group. In addition, the rETR, Ik and qP were obviously enhanced between the +CO\u003csub\u003e2\u003c/sub\u003e and +CN groups. Similar results were reported that increased CO\u003csub\u003e2\u003c/sub\u003e benefits the photosynthetic production of \u003cem\u003ePorphyra haitanensis \u003c/em\u003e[36]\u003cem\u003e.\u003c/em\u003e \u003c/p\u003e\n\u003cp\u003eThe RGR of algae serves as a direct indicator for evaluating how environmental factors influence algal growth. For example, \u003cem\u003eG. lemaneiformis\u003c/em\u003e exhibited a significantly lower RGR under low CO₂ levels compared to high CO₂ conditions [37]. Previous studies have also reported increased RGR in \u003cem\u003eG. lemaneiformis\u003c/em\u003e and \u003cem\u003eGracilaria\u003c/em\u003e sp\u003cem\u003e.\u003c/em\u003e under elevated CO₂ conditions [38, 39]. We observed consistent trend that the RGR increased in high-carbon treatments (+C and +CN groups). The algae efficiently utilized the inorganic carbon (HCO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e) driving photosynthetic carbon concentration mechanisms (CCMs). Under high CO\u003csub\u003e2\u003c/sub\u003e conditions, CCMs enhance carbon utilization for photosynthesis, promoting growth and increasing RGR in \u003cem\u003eSargassum\u003c/em\u003e. In the present study, the enrichment of CO\u003csub\u003e2\u003c/sub\u003e enhanced the nitrogen absorption rate in different algae, for example the green algae \u003cem\u003eUlva Lactuca\u003c/em\u003e, the red algae \u003cem\u003eGracilaria\u003c/em\u003e sp\u003cem\u003e.\u003c/em\u003e and the brown algae \u003cem\u003eHizikila fusiforme\u003c/em\u003e [40-42]. These results are similar with our previous study that high CO\u003csub\u003e2\u003c/sub\u003e concentrations stimulate the rate of NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e absorption. NR is a substrate-inducing enzyme and is positively correlated with the increase in intracellular NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e\u0026minus;\u003c/sup\u003e concentration [39]. However, our data showed that NRase activity were significantly decreased in different treatment environments, and both transcriptomic and quantitative results showed the same trends. When a relatively high concentration of nitrogen the algae obtains sufficient nutrients for physiological metabolism, and the CAT and SOD activities of the algal cells increase [37]. However high nitrogen did not increase their activity in our study (Supplementary Fig. S2).\u003c/p\u003e\n\u003cp\u003eIn previous studies, transcriptome analysis was used in algae molecular response to varying carbon or nitrogen environments [43, 44]. In this study, transcriptomic analysis was conducted to investigate the responses of \u003cem\u003eSargassum\u003c/em\u003e to different C and N treatments. The analysis of DEGs highlighted the oxidative phosphorylation, photosynthesis and carbon fixation, the TCA cycle and nitrogen metabolic pathways.\u003c/p\u003e\n\u003cp\u003eThe oxidative phosphorylation in cells is the main energy generation to sustain vital life activities. There are four main membrane-bound complexes-complex I [45], complex II [46], complex III [47, 48], and complex IV [49] participate in mitochondrial oxidative phosphorylation. Transcriptome analysis revealed that both the +N and +CN groups significantly upregulated genes related to NADH dehydrogenase, succinate dehydrogenase complex, cytochrome c complex and ATPase. The expression of V-type proton ATPase was increased when \u003cem\u003eChlorococcum littorale\u003c/em\u003e was exposed to high CO\u003csub\u003e2\u003c/sub\u003e levels [50]. We also found that the expression of ATPases (ATP1 and ATP15) was significantly improved in the +C treated group (Fig. 5). \u003c/p\u003e\n\u003cp\u003eThe Calvin cycle is an important process of carbon fixation in photosynthesis, where chloroplasts convert CO\u003csub\u003e2 \u003c/sub\u003einto carbohydrates [51]. Ribulose bisphosphate carboxylase oxygenase (Rubisco) is an important enzyme in the Calvin cycle and plays a crucial role in photosynthesis carbon fixation [52]. Previous studies have yielded mixed results on the effects of CO\u003csub\u003e2 \u003c/sub\u003eon Rubisco in algae [53-57]. McCarthy et al observed an increase in Rubisco content under elevated CO\u003csub\u003e2\u003c/sub\u003e concentrations in \u003cem\u003eThalassiosira pseudonana\u003c/em\u003e and\u003cem\u003e Emiliania huxleyi\u003c/em\u003e [54], while other studies found that Rubisco content either decreases or remains unchanged [55, 56]. In our experiments, Rubisco activity and gene expression decreased at high CO\u003csub\u003e2 \u003c/sub\u003econditions (Fig. 5 and Fig. 7). This result seemingly implies that the regulation of the carbon concentrating mechanism (CCM) does not maintain a saturating CO\u003csub\u003e2\u003c/sub\u003e concentration at the site of Rubisco fixation. In addition, other genes related to the Calvin cycle (\u003cem\u003ePGK, GAPDH, Aldolase, FBPase, tktA/B, PRK\u003c/em\u003e) were significantly upregulated. It suggests that more carbon is fixed through the Calvin cycle to sustain rapid cell growth under high CO\u003csub\u003e2\u003c/sub\u003e concentration. Enhanced carbon fixation raises the demand for ATP and NADPH. Light-harvesting complex antenna proteins (Lhca1, Lhca2 and Lhca5) were upregulated in the +C, +N and +CN groups. The photon energy harvested by LHCs in the thylakoid membrane of chloroplast flows through to the electron transport chain for NADPH reduction, while the proton gradient generated across the membrane drives ATP synthesis [58]. Lowering of linear electron transport could be a mechanism to retain more reductant (NADPH) than energy in the form of ATP [59].