Genome-Wide Identification, Characterization and Expression Analysis of the Lhc Gene Family in Tetradesmus obliquus

Genome-Wide Identification, Characterization and Expression Analysis of the Lhc Gene Family in Tetradesmus obliquus | 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 Article Genome-Wide Identification, Characterization and Expression Analysis of the Lhc Gene Family in Tetradesmus obliquus Hailing Tan, Yonghua Ma, Gaolei Cai, Hui Tan, Deyu You, Yuhang Yin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4429243/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The process of photosynthesis depends heavily on the light-harvesting chlorophyll a / b -binding proteins ( Lhc ). However, to date, there has been a lack of systematic understanding of the Lhc gene family members in T. obliquus . This study conducted a systematic identification and analysis of the Lhc family genes in T. obliquus using bioinformatics. The findings show that 33 ToLhc genes in total, dispersed unevenly over 14 chromosomes, were found in T. obliquus . Most ToLhc genes encode stable proteins, with the majority predicted to localize in the chloroplast. The most prevalent cis-acting elements were those linked to both biotic and abiotic stress responses, according to analysis.RT-qPCR analysis showed that all ToLhc genes were down-regulated under 6 mg/L Cr 6+ conditions, except for ToLhca1 / 5.3 and ToLhcb1.1 , which maintained expression levels. This study systematically identified and characterized members of the ToLhc gene family in the green algae T. obliquus . Additionally, it offered an initial comprehension of the expression patterns of 33 genes under Cr 6+ heavy metal stress. The aim was to assess and predict the ecological risk of heavy metal Cr 6+ pollution to aquatic organisms, and to offer a theoretical framework for assessing how Cr 6+ affects algae. Biological sciences/Molecular biology Biological sciences/Plant sciences Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Algae are thought to be the principal producers and play a significant part in the food chain. Due to their short life cycle and susceptibility to aquatic environmental changes, their unique structure and abundance can serve as indicators of water conditions 1 , Tetradesmus obliquus ( T. obliquus ), belonging to the division Chlorophyta, class Chlorophyceae, is a common freshwater eukaryotic unicellular microalga. Its evaluation of the effects of chemical substances on many generations and at the population level can be obtained in a short period. It is mainly distributed in stagnant water environments such as ponds, lakes, and swamps 2 , T. obliquus , characterized by its advantages of easy isolation, rapid reproduction, and strong responsiveness, has been widely utilized as an indicator organism for water quality assessment 3,4 . Its distinctive cell wall structure allows for direct observation of both morphology and functionality at the cellular level 5 . Currently, with the increasing extraction and utilization of heavy metals, the problem of heavy metal pollution is becoming more and more intense. As the primary producer in aquatic ecosystems, T. obliquus engages in photosynthesis, converting inorganic substances (such as CO 2 , nitrogen, and phosphorus) into organic compounds and releasing oxygen in water. Furthermore, it is essential for preserving the composition and efficiency of aquatic ecosystems 6 . Trace heavy metal ions are harmful to algae, which can interfere with their normal development and metabolism, reduce chlorophyll production, and lower photosynthetic efficiency. These ions may cause genetic changes, altering the species composition of algae in their native habitat and leading to abnormal algal cell shapes 7 . For example, CdS QDs nanomaterials can improve the capture excitation energy efficiency of chlorophyll a in T. obliquus and enhance its sensitivity to photoinhibition. Under CdS QDs exposure, the growth and photosynthetic pigment content of T. obliquus exhibited a dose-effect relationship, indicating that the inhibitory effect became more pronounced as the treatment concentration increased 8 . Chlorella vulgaris exhibited decreased light-harvesting capability, PSII photosynthesis, and oxygen evolution under heavy metal stress caused by cadmium (Cd) and PFOA + Cd. Moreover, the number of genes involved in photosynthesis significantly and generally decreased 9 . Chlorella and Scenedesmus were inhibited by exposure to 20 nm PS particles, which reduced the amount of chlorophyll produced by algal cells. Furthermore, the presence of nanoplastics encourages algae to produce reactive oxygen species, which initiate oxidative stress reactions 10 . The group of thylakoid membrane proteins encoded by nuclear genes is known as the light-harvesting chlorophyll a / b -binding protein complex ( Lhc ) 11 , which plays a crucial role in photosynthesis. Chlorophyll a/b is highly abundant in the chloroplast membranes of certain algae, including Chlorophyta with chlorophyll a/b , Chromophyta with chlorophyll a/c , and Rhodophyta with chlorophyll a 12 . This complex efficiently absorbs light energy and transfers it to the reaction centers of photosystem I (PSI) and photosystem II (PSII), where photochemical reactions begin and light energy is converted to chemical energy. The PSI complex in higher plants and green algae is composed of the PSI core complex (PSI-CC) and light-harvesting complex I (LHCI). These two complexes cooperate to liberate electrons from water molecules and transport them to the process of generating NADPH 13 . This protein family is a complex transmembrane protein composed of three transmembrane helical structures. Each transmembrane helix contains a conserved chlorophyll-binding domain 14 . This protein family can maintain the structure of the thylakoid membrane, as well as light absorption and energy distribution. The stable channels are formed through its transmembrane helical structures, allowing chlorophyll to be transported and localized on the cell membrane. Furthermore, the plant's reaction to environmental stressors is influenced by this protein 15 . The investigation of light energy capture in photosynthesis was first revealed in Pisum sativum , where the Lhc gene was identified by cloning and sequencing the DNA sequence that encodes the mRNA precursor of the chlorophyll a/b -binding polypeptide 16 . In model plants, the Lhc gene family has been methodically identified. such as wheat ( Triticum aestivum ) 17 , Arabidopsis thaliana 18 . The whole-genome sequencing and assembly of many biological species have advanced significantly with the quick development of high-throughput sequencing technology, offering a strong basis for the precise identification of gene family members. Systematic analyses have been conducted in recent years on some economic crops, such as tea ( Camellia sinensis ) 19 , apple ( Malus domestica ) 20 , tomato ( Solanum lycopersicum ) 21 , and cotton ( Gossypium hirsutum ) 22 . However, there is no related report on T. obliquus . The Lhc gene family of T. obliquus was investigated thoroughly in this work, which included gene structure, conserved motifs, evolutionary connections, chromosomal distribution, and putative cis-elements analysis. Furthermore, the expression patterns of Lhc gene family were examined. Crucially, a thorough analysis was conducted on how the Lhc family genes react to various stresses. The results of this study offer theoretical understandings of the regulatory functions of this gene family in algae and are crucial for understanding the functions and mechanisms of the Lhc gene family in T. obliquus stress responses, growth, and development. Results Identification and phylogenetic analysis of the ToLhc gene in T. obliquus Through BLASTp alignment and conserved domain analysis, a total of 33 members of the Lhc gene family were identified in T. obliquus . Based on the evolutionary relationships indicated by the phylogenetic tree, the Lhc gene family was categorized into five subfamilies: Lhca, Lhcb, CP269 (Lhcb5), CP24 (Lhcb6), and CP29 (Lhcb4) 20 . The Lhca subfamily has the highest number of members ( 21 ) in the T. obliquus , followed by the Lhcb subfamily ( 8 ), CP26 (Lhcb5), and CP24 (Lhcb6) subfamilies (1 each), while the CP29 (Lhcb4) subfamily has the fewest members (0) (Fig. 1 ). CP24 (Lhcb6), CP26 (Lhcb5), and CP29 (Lhcb4) were considered minor Lhcb proteins, with CP24 and CP29 combining independently of the Lhcb-CP26 subfamily, clustering with the Lhca subfamily on the same branch. Figure 1 . Evolutionary Relationship of the Lhc Gene Family in T. obliquus , Arabidopsis thaliana , and Chlorella vulgaris. Chromosomal Distribution of ToLhc Genes in T. obliquus Using MapInspect, a chromosome distribution map was produced based on the gene annotation data of ToLhc genes in T. obliquus , as illustrated in Fig. 2 . In Chr01-Chr14, Chr03 and Chr04 each have 5 genes, corresponding to ToLhca1.3 , ToLhca5.8 , ToLhca5.7 , ToLhca6.1 , ToLhca5.6 , ToLhca4.2 , ToLhca3, ToLhca1.2 , ToLhcb2.6 , ToLhca1.1 ; Chr01 has 4 chromosomes, corresponding to ToLhca6.3 , ToLhca2.1 , ToLhca1.1 , ToLhca5.5 ; Chr11 and Chr13 each have three chromosomes, corresponding to ToLhca5.3 , ToLhcb5.4 , ToLhca3.1 and ToLhca6.2 , ToLhcb4.1 , ToLhca5 . There was only one ToLhc gene on other chromosomes, while two were found on Chr02, Chr07, Chr09, and Chr12. Figure 2 . Chromosomal arrangement of the ToLhc gene Chr represented chromosome, the scale on the left represented the physical length of chromosome (Mb). Gene Structure and Conserved Motif Analysis of ToLhc Genes in T. obliquus According to the GFF3 annotation information, a ToLhc gene structure diagram was generated using TBtools and sorted according to the phylogenetic tree order (Fig. 3 A). In the gene structure analysis, most of the ToLhc genes contain 1–11 introns, with 15 introns in ToLhca5.3 , while the number of exons is mostly 2–12, with 16 exons in all ToLhc genes except ToLhca5.3 . None of the ToLhc genes contain UTR regions (Fig. 3 B). In the conserved motif analysis, each ToLhc gene contains 2–7 conserved motifs. Among them, ToLhcb6 , ToLhca6.2 , ToLhca2.1 , and ToLhcb2.3 have 2 motifs, while ToLhca5.5 / 5.6 / 5.7 / 5.8 / 1.1 / 2.2 and ToLhcb2.1 / 2.2 / 2.5 / 2.6 has the most motifs, with 7 motifs (Fig. 3 C). Motif1/2/3/4 constitute key functional domains, with Motif1 containing Chloroa_b-bind and Chlloro_AB-bd_pln domains, Motif2/3 containing Chloroa_b-bind domain, and Motif4 containing Chlloro_AB-bd_pln domain (Fig. 3 D). Figure 3 . Translation: Gene Structure Analysis and Conserved Motif Analysis of the ToLhc Gene Family. Protein Characteristics and Three-Dimensional Structure of ToLhc in T. obliquus As indicated in Table 1 , the amino acid number of ToLhc proteins was found to be distributed from 187 to 1101, with an average of 278. It was discovered that the proteins' molecular weights ranged from 11.05 to 11.23 kDa, with an average of 29 kDa. Among them, ToLhca 3 was found to have the largest molecular weight, at 117.23 kDa, while the remaining proteins were all found to be less than 50 kDa. The theoretical isoelectric points of the proteins were found to be distributed from 4.87 to 9.55, with an average of 7.09. Among the 25 Lhc proteins, it was found that 25 had isoelectric points less than 8, making them acidic proteins, while the remaining ToLhc proteins were found to be alkaline. The average grand average of hydropathicity (GRAVY) for the ToLhc protein was determined to be -0.06. Except for ToLhca1 , ToLhca3.1 , ToLhca6.2 , ToLhca5.5 , ToLhca5.7 , ToLhca5.8 , ToLhca5.8 , ToLhca1.2 , ToLhca1.3 , ToLhcb2.1 , and ToLhcb2.3 proteins, it was found that the rest of the ToLhc proteins were hydrophilic (GRAVY < 0). The protein instability index was found to be distributed from 18.16 to 55, with an average of 32.74. Among them, it was found that ToLhca1 / 5.2 / 6.3 / 5.3 / 1.2/2.1 and ToLhcb2.5 were unstable proteins, while the rest were found to be stable. Subcellular localization prediction revealed that among the members of the ToLhc family, 7 were situated within the cellular membrane, 12 within the chloroplast, 2 within both the chloroplast and the nucleus, and 3 within both the chloroplast and the cellular membrane. Among them, it was observed that ToLhca1.1 might be located within the cellular membrane, cytoplasm, and mitochondria; ToLhca2.1 might be located within mitochondria and peroxisomes; ToLhca5.6 might be located within the cellular membrane, chloroplast, and peroxisomes; ToLhca6.1 might be located within the cellular membrane, chloroplast, mitochondria, and nucleus; ToLhcb7 might be located within the cellular membrane, chloroplast, and nucleus; ToLhca2.2 was found to be exclusively located within the cell wall; ToLhca3.1 might be located within the cellular membrane, cell wall, chloroplast, mitochondria, and peroxisomes; ToLhca6.2 was found to be exclusively located within the nucleus; ToLhca4.1 might be located within the cell wall and chloroplast. Table 1 Protein characterization of ToLhc. Len: Length of amino acid; MW: Molecular weight; pl: Ilsoeleti point; Ins: lnstability index; GRAVY: Grand average of hydropathicity; Sub Loc: Subcellular localization. Protein name Protein ID Len (aa) MW (kDa) pI Ins GRAVY Sub Loc ToLhcb6 WIA39984.1 277 29.44 6.84 22.22 -0.101 Chloroplast. ToLhca1 WIA37648.1 225 23.88 5.04 55.00 0.028 Cell membrane. ToLhca3.1 WIA41895.1 265 28.94 7.68 31.30 0.001 Cell membrane. Cell wall. Chloroplast. Mitochondrion. ToLhca3 WIA34115.1 1101 117.23 6.85 20.02 -0.224 Cell membrane. Chloroplast. ToLhca4.1 WIA4366.1 208 23.28 9.55 30.49 -0.139 Cell wall. Chloroplast. ToLhca4.2 WIA34029.1 259 28.08 7.69 28.19 -0.101 Chloroplast. ToLhca5.2 WIA39759.1 210 23.04 8.55 42.63 -0.030 Chloroplast. ToLhca5.1 WIA31012.1 187 19.47 7.94 32.78 -0.218 Chloroplast. ToLhca6.1 WIA33860.1 233 23.77 9.55 30.49 -0.139 Cell membrane. Chloroplast. Mitochondrion.Nucleus. ToLhca6.2 WIA43474.1 482 48.85 8.43 33.61 0.037 Nucleus. ToLhca6.3 WIA28189.1 103 11.05 8.55 42.63 -0.030 Cell membrane. ToLhca5.3 WIA41571.1 233 25.09 6.67 52.05 -0.049 Chloroplast. Nucleus. ToLhca5.4 WIA41658.1 306 33.88 7.94 32.78 -0.218 Cell membrane. ToLhca5.5 WIA29867.1 234 26.21 5.50 30.34 0.023 Chloroplast. ToLhca5.6 WIA33808.1 221 24.32 6.84 31.02 -0.048 Cell membrane. Chloroplast. Peroxisome. ToLhca5.7 WIA33810.1 245 26.64 8.43 33.61 0.037 Chloroplast. ToLhca5.8 WIA33809.1 201 21.79 8.31 27.86 0.030 Chloroplast. Nucleus. ToLhca1.2 WIA34426.1 247 27.12 6.92 47.52 0.038 Cell membrane. ToLhca1.1 WIA28655.1 215 23.13 6.84 22.22 -0.101 Cell membrane. Cytoplasm. Mitochondrion. ToLhca1.4 WIA39615.1 256 28.09 5.96 20.61 -0.082 Cell membrane. Chloroplast. ToLhca1.3 WIA33119.1 262 28.48 7.68 31.30 0.001 Cell membrane. ToLhca2.1 WIA30592.1 254 27.64 6.67 52.05 -0.049 Mitochondrion. Peroxisome. ToLhca2.3 WIA38668.1 242 25.52 5.56 18.16 -0.102 Cell membrane. Chloroplast. ToLhca2.2 WIA36289.1 251 27.38 4.98 20.14 -0.011 Cell wall. Chloroplast. Mitochondrion. ToLhcb7 WIA35708.1 393 41.43 4.87 22.70 -0.024 Cell membrane. Chloroplast. Nucleus ToLhcb1.1 WIA34922.1 261 27.71 6.74 37.81 -0.172 Cell membrane. ToLhcb5 WIA43630.1 289 30.55 7.69 28.19 -0.101 Cell membrane. ToLhcb2.1 WIA40812.1 274 29.24 5.50 30.34 0.023 Chloroplast. ToLhcb2.2 WIA43838.1 259 27.97 6.85 20.02 -0.224 Chloroplast. ToLhcb2.3 WIA42836.1 248 26.45 8.31 27.86 0.030 Chloroplast. ToLhcb2.4 WIA42835.1 249 26.44 6.84 31.02 -0.048 Chloroplast. ToLhcb2.5 WIA37845.1 255 27.08 6.84 61.83 -0.054 Chloroplast. ToLhcb2.6 WIA34487.1 256 27.34 5.64 31.84 -0.046 Chloroplast. The results of three-dimensional structure prediction indicate that the ToLhc protein is primarily composed of α-helices, β-turns, extended strands, and random coils (Fig. 4 ). The percentages of random coils, β-turns, extended strands, and α-helices in Group I are 45.85%, 3.97%, 8.66%, and 41.52%, respectively. In Group II, α-helices dominate (35.06–61.17%, with an average of 44.78%), followed by random coils (28.34–57.77%, with an average of 43.15%), extended strands (2.91–15.56%, with an average of 7.48%), and β-turns (1.95–8.51%, with an average of 4.59%). In Group III, the percentages of α-helices, β-turns, extended strands, and random coils are 36.33%, 7.27%, 8.65%, and 47.75%, respectively. In Group IV, random coils have the highest percentage (36.13–52.9%, with an average of 45.89%), followed by α-helices (35.77–46.59%, with an average of 41.83%), extended strands (3.91–12.98%, with an average of 7.25%), and β-turns (2.82–7.03%, with an average of 5.03%). Combined with phylogenetic analysis, it is observed that proteins on the same branch have more similar structural characteristics. Figure 4 . 3D model prediction of the ToLhc gene family. The cis-acting element of the ToLhc gene family Within the upstream 1500 bp promoter region of the 33 ToLhc genes, 48 cis-acting elements were found. These cis-acting elements can be classified into three groups: 10 elements associated with growth and development, 19 elements related to plant hormone responses, and 24 elements associated with biotic and abiotic stress (Fig. 5 ). In the promoter regions of all 33 ToLhc genes, elements associated with growth and development, such as TATA-box and CAAT-box, were abundant. The promoter regions of ToLhc genes were also found to contain a number of plant hormone-responsive elements, such as auxin (IAA) (TGA-element), gibberellin (GA3) (TATC-box), salicylic acid (SA) (TCA-element), jasmonic acid (JA) (TGACG-motif and CGTCA-motif), and ABA (ABRE). Furthermore, stress-responsive elements linked to both biotic and abiotic stimuli were identified. These included light-responsive elements (GATA-motif, ATC-motif, Box 4, CAG-motif, chs-CMA2a, chs-CMA2b, GA-motif, GATA-motif, GTGGC-motif, I-box, LAMP-element, Pc-CMA2c, TCCC-motif, TCT-motif, AE-box), anaerobic induction regulatory elements (ARE), low-temperature-responsive elements (LTR), defense and stress-responsive elements (TC-rich repeats). Figure 5 . Results of cis-acting element analysis of the ToLhc gene family promoters. qRT-PCR validation of the ToLhc gene family In order to further understand the expression characteristics of ToLhc genes under different Cr 6+ concentrations, this study selected 33 ToLhc genes and analyzed their relative expression levels under heavy metal Cr 6+ stress using RT-qPCR (Fig. 6 ). The results showed that under different concentrations of Cr 6+ , 12 ToLhc genes, including ToLhcb5 / 2.6 / 2.3 / 2.4 / 2.5 , as well as ToLhca1.1 / 2.1 / 2.3 / 2.2 / 1.4 / 1.2 / 1.3 , exhibited a downregulation trend. Among them, under no treatment (0 mg/L), the expression levels of these 9 ToLhc genes were the highest, and with the increase of stress concentration, the expression levels approached the lowest value at Cr 6+ concentration of 3 mg/L (Fig. 6 A). Three genes, ToLhca1 / 5.1/4.1 , showed a trend of initially downregulation, then upregulation, and then downregulation again. The expression levels decreased at 0.1 mg/L but increased again at 1.5 mg/L, and they still had expression levels at a Cr 6+ concentration of 6 mg/L (Fig. 6 B). ToLhcb.1 / 6 and ToLhca3.1 genes exhibited a downregulation trend, but still had corresponding expression levels at different Cr 6+ concentrations (Fig. 6 C). This shows that the way algae react to Cr 6+ stress may be influenced by Lhc genes. Figure 6 . Relative expression levels of ToLhc under different concentrations of Cr 6+ stress. Discussion Algae are primary producers in aquatic ecosystems, typically existing as single cells or multicellular colonies (such as filaments or sheets). They lack differentiation into roots, stems, and leaves but possess the ability to perform photosynthesis 23 . The toxic effects of heavy metals on microalgae are manifested in several ways: they can affect algal growth and metabolism, inhibit photosynthesis, reduce cellular pigments, and induce cellular deformation 24 . One of the most vulnerable organisms to heavy metal pollution after it enters aquatic environments through various pathways is microalgae. Under heavy metal ion stress, photosynthesis is one of the important physiological indicators used to assess the extent of damage to plants 25 . Different plants and algae exhibit distinct metal accumulation patterns under heavy metal stress, leading to varying degrees of impact on chlorophyll production 26 . Chromium (Cr) is one of the heavy metal pollutants, and emissions from fuel combustion, wastewater, and waste residues containing chromium are the main sources of pollution in industrial development 27 . In aquatic environments, Cr 3+ and Cr 6+ are the most common forms of chromium, with Cr 6+ being difficult to degrade and possessing characteristics such as carcinogenicity and mutagenicity 28 . This experiment, through systematic identification and analysis of the Lhc genes in T. obliquus , reveals their response patterns under different concentrations of Cr 6+ stress. The purpose is to assess and predict the ecological risk of heavy metal Cr 6+ pollution on aquatic organisms, providing the theoretical basis for the ecological impact assessment of Cr 6+ on algae. This study identified 33 ToLhc genes from T. obliquus and classified them into 5 subfamilies based on phylogenetic analysis (Fig. 1 ), namely Lhca, Lhcb, CP269 (Lhcb5), CP24 (Lhcb6), and CP29 (Lhcb4). The research indicates that the majority of ToLhc genes are located in the chloroplast, with a smaller portion distributed in other cellular organelles. In maize ( Zea mays ), over 94% of Lhc genes are localized in the chloroplast, with the remaining genes found in the mitochondria. This suggests that Lhc proteins primarily play a role in the photosynthetic pathway 29 . The predictive analysis of this study indicates that among the members of the ToLhc family, 7 ToLhc genes are distributed in the cell membrane, 12 ToLhc genes are distributed in the chloroplast, 2 ToLhc genes are simultaneously distributed in both the chloroplast and the nucleus, and 3 ToLhc genes are simultaneously distributed in both the chloroplast and the cell membrane. Therefore, ToLhc genes may primarily function in the cell membrane and chloroplast. Genes on the same branch of the evolutionary tree display similar patterns, but there are notable differences between subfamilies, according to gene structure and conserved motif analysis. Compared to Lhcb, the gene structure of Lhca is more complex, containing a greater number of introns. The introns of genes TaLhca5.3 / 5.6 / 2.2 are relatively long, which may be related to the gene's expression levels 30 . In this investigation, the examination of diverse gene expression patterns demonstrated that the expression levels of three genes, TaLhca5.3 / 5.6 / 2.2 , were low or undetectable, supporting this observation. Lhc genes are typical light-dependent genes, playing a crucial role in light capture, transfer, and protection. They are essential for the photosynthetic process in plants 31 . The promoter's analysis cis-acting elements of Lhc genes in T. obliquus in this study indicates that each upstream promoter region of Lhc genes in T. obliquus contains light-responsive elements. This suggests their crucial regulatory role in the expression of Lhc genes in response to light in T. obliquus . The study reveals that the expression of Lhc genes is associated with various abiotic stresses. After exposure to CdS ODs for 10 days, the genes related to the Light-Harvesting Complex (LHC) in T. obliquus were upregulated 8 . Overexpression of the non-gene Lhcb2 can enhance the tolerance of tobacco to low temperatures 32 . The phosphorylation of Lhcb proteins in spinach was markedly inhibited by NaCl treatment, and the sensitivity to NaCl changed in light and dark environments 33 . These hormone response elements are mostly associated with the plant's response to environmental stress. Heavy metals hurt the growth, enzyme activity, and photosynthetic activities of microalgae, particularly on PSⅠ and PSⅡ. This impact is dose-dependent, demonstrating a clear dose-response relationship 34 . The 33 ToLhc genes were subjected to qRT-PCR validation. The results indicate that under different concentrations of Cr 6+ stress, the expression levels of ToLhcb5 / 2.6 / 2.3 / 2.4 / 2.5 and ToLhca1.1 / 2.1 / 2.3 / 2.2 / 1.4 / 1.2 / 1.3 were highest under no treatment (0 mg/L), and then the expression levels showed a dose-response relationship. However, the gene expression levels of ToLhcb5 / 2.6 / 2.3 / 2.4 / 2.5 and ToLhca1.1 / 2.1 / 2.3 / 2.2 / 1.4 / 1.2 / 1.3 reached the lowest point at 3 mg/L. This may be related to the defense mechanism of T. obliquus , reducing gene expression under stress to resist damage. In contrast, ToLhca1 / 5.1 / 4.1 showed an increase at 1.5 mg/L, possibly due to a low-dose stimulation effect, and some expression levels were still detected at 3 mg/L, indicating partial recovery of photosynthetic capacity after adaptation for a certain period. For ToLhca1 / 5.1 / 4.1 , there was no significant difference between 0 mg/L and 0.1 mg/L, indicating that the impact of low concentration on T. obliquus is minimal. However, as the concentration increased, the expression levels showed a decreasing trend, and ToLhca3.1 and ToLhcb1.1 still exhibited partial expression at 6 mg/L. Conclusions This study identified a total of 33 members of the Lhc gene family in T. obliquus , distributed across its 14 chromosomes. The Lhc genes of T. obliquus were grouped into 5 subfamilies in the phylogenetic tree, and genes in the same subfamily displayed conserved motifs and similar gene structures. The expression of Lhc genes showed a dose-response relationship under Cr treatment. It has been proposed that Lhc genes are involved in how algae react to heavy metal stress. Methods Identification and Sequence Analysis of ToLhc Genes in T. obliquus Through the NCBI ( https://www.ncbi.nlm.nih.gov/ ) database, the full-genome protein sequences of Light-Harvesting Complex (Lhc ) in T. obliquus , Arabidopsis thaliana , and Chlorella vulgaris were retrieved. The PF00504 (Ethylene-insensitive 2) domain was downloaded using HMMER3.0, and validation was performed through Pfam ( http://Pfam.xfam.org ) and InterProScan ( http://www.ebi.ac.uk/InterProScan ) 35 . he protein sequences of ToLhc without the PF00504 (Ethylene-insensitive 2) domain, as well as those with redundancy, incompleteness, and annotations were removed 36 . Phylogenetic Analysis of ToLhc Genes in T. obliquus ToLhc genes were retrieved from T. obliquus , Arabidopsis thaliana , and Chlorella vulgaris . Multiple sequence alignment (MSA) was performed using ClustalW2, aligning the ToLhc protein sequences from T. obliquus with those from A. thaliana and C. vulgaris 37 , The ITOL website ( https://itol.embl.de/login.cgi ) was used to visualize and annotate the phylogenetic tree, which was created using the neighbor-joining method. This allowed for the grouping and naming of Lhc genes according to their evolutionary relationships 38 . Chromosomal localization analysis of the ToLhc genes in T. obliquus The GFF3 annotation file of the reference genome of T. obliquus was downloaded from the NCBI database ( https://www.ncbi.nlm.nih.gov/ ). From the GFF3 file, gene structure annotations for members of the ToLhc family were extracted. The chromosomal distribution map was created using the start and end position information of these genes on the chromosomes with the MapInspect tool, followed by refinement and adjustments for visual clarity 39 . Analysis of ToLhc Motifs and Gene Structure in T. obliquus Based on the genome annotation information of T. obliquus , the gene structure analysis diagram was drawn using TBtools. Next, the Lhc protein sequences were uploaded to MEME ( http://memesuite.org/index.html ) 40 and to find their conserved motifs, Smart Motif ( http://smart.embl-heidelberg.de/ ) was used 41 . The motif analysis was set with a motif quantity of 10 and a maximum width of 50. TBtools was employed to draw the Motif analysis diagram, achieving the visualization of conserved motif sequences 42 . Protein Characteristics and Tertiary Structure Prediction of ToLhc Genes in T. obliquus The protein characteristics of ToLhc were analyzed using the protein analysis tool ExPASy Server10 ( https://prosite.expasy.ory/PS50011 ). These properties include stability, average hydrophobicity (GRAVY), amino acid count, isoelectric point (pI), and relative molecular weight (MW). Furthermore, the relevant data were arranged and the subcellular localization of Lhc genes was predicted using the online tool Plant-mPLoc ( http://www.csbio.sjtu.edu.cn/cgibin/PlantPLoc.cgi ) 43 . Homology modeling of ToLhc was performed using SWISS-MODEL ( https://www.swissmodel.expasy.org/ ) 44 . Analysis of cis-acting elements in the promoter regions of the ToLhc gene family in T. obliquus To analyze the cis-acting elements in the promoter regions of the ToLhc gene family in T. obliquus , the upstream sequences (1 ~ 1500 bp) of the ToLhc family members were manually extracted from the genome sequence. These sequences were then submitted to PlantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) for the identification of cis-acting elements in the promoter region 45 . The analysis results were organized and visualized using the R software package heatmap. Chromium Toxicity Effects in T. obliquus T. obliquus was acquired from the Institute of Aquatic Biology, Chinese Academy of Sciences, Freshwater Algae Species Bank. The supplier of potassium dichromate (K 2 Cr 2 O 7 ) was Tianjin Kemiou Chemical Reagent Co., Ltd.; the remaining reagents were analytical grade. Algae cell culture and Cr 6+ exposure protocol: The algae cells were initially cultivated following the protocol outlined by Wen et al 46 . T. obliquus in the logarithmic growth phase were inoculated into 250mL conical flasks. At the beginning of the experiment, the density of the algae cells was approximately 1×10 6 cells/mL (initial OD 680nm = 0.087) with a total solution volume of 200 mL. Cr 6+ concentrations were 0 (CK), 0.1, 1.5, 3, 4.5, and 6 mg/L, and the experimental period was 96 hours 47 . RNA extraction and RT-qPCR analysis in T. obliquus Using the TRizol reagent (GenStar, Beijing, China), total RNA was extracted, and the DNaseI enzyme (Vazyme, Nanjing, China) was used to remove DNA. RevertAid reverse transcriptase (Vazyme, Nanjing, China) was used to reverse transcribe RNA into complementary DNA (cDNA). After that, cDNA was diluted using water free of enzymes. Using Primer 5.0 software, create gene-specific primers with tub serving as the internal reference gene 48 . The RT-qPCR analysis was conducted according to the manufacturer's instructions (Vazyme, Nanjing, China). The quantitative real-time polymerase chain reaction was performed using the 2 × SYBR Premix Extaq 10 µL forward and reverse primers, 0.4 µL each. Two milliliters (µL) of ddH 2 O were used to dilute the cDNA There are three steps in the program: Step 1 involves pre-denaturation at 95°C for 30 s; step 2 involves denaturation at 95°C for 5 s; and step 3 involves primer annealing, extension, and fluorescence signal collection at 60°C for 30 s; cycles of step 2 to step 3 were repeated 40 times. The relative expression level was calculated using the 2 −ΔΔCt method, and GraphPad Prism 8.0 software was used for differential significance analysis 49 . Declarations Ethics approval and consent to participate This study does not include human or animal subjects. Statement on guidelines All experimental studies and experimental materials involved in this research are in full compliance with relevant institutional, national, and international guidelines and legislation. Acknowledgments (not compulsory) In this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments). Data availability All data generated or analyzed during this study are included in this published article [and its supplementary information files]. Author contributions statem ent Hailing Tan and Yonghua Ma conceived and designed the experiments; Hailing Tan, Gaolei Cai, Hui Tan and Yuhang Yin performed the experiment; Hailing Tan conducted the bioinformatics analysis and wrote the manuscript; Jun Yang, Fengxia Tan and Yi Chai revised the manuscript. Competing interests The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding: This work was partially supported by the Key Lab of Freshwater Biodiversity Conservation Ministry of Agriculture and Rural Affairs of China, Yangtze River Fisheries Research Institute(2024LFBC1113). References Wang, S., Li, Q., Huang, S., Zhao, W., & Zheng, Z. Single and combined effects of microplastics and lead on the freshwater algae Microcystis aeruginosa. Ecotoxicology and Environmental Safety 208, 111664, DOI: https://doi.org/10.1016/j.ecoenv.2020.111664 (2021). Salazar, J., Santana-Sánchez, A., Näkkilä, J., Sirin, S., & Allahverdiyeva, Y. Complete N and P removal from hydroponic greenhouse wastewater by Tetradesmus obliquus : A strategy for algal bioremediation and cultivation in Nordic countries. 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Molecular complexation properties of Cd 2+ by algal organic matter from Scenedesmus obliquus . Ecotoxicology and Environmental Safety 263, 115378, DOI: https://doi.org/10.1016/j.ecoenv.2023.115378 (2023). Fan, H. Toxic effects and molecular mechanism of different methyl-substituted ionic liquid on scenedesmus obliquus , Zhejiang Gongshang University, (2020). in Chinese. Yin, J. et al. Identification of circular RNAs and their targets during tomato fruit ripening. Postharvest Biology and Technology 136, 90–98, DOI: https://doi.org/10.1016/j.postharvbio.2017.10.013 (2018). Additional Declarations No competing interests reported. Supplementary Files SupplementalTableS1.xlsx Cite Share Download PDF Status: Posted Version 1 posted 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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11:18:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116137,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal arrangement of the\u003cem\u003e ToLhc\u003c/em\u003egene Chr represented chromosome, the scale on the left represented the physical length of chromosome (Mb).\u003c/p\u003e","description":"","filename":"floatimage2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4429243/v1/49f46d1ce7e0833ab065a1ba.jpg"},{"id":57490550,"identity":"e3e85b5f-3c4d-4ac9-8fc2-08f6fd39e737","added_by":"auto","created_at":"2024-05-31 11:18:29","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":344457,"visible":true,"origin":"","legend":"\u003cp\u003eTranslation: Gene Structure Analysis and Conserved Motif Analysis of the \u003cem\u003eToLhc\u003c/em\u003e Gene 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\u003cem\u003eToLhc\u003c/em\u003e gene family promoters.\u003c/p\u003e","description":"","filename":"floatimage5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4429243/v1/3362ff217404bb0267204378.jpg"},{"id":57490555,"identity":"fcc1f6a5-8c70-4d70-83f5-17e6f6b31b91","added_by":"auto","created_at":"2024-05-31 11:18:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":448583,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of \u003cem\u003eToLhc\u003c/em\u003e under different concentrations of Cr\u003csup\u003e6+\u003c/sup\u003e stress.\u003c/p\u003e","description":"","filename":"floatimage6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4429243/v1/7719fc2b7040cad6188a94d1.jpg"},{"id":64213162,"identity":"db22814e-a627-446c-8cd1-098a32bb69c8","added_by":"auto","created_at":"2024-09-10 08:23:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2798336,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4429243/v1/4b310909-2f0a-44c2-b2e0-eee60342e338.pdf"},{"id":57491131,"identity":"a6d4df1f-74f0-4a74-a974-d1f892f97140","added_by":"auto","created_at":"2024-05-31 11:26:29","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15555,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4429243/v1/715e7a7bbe45409739d93f2c.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Identification, Characterization and Expression Analysis of the Lhc Gene Family in Tetradesmus obliquus","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAlgae are thought to be the principal producers and play a significant part in the food chain. Due to their short life cycle and susceptibility to aquatic environmental changes, their unique structure and abundance can serve as indicators of water conditions\u003csup\u003e1\u003c/sup\u003e, \u003cem\u003eTetradesmus obliquus\u003c/em\u003e(\u003cem\u003eT. obliquus\u003c/em\u003e), belonging to the division Chlorophyta, class Chlorophyceae, is a common freshwater eukaryotic unicellular microalga. Its evaluation of the effects of chemical substances on many generations and at the population level can be obtained in a short period. It is mainly distributed in stagnant water environments such as ponds, lakes, and swamps\u003csup\u003e2\u003c/sup\u003e, \u003cem\u003eT. obliquus\u003c/em\u003e, characterized by its advantages of easy isolation, rapid reproduction, and strong responsiveness, has been widely utilized as an indicator organism for water quality assessment\u003csup\u003e3,4\u003c/sup\u003e. Its distinctive cell wall structure allows for direct observation of both morphology and functionality at the cellular level\u003csup\u003e5\u003c/sup\u003e. Currently, with the increasing extraction and utilization of heavy metals, the problem of heavy metal pollution is becoming more and more intense. As the primary producer in aquatic ecosystems, \u003cem\u003eT. obliquus\u003c/em\u003e engages in photosynthesis, converting inorganic substances (such as CO\u003csub\u003e2\u003c/sub\u003e, nitrogen, and phosphorus) into organic compounds and releasing oxygen in water. Furthermore, it is essential for preserving the composition and efficiency of aquatic ecosystems\u003csup\u003e6\u003c/sup\u003e. Trace heavy metal ions are harmful to algae, which can interfere with their normal development and metabolism, reduce chlorophyll production, and lower photosynthetic efficiency. These ions may cause genetic changes, altering the species composition of algae in their native habitat and leading to abnormal algal cell shapes\u003csup\u003e7\u003c/sup\u003e. For example, CdS QDs nanomaterials can improve the capture excitation energy efficiency of chlorophyll a in \u003cem\u003eT. obliquus\u003c/em\u003e and enhance its sensitivity to photoinhibition. Under CdS QDs exposure, the growth and photosynthetic pigment content of \u003cem\u003eT. obliquus\u003c/em\u003e exhibited a dose-effect relationship, indicating that the inhibitory effect became more pronounced as the treatment concentration increased\u003csup\u003e8\u003c/sup\u003e. \u003cem\u003eChlorella vulgaris\u003c/em\u003e exhibited decreased light-harvesting capability, PSII photosynthesis, and oxygen evolution under heavy metal stress caused by cadmium (Cd) and PFOA\u0026thinsp;+\u0026thinsp;Cd. Moreover, the number of genes involved in photosynthesis significantly and generally decreased\u003csup\u003e9\u003c/sup\u003e. \u003cem\u003eChlorella\u003c/em\u003e and \u003cem\u003eScenedesmus\u003c/em\u003e were inhibited by exposure to 20 nm PS particles, which reduced the amount of chlorophyll produced by algal cells. Furthermore, the presence of nanoplastics encourages algae to produce reactive oxygen species, which initiate oxidative stress reactions\u003csup\u003e10\u003c/sup\u003e. The group of thylakoid membrane proteins encoded by nuclear genes is known as the light-harvesting chlorophyll \u003cem\u003ea\u003c/em\u003e/\u003cem\u003eb\u003c/em\u003e-binding protein complex (\u003cem\u003eLhc\u003c/em\u003e)\u003csup\u003e11\u003c/sup\u003e, which plays a crucial role in photosynthesis. Chlorophyll \u003cem\u003ea/b\u003c/em\u003e is highly abundant in the chloroplast membranes of certain algae, including Chlorophyta with chlorophyll \u003cem\u003ea/b\u003c/em\u003e, Chromophyta with chlorophyll \u003cem\u003ea/c\u003c/em\u003e, and Rhodophyta with chlorophyll \u003cem\u003ea\u003c/em\u003e\u003csup\u003e\u003cem\u003e12\u003c/em\u003e\u003c/sup\u003e. This complex efficiently absorbs light energy and transfers it to the reaction centers of photosystem I (PSI) and photosystem II (PSII), where photochemical reactions begin and light energy is converted to chemical energy. The PSI complex in higher plants and green algae is composed of the PSI core complex (PSI-CC) and light-harvesting complex I (LHCI). These two complexes cooperate to liberate electrons from water molecules and transport them to the process of generating NADPH\u003csup\u003e13\u003c/sup\u003e. This protein family is a complex transmembrane protein composed of three transmembrane helical structures. Each transmembrane helix contains a conserved chlorophyll-binding domain\u003csup\u003e14\u003c/sup\u003e. This protein family can maintain the structure of the thylakoid membrane, as well as light absorption and energy distribution. The stable channels are formed through its transmembrane helical structures, allowing chlorophyll to be transported and localized on the cell membrane. Furthermore, the plant's reaction to environmental stressors is influenced by this protein\u003csup\u003e15\u003c/sup\u003e. The investigation of light energy capture in photosynthesis was first revealed in \u003cem\u003ePisum sativum\u003c/em\u003e, where the \u003cem\u003eLhc\u003c/em\u003e gene was identified by cloning and sequencing the DNA sequence that encodes the mRNA precursor of the chlorophyll \u003cem\u003ea/b\u003c/em\u003e-binding polypeptide\u003csup\u003e16\u003c/sup\u003e. In model plants, the \u003cem\u003eLhc\u003c/em\u003e gene family has been methodically identified. such as wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e)\u003csup\u003e17\u003c/sup\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003csup\u003e18\u003c/sup\u003e. The whole-genome sequencing and assembly of many biological species have advanced significantly with the quick development of high-throughput sequencing technology, offering a strong basis for the precise identification of gene family members. Systematic analyses have been conducted in recent years on some economic crops, such as tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e)\u003csup\u003e19\u003c/sup\u003e, apple (\u003cem\u003eMalus domestica\u003c/em\u003e)\u003csup\u003e20\u003c/sup\u003e, tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e)\u003csup\u003e21\u003c/sup\u003e, and cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e)\u003csup\u003e22\u003c/sup\u003e. However, there is no related report on \u003cem\u003eT. obliquus\u003c/em\u003e. The \u003cem\u003eLhc\u003c/em\u003e gene family of \u003cem\u003eT. obliquus\u003c/em\u003e was investigated thoroughly in this work, which included gene structure, conserved motifs, evolutionary connections, chromosomal distribution, and putative cis-elements analysis. Furthermore, the expression patterns of \u003cem\u003eLhc\u003c/em\u003e gene family were examined. Crucially, a thorough analysis was conducted on how the \u003cem\u003eLhc\u003c/em\u003e family genes react to various stresses. The results of this study offer theoretical understandings of the regulatory functions of this gene family in algae and are crucial for understanding the functions and mechanisms of the \u003cem\u003eLhc\u003c/em\u003e gene family in \u003cem\u003eT. obliquus\u003c/em\u003e stress responses, growth, and development.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification and phylogenetic analysis of the\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003egene in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThrough BLASTp alignment and conserved domain analysis, a total of 33 members of the \u003cem\u003eLhc\u003c/em\u003e gene family were identified in \u003cem\u003eT. obliquus\u003c/em\u003e. Based on the evolutionary relationships indicated by the phylogenetic tree, the \u003cem\u003eLhc\u003c/em\u003e gene family was categorized into five subfamilies: Lhca, Lhcb, CP269 (Lhcb5), CP24 (Lhcb6), and CP29 (Lhcb4)\u003csup\u003e20\u003c/sup\u003e. The Lhca subfamily has the highest number of members (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e) in the \u003cem\u003eT. obliquus\u003c/em\u003e, followed by the Lhcb subfamily (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), CP26 (Lhcb5), and CP24 (Lhcb6) subfamilies (1 each), while the CP29 (Lhcb4) subfamily has the fewest members (0) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). CP24 (Lhcb6), CP26 (Lhcb5), and CP29 (Lhcb4) were considered minor Lhcb proteins, with CP24 and CP29 combining independently of the Lhcb-CP26 subfamily, clustering with the Lhca subfamily on the same branch.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Evolutionary Relationship of the \u003cem\u003eLhc\u003c/em\u003e Gene Family in \u003cem\u003eT. obliquus\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and \u003cem\u003eChlorella vulgaris.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosomal Distribution of\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003eGenes in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing MapInspect, a chromosome distribution map was produced based on the gene annotation data of \u003cem\u003eToLhc\u003c/em\u003e genes in \u003cem\u003eT. obliquus\u003c/em\u003e, as illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. In Chr01-Chr14, Chr03 and Chr04 each have 5 genes, corresponding to \u003cem\u003eToLhca1.3\u003c/em\u003e, \u003cem\u003eToLhca5.8\u003c/em\u003e, \u003cem\u003eToLhca5.7\u003c/em\u003e, \u003cem\u003eToLhca6.1\u003c/em\u003e, \u003cem\u003eToLhca5.6\u003c/em\u003e, \u003cem\u003eToLhca4.2\u003c/em\u003e, \u003cem\u003eToLhca3, ToLhca1.2\u003c/em\u003e, \u003cem\u003eToLhcb2.6\u003c/em\u003e, \u003cem\u003eToLhca1.1\u003c/em\u003e; Chr01 has 4 chromosomes, corresponding to \u003cem\u003eToLhca6.3\u003c/em\u003e, \u003cem\u003eToLhca2.1\u003c/em\u003e, \u003cem\u003eToLhca1.1\u003c/em\u003e, \u003cem\u003eToLhca5.5\u003c/em\u003e; Chr11 and Chr13 each have three chromosomes, corresponding to \u003cem\u003eToLhca5.3\u003c/em\u003e, \u003cem\u003eToLhcb5.4\u003c/em\u003e, \u003cem\u003eToLhca3.1\u003c/em\u003e and \u003cem\u003eToLhca6.2\u003c/em\u003e, \u003cem\u003eToLhcb4.1\u003c/em\u003e, \u003cem\u003eToLhca5\u003c/em\u003e. There was only one \u003cem\u003eToLhc\u003c/em\u003e gene on other chromosomes, while two were found on Chr02, Chr07, Chr09, and Chr12.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Chromosomal arrangement of the \u003cem\u003eToLhc\u003c/em\u003e gene Chr represented chromosome, the scale on the left represented the physical length of chromosome (Mb).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGene Structure and Conserved Motif Analysis of \u003cem\u003eToLhc\u003c/em\u003e Genes in \u003cem\u003eT. obliquus\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eAccording to the GFF3 annotation information, a \u003cem\u003eToLhc\u003c/em\u003e gene structure diagram was generated using TBtools and sorted according to the phylogenetic tree order (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In the gene structure analysis, most of the \u003cem\u003eToLhc\u003c/em\u003e genes contain 1\u0026ndash;11 introns, with 15 introns in \u003cem\u003eToLhca5.3\u003c/em\u003e, while the number of exons is mostly 2\u0026ndash;12, with 16 exons in all \u003cem\u003eToLhc\u003c/em\u003e genes except \u003cem\u003eToLhca5.3\u003c/em\u003e. None of the \u003cem\u003eToLhc\u003c/em\u003e genes contain UTR regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). In the conserved motif analysis, each \u003cem\u003eToLhc\u003c/em\u003e gene contains 2\u0026ndash;7 conserved motifs. Among them, \u003cem\u003eToLhcb6\u003c/em\u003e, \u003cem\u003eToLhca6.2\u003c/em\u003e, \u003cem\u003eToLhca2.1\u003c/em\u003e, and \u003cem\u003eToLhcb2.3\u003c/em\u003e have 2 motifs, while \u003cem\u003eToLhca5.5\u003c/em\u003e/\u003cem\u003e5.6\u003c/em\u003e/\u003cem\u003e5.7\u003c/em\u003e/\u003cem\u003e5.8\u003c/em\u003e/\u003cem\u003e1.1\u003c/em\u003e/\u003cem\u003e2.2\u003c/em\u003e and \u003cem\u003eToLhcb2.1\u003c/em\u003e/\u003cem\u003e2.2\u003c/em\u003e/\u003cem\u003e2.5\u003c/em\u003e/\u003cem\u003e2.6\u003c/em\u003e has the most motifs, with 7 motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Motif1/2/3/4 constitute key functional domains, with Motif1 containing Chloroa_b-bind and Chlloro_AB-bd_pln domains, Motif2/3 containing Chloroa_b-bind domain, and Motif4 containing Chlloro_AB-bd_pln domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Translation: Gene Structure Analysis and Conserved Motif Analysis of the \u003cem\u003eToLhc\u003c/em\u003e Gene Family.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein Characteristics and Three-Dimensional Structure of\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003ein\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAs indicated in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the amino acid number of \u003cem\u003eToLhc\u003c/em\u003e proteins was found to be distributed from 187 to 1101, with an average of 278. It was discovered that the proteins' molecular weights ranged from 11.05 to 11.23 kDa, with an average of 29 kDa. Among them, \u003cem\u003eToLhca\u003c/em\u003e 3 was found to have the largest molecular weight, at 117.23 kDa, while the remaining proteins were all found to be less than 50 kDa. The theoretical isoelectric points of the proteins were found to be distributed from 4.87 to 9.55, with an average of 7.09. Among the 25 \u003cem\u003eLhc\u003c/em\u003e proteins, it was found that 25 had isoelectric points less than 8, making them acidic proteins, while the remaining \u003cem\u003eToLhc\u003c/em\u003e proteins were found to be alkaline. The average grand average of hydropathicity (GRAVY) for the \u003cem\u003eToLhc\u003c/em\u003e protein was determined to be -0.06. Except for \u003cem\u003eToLhca1\u003c/em\u003e, \u003cem\u003eToLhca3.1\u003c/em\u003e, \u003cem\u003eToLhca6.2\u003c/em\u003e, \u003cem\u003eToLhca5.5\u003c/em\u003e, \u003cem\u003eToLhca5.7\u003c/em\u003e, \u003cem\u003eToLhca5.8\u003c/em\u003e, \u003cem\u003eToLhca5.8\u003c/em\u003e, \u003cem\u003eToLhca1.2\u003c/em\u003e, \u003cem\u003eToLhca1.3\u003c/em\u003e, \u003cem\u003eToLhcb2.1\u003c/em\u003e, and \u003cem\u003eToLhcb2.3\u003c/em\u003e proteins, it was found that the rest of the \u003cem\u003eToLhc\u003c/em\u003e proteins were hydrophilic (GRAVY\u0026thinsp;\u0026lt;\u0026thinsp;0). The protein instability index was found to be distributed from 18.16 to 55, with an average of 32.74. Among them, it was found that \u003cem\u003eToLhca1\u003c/em\u003e/\u003cem\u003e5.2\u003c/em\u003e/\u003cem\u003e6.3\u003c/em\u003e/\u003cem\u003e5.3\u003c/em\u003e/\u003cem\u003e1.2/2.1\u003c/em\u003e and \u003cem\u003eToLhcb2.5\u003c/em\u003e were unstable proteins, while the rest were found to be stable. Subcellular localization prediction revealed that among the members of the \u003cem\u003eToLhc\u003c/em\u003e family, 7 were situated within the cellular membrane, 12 within the chloroplast, 2 within both the chloroplast and the nucleus, and 3 within both the chloroplast and the cellular membrane. Among them, it was observed that \u003cem\u003eToLhca1.1\u003c/em\u003e might be located within the cellular membrane, cytoplasm, and mitochondria; \u003cem\u003eToLhca2.1\u003c/em\u003e might be located within mitochondria and peroxisomes; \u003cem\u003eToLhca5.6\u003c/em\u003e might be located within the cellular membrane, chloroplast, and peroxisomes; \u003cem\u003eToLhca6.1\u003c/em\u003e might be located within the cellular membrane, chloroplast, mitochondria, and nucleus; \u003cem\u003eToLhcb7\u003c/em\u003e might be located within the cellular membrane, chloroplast, and nucleus; \u003cem\u003eToLhca2.2\u003c/em\u003e was found to be exclusively located within the cell wall; \u003cem\u003eToLhca3.1\u003c/em\u003e might be located within the cellular membrane, cell wall, chloroplast, mitochondria, and peroxisomes; \u003cem\u003eToLhca6.2\u003c/em\u003e was found to be exclusively located within the nucleus; \u003cem\u003eToLhca4.1\u003c/em\u003e might be located within the cell wall and chloroplast.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eProtein characterization of ToLhc. Len: Length of amino acid; MW: Molecular weight; pl: Ilsoeleti point; Ins: lnstability index; GRAVY: Grand average of hydropathicity; Sub Loc: Subcellular localization.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProtein name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eProtein ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLen\u003c/p\u003e \u003cp\u003e(aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMW\u003c/p\u003e \u003cp\u003e(kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003epI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eIns\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eGRAVY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSub Loc\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA39984.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e277\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA37648.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e225\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e55.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca3.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA41895.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Cell wall. Chloroplast. Mitochondrion.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA34115.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e117.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Chloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca4.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA4366.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e208\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell wall. Chloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca4.2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA34029.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e28.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA39759.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e210\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA31012.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e187\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e19.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e32.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca6.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA33860.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.139\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Chloroplast. Mitochondrion.Nucleus.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca6.2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA43474.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e482\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e48.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNucleus.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca6.3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA28189.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e103\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA41571.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e233\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast. Nucleus.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA41658.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e306\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e32.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA29867.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA33808.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e221\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Chloroplast. Peroxisome.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA33810.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e245\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca5.8\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA33809.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e201\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast. Nucleus.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca1.2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA34426.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e247\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e47.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.038\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca1.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA28655.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e215\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Cytoplasm. Mitochondrion.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca1.4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA39615.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.082\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Chloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca1.3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA33119.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e262\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca2.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA30592.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e254\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e52.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.049\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eMitochondrion. Peroxisome.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca2.3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA38668.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e242\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e18.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Chloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhca2.2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA36289.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.011\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell wall. Chloroplast. Mitochondrion.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb7\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA35708.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e393\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e22.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.024\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane. Chloroplast. Nucleus\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb1.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA34922.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e37.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA43630.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e30.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e28.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eCell membrane.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb2.1\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA40812.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.023\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb2.2\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA43838.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e20.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb2.3\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA42836.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e27.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.030\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb2.4\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA42835.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e249\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb2.5\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA37845.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e255\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e61.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.054\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eToLhcb2.6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWIA34487.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e27.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e31.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e-0.046\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eChloroplast.\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results of three-dimensional structure prediction indicate that the \u003cem\u003eToLhc\u003c/em\u003e protein is primarily composed of α-helices, β-turns, extended strands, and random coils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The percentages of random coils, β-turns, extended strands, and α-helices in Group I are 45.85%, 3.97%, 8.66%, and 41.52%, respectively. In Group II, α-helices dominate (35.06\u0026ndash;61.17%, with an average of 44.78%), followed by random coils (28.34\u0026ndash;57.77%, with an average of 43.15%), extended strands (2.91\u0026ndash;15.56%, with an average of 7.48%), and β-turns (1.95\u0026ndash;8.51%, with an average of 4.59%). In Group III, the percentages of α-helices, β-turns, extended strands, and random coils are 36.33%, 7.27%, 8.65%, and 47.75%, respectively. In Group IV, random coils have the highest percentage (36.13\u0026ndash;52.9%, with an average of 45.89%), followed by α-helices (35.77\u0026ndash;46.59%, with an average of 41.83%), extended strands (3.91\u0026ndash;12.98%, with an average of 7.25%), and β-turns (2.82\u0026ndash;7.03%, with an average of 5.03%). Combined with phylogenetic analysis, it is observed that proteins on the same branch have more similar structural characteristics.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. 3D model prediction of the \u003cem\u003eToLhc\u003c/em\u003e gene family.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe cis-acting element of the\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWithin the upstream 1500 bp promoter region of the 33 \u003cem\u003eToLhc\u003c/em\u003e genes, 48 cis-acting elements were found. These cis-acting elements can be classified into three groups: 10 elements associated with growth and development, 19 elements related to plant hormone responses, and 24 elements associated with biotic and abiotic stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). In the promoter regions of all 33 \u003cem\u003eToLhc\u003c/em\u003e genes, elements associated with growth and development, such as TATA-box and CAAT-box, were abundant. The promoter regions of \u003cem\u003eToLhc\u003c/em\u003e genes were also found to contain a number of plant hormone-responsive elements, such as auxin (IAA) (TGA-element), gibberellin (GA3) (TATC-box), salicylic acid (SA) (TCA-element), jasmonic acid (JA) (TGACG-motif and CGTCA-motif), and ABA (ABRE). Furthermore, stress-responsive elements linked to both biotic and abiotic stimuli were identified. These included light-responsive elements (GATA-motif, ATC-motif, Box 4, CAG-motif, chs-CMA2a, chs-CMA2b, GA-motif, GATA-motif, GTGGC-motif, I-box, LAMP-element, Pc-CMA2c, TCCC-motif, TCT-motif, AE-box), anaerobic induction regulatory elements (ARE), low-temperature-responsive elements (LTR), defense and stress-responsive elements (TC-rich repeats).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Results of cis-acting element analysis of the \u003cem\u003eToLhc\u003c/em\u003e gene family promoters.\u003c/p\u003e \u003cp\u003e \u003cb\u003eqRT-PCR validation of the\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eIn order to further understand the expression characteristics of \u003cem\u003eToLhc\u003c/em\u003e genes under different Cr\u003csup\u003e6+\u003c/sup\u003e concentrations, this study selected 33 \u003cem\u003eToLhc\u003c/em\u003e genes and analyzed their relative expression levels under heavy metal Cr\u003csup\u003e6+\u003c/sup\u003e stress using RT-qPCR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The results showed that under different concentrations of Cr\u003csup\u003e6+\u003c/sup\u003e, 12 \u003cem\u003eToLhc\u003c/em\u003e genes, including \u003cem\u003eToLhcb5\u003c/em\u003e/\u003cem\u003e2.6\u003c/em\u003e/\u003cem\u003e2.3\u003c/em\u003e/\u003cem\u003e2.4\u003c/em\u003e/\u003cem\u003e2.5\u003c/em\u003e, as well as \u003cem\u003eToLhca1.1\u003c/em\u003e/\u003cem\u003e2.1\u003c/em\u003e/\u003cem\u003e2.3\u003c/em\u003e/\u003cem\u003e2.2\u003c/em\u003e/\u003cem\u003e1.4\u003c/em\u003e/\u003cem\u003e1.2\u003c/em\u003e/\u003cem\u003e1.3\u003c/em\u003e, exhibited a downregulation trend. Among them, under no treatment (0 mg/L), the expression levels of these 9 \u003cem\u003eToLhc\u003c/em\u003e genes were the highest, and with the increase of stress concentration, the expression levels approached the lowest value at Cr\u003csup\u003e6+\u003c/sup\u003e concentration of 3 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Three genes, \u003cem\u003eToLhca1\u003c/em\u003e/\u003cem\u003e5.1/4.1\u003c/em\u003e, showed a trend of initially downregulation, then upregulation, and then downregulation again. The expression levels decreased at 0.1 mg/L but increased again at 1.5 mg/L, and they still had expression levels at a Cr\u003csup\u003e6+\u003c/sup\u003e concentration of 6 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). \u003cem\u003eToLhcb.1\u003c/em\u003e/\u003cem\u003e6\u003c/em\u003e and \u003cem\u003eToLhca3.1\u003c/em\u003e genes exhibited a downregulation trend, but still had corresponding expression levels at different Cr\u003csup\u003e6+\u003c/sup\u003e concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This shows that the way algae react to Cr\u003csup\u003e6+\u003c/sup\u003e stress may be influenced by \u003cem\u003eLhc\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Relative expression levels of \u003cem\u003eToLhc\u003c/em\u003e under different concentrations of Cr\u003csup\u003e6+\u003c/sup\u003e stress.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlgae are primary producers in aquatic ecosystems, typically existing as single cells or multicellular colonies (such as filaments or sheets). They lack differentiation into roots, stems, and leaves but possess the ability to perform photosynthesis\u003csup\u003e23\u003c/sup\u003e. The toxic effects of heavy metals on microalgae are manifested in several ways: they can affect algal growth and metabolism, inhibit photosynthesis, reduce cellular pigments, and induce cellular deformation\u003csup\u003e24\u003c/sup\u003e. One of the most vulnerable organisms to heavy metal pollution after it enters aquatic environments through various pathways is microalgae. Under heavy metal ion stress, photosynthesis is one of the important physiological indicators used to assess the extent of damage to plants\u003csup\u003e25\u003c/sup\u003e. Different plants and algae exhibit distinct metal accumulation patterns under heavy metal stress, leading to varying degrees of impact on chlorophyll production\u003csup\u003e26\u003c/sup\u003e. Chromium (Cr) is one of the heavy metal pollutants, and emissions from fuel combustion, wastewater, and waste residues containing chromium are the main sources of pollution in industrial development\u003csup\u003e27\u003c/sup\u003e. In aquatic environments, Cr\u003csup\u003e3+\u003c/sup\u003e and Cr\u003csup\u003e6+\u003c/sup\u003e are the most common forms of chromium, with Cr\u003csup\u003e6+\u003c/sup\u003e being difficult to degrade and possessing characteristics such as carcinogenicity and mutagenicity\u003csup\u003e28\u003c/sup\u003e. This experiment, through systematic identification and analysis of the \u003cem\u003eLhc\u003c/em\u003e genes in \u003cem\u003eT. obliquus\u003c/em\u003e, reveals their response patterns under different concentrations of Cr\u003csup\u003e6+\u003c/sup\u003e stress. The purpose is to assess and predict the ecological risk of heavy metal Cr\u003csup\u003e6+\u003c/sup\u003e pollution on aquatic organisms, providing the theoretical basis for the ecological impact assessment of Cr\u003csup\u003e6+\u003c/sup\u003e on algae. This study identified 33 \u003cem\u003eToLhc\u003c/em\u003e genes from \u003cem\u003eT. obliquus\u003c/em\u003e and classified them into 5 subfamilies based on phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), namely Lhca, Lhcb, CP269 (Lhcb5), CP24 (Lhcb6), and CP29 (Lhcb4). The research indicates that the majority of \u003cem\u003eToLhc\u003c/em\u003e genes are located in the chloroplast, with a smaller portion distributed in other cellular organelles. In maize (\u003cem\u003eZea mays\u003c/em\u003e), over 94% of \u003cem\u003eLhc\u003c/em\u003e genes are localized in the chloroplast, with the remaining genes found in the mitochondria. This suggests that \u003cem\u003eLhc\u003c/em\u003e proteins primarily play a role in the photosynthetic pathway\u003csup\u003e29\u003c/sup\u003e. The predictive analysis of this study indicates that among the members of the \u003cem\u003eToLhc\u003c/em\u003e family, 7 \u003cem\u003eToLhc\u003c/em\u003e genes are distributed in the cell membrane, 12 \u003cem\u003eToLhc\u003c/em\u003e genes are distributed in the chloroplast, 2 \u003cem\u003eToLhc\u003c/em\u003e genes are simultaneously distributed in both the chloroplast and the nucleus, and 3 \u003cem\u003eToLhc\u003c/em\u003e genes are simultaneously distributed in both the chloroplast and the cell membrane. Therefore, \u003cem\u003eToLhc\u003c/em\u003e genes may primarily function in the cell membrane and chloroplast. Genes on the same branch of the evolutionary tree display similar patterns, but there are notable differences between subfamilies, according to gene structure and conserved motif analysis. Compared to Lhcb, the gene structure of Lhca is more complex, containing a greater number of introns. The introns of genes \u003cem\u003eTaLhca5.3\u003c/em\u003e/\u003cem\u003e5.6\u003c/em\u003e/\u003cem\u003e2.2\u003c/em\u003e are relatively long, which may be related to the gene's expression levels\u003csup\u003e30\u003c/sup\u003e. In this investigation, the examination of diverse gene expression patterns demonstrated that the expression levels of three genes, \u003cem\u003eTaLhca5.3\u003c/em\u003e/\u003cem\u003e5.6\u003c/em\u003e/\u003cem\u003e2.2\u003c/em\u003e, were low or undetectable, supporting this observation. \u003cem\u003eLhc\u003c/em\u003e genes are typical light-dependent genes, playing a crucial role in light capture, transfer, and protection. They are essential for the photosynthetic process in plants\u003csup\u003e31\u003c/sup\u003e. The promoter's analysis cis-acting elements of \u003cem\u003eLhc\u003c/em\u003e genes in \u003cem\u003eT. obliquus\u003c/em\u003e in this study indicates that each upstream promoter region of \u003cem\u003eLhc\u003c/em\u003e genes in T. obliquus contains light-responsive elements. This suggests their crucial regulatory role in the expression of \u003cem\u003eLhc\u003c/em\u003e genes in response to light in \u003cem\u003eT. obliquus\u003c/em\u003e. The study reveals that the expression of \u003cem\u003eLhc\u003c/em\u003e genes is associated with various abiotic stresses. After exposure to CdS ODs for 10 days, the genes related to the Light-Harvesting Complex (LHC) in \u003cem\u003eT. obliquus\u003c/em\u003e were upregulated\u003csup\u003e8\u003c/sup\u003e. Overexpression of the non-gene Lhcb2 can enhance the tolerance of tobacco to low temperatures\u003csup\u003e32\u003c/sup\u003e. The phosphorylation of Lhcb proteins in