\u003c/p\u003e\n\u003cp\u003eThe glycolysis is a universal cytosolic pathway for the degradation of hexoses to generate pyruvate [60]. It is first oxidized by the mitochondrial pyruvate dehydrogenase complex (PDC) to form acetyl-CoA, which enters the TCA cycle to generate reductant for ATP production [61, 62]. In the +N vs control group, most genes in glycolysis and the TCA cycle showed an upward-regulation trend. The concerted upregulation of these genes could accelerate the TCA cycle, leading to the generation of more NADH and GTP/ATP. This suggests that more metabolic energy and intermediates are generated through accelerated glycolysis and the TCA cycle, supporting robust cell growth under high NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e concentration.\u003c/p\u003e\n\u003cp\u003eThe nitrogen metabolism of macroalgae is a complex and intricate physiological process that involves the uptake of nitrogen sources through cellular absorption systems. Nitrogen enters the cells via transporter proteins on the cell membrane, providing raw materials for subsequent nitrogen assimilation processes. Nitrate is one of the important inorganic nitrogen sources for macroalgae. Under the action of nitrate reductase (NR) and nitrite reductase (NiR), nitrate is first reduced to nitrite and then further reduced to ammonium. Assimilation of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e, either derived from direct uptake or from reduction of NO\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e, occurs via a series of reactions involving the enzymes glutamine (Gln) synthetase (GS) and glutamate (Glu) synthase (GOGAT) [63, 64]. In transcriptome data, NR and NiR were decreased in +N, +C and +CN treatments than control, respectively. Meanwhile, the expression level of NR showed the same results that expression in the three different conditions were significantly lower than in the control. Compared to the control group, the GS and GOGAT were significantly increased in different treatment groups. These results indicated that exogenous carbon and nitrogen did not promote the expression of NR and NiR, the accumulation of NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e within the cells, which promoted the expression of GS and GOGAT (Fig. 7).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe continuous emission of CO\u003csub\u003e2\u003c/sub\u003e and enrichment of nitrogen nutrients are causing irreversible changes in oceans, significantly impacting marine ecosystems. Currently, little is known about how algae adapt to ocean acidification and varying nitrogen content. Our study demonstrates that ocean acidification and nitrogen content synergistically influence the development and growth of \u003cem\u003eS. hemiphyllum.\u003c/em\u003e The combination of physiological and transcriptomic approaches, we have systematically demonstrated that photosynthesis, the antioxidant system, energy metabolism and carbohydrate synthesis were involved in responses of the \u003cem\u003eS. hemiphyllum\u003c/em\u003e in different treatment conditions (+N, +CO\u003csub\u003e2\u003c/sub\u003e and +CN). This study is the first to investigate the transcriptional response of \u003cem\u003eS. hemiphyllum \u003c/em\u003eunder high CO\u003csub\u003e2\u003c/sub\u003e and high NO\u003csub\u003e3\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e concentrations using RNA-Seq. It provides a theoretical and experimental basis for understanding macroalgae adaptation to ocean acidification and nitrogen enrichment. Therefore, subsequent research will focus on analyzing changes in algal metabolites and metabolic pathways to explore the regulatory mechanisms of algae responses to environmental changes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCCMs Carbon dioxide concentrating mechanisms\u003c/p\u003e\n\u003cp\u003eDIC dissolved inorganic carbon\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRGR Relative growth rates\u0026nbsp;\u003c/p\u003e\n\u003cp\u003erETR relative electron transport rate\u003c/p\u003e\n\u003cp\u003ePCA Principal Component Analysis\u003c/p\u003e\n\u003cp\u003eDEGs Differentially expressed genes\u003c/p\u003e\n\u003cp\u003eGO Gene Ontology\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eKEGG Kyoto Encyclopedia of Genes and Genomes\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw sequence data reported in this paper have been deposited in the Genome Sequence Archive in National Genomics Data Center (Nucleic Acids Res 2024), China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA024761) that are publicly accessible at https://bigd.big.ac.cn/gsa/browse/CRA024761.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key R\u0026amp;D Program of China (2023YFD2400100); Research on breeding technology of candidate species for Guangdong modern marine ranching (2024-MRB-00-001); Supported by China Agriculture Research System of MOF and MARA (CARS-50); Science and Technology Plan Projects of Guangdong Province (No. 2021B1212050025); the Program for University Innovat ion Team of Guangdong Province (2022KCXTD008); and the STU Scientific Research Initiation Grant (NTF23030T).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.C. designed the experiments and H.D. supervised the overall project. X.K. collected and curated the data. X.K. and H.L. conducted the experiments. J.Z. and J.W. and Y.W. prepared figures1-4. J.C. performed the formal analysis and wrote the original draft of the manuscript. T.L. critically reviewed the manuscript for intellectual content. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJing Chen: [email protected]\u003c/p\u003e\n\u003cp\u003eXiao Ke: [email protected]\u003c/p\u003e\n\u003cp\u003eJinhui Wu: [email protected]\u003c/p\u003e\n\u003cp\u003eYurong Wang: [email protected]\u003c/p\u003e\n\u003cp\u003eHonghao Liang: [email protected]\u003c/p\u003e\n\u003cp\u003eJie Zheng: [email protected]\u003c/p\u003e\n\u003cp\u003eTangcheng Li: [email protected]\u003c/p\u003e\n\u003cp\u003eHong Du: [email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author thanks the lab members for assistance. This study was carried out with the support of Guangdong Provincial Key Laboratory of Marine Disaster Prediction and Prevention, Shantou University.\u003c/p\u003e\n\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYu Z, Hu C, Sun H, Li H, Peng P. Pond culture of seaweed Sargassum hemiphyllum in southern China. \u003cem\u003eChinese Journal of Oceanology and Limnology.\u003c/em\u003e2013, 31(2):300-305.\u003c/li\u003e\n\u003cli\u003eRivera M, Scrosati R. Population dynamics of Sargassum lapazeanum (Fucales, Phaeophyta) from the Gulf of California, Mexico. \u003cem\u003ePhycologia. \u003c/em\u003e2006, 45(2):178-189.\u003c/li\u003e\n\u003cli\u003eHan T, Shi R, Qi Z, Huang H. 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Aspects of marine cyanobacterial nitrogen physiology and connection to the nitrogen cycle. \u003cem\u003eNitrogen in the marine environment\u003c/em\u003e. 2008:1073-1095.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Supplementary Table 1","content":"\u003cp\u003eSupplementary Table 1 is not available with this version\u003c/p\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":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Sargassum hemiphyllum, acidification, nitrate nitrogen, physiology, transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-6499830/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6499830/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Sargassum hemiphyllum is a major brown macroalga and has important ecological and economic significance. Ocean acidification and nitrogen enrichment are serious threats to marine ecosystems primarily by altering the physiology of organisms. However, the response of S. hemiphyllum to the combined effects of ocean acidification and elevated nitrogen levels remains unclear. This study conducted a 7-day dual-factor experiment to investigate the physiological and transcriptional responses of S. hemiphyllum under two CO2 levels (400 μatm and 1000 μatm) and two NO3⁻ levels (50 μmol/L and 300 μmol/L). The results showed that high CO2 and NO3- concentrations promoted the synthesis of photosynthetic pigments including qN and NPQ. Physiological results showed that high CO2 and the combined high NO3- and CO2 treatments enhanced growth rate and NO3- uptake rate, but NR activity was significantly decreased. Transcriptome analysis identified differentially expressed genes involved in oxidative phosphorylation, carbon metabolism, the TCA cycle, and nitrogen metabolic pathways. Notably, genes related to oxidative phosphorylation and TCA cycle were significantly up-regulated under high NO3- and dual-factor treatments, suggesting that carbohydrate metabolism and energy metabolism of S. hemiphyllum were significantly enhanced. The qRT-PCR analysis revealed that the expression levels of key genes involved in carbon fixation and nitrogen metabolism, including PFK, PRK, GAPDH, Rubisco, NR, and MDH, were significantly downregulated. These findings elucidate the molecular mechanisms by which S. hemiphyllum adapts to ocean acidification and nitrogen enrichment, offering valuable insights for understanding its capacity to withstand changing marine environments.","manuscriptTitle":"Physiological and Transcriptomic Responses of Sargassum hemiphyllum to Ocean Acidification and Nitrogen Enrichment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 18:40:22","doi":"10.21203/rs.3.rs-6499830/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-15T02:48:57+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-15T02:41:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"179285851818219339401413292391983755318","date":"2025-06-17T02:13:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-11T12:45:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"218709462231329773236193349848348094050","date":"2025-05-11T05:27:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-09T04:38:46+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-23T21:01:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-22T22:11:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-22T22:09:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-04-22T03:18:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a4359d0e-a45a-4701-aaa9-e921a6f264f0","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:00:30+00:00","versionOfRecord":{"articleIdentity":"rs-6499830","link":"https://doi.org/10.1186/s12864-025-12157-w","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-11-13 15:57:27","publishedOnDateReadable":"November 13th, 2025"},"versionCreatedAt":"2025-05-13 18:40:22","video":"","vorDoi":"10.1186/s12864-025-12157-w","vorDoiUrl":"https://doi.org/10.1186/s12864-025-12157-w","workflowStages":[]},"version":"v1","identity":"rs-6499830","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6499830","identity":"rs-6499830","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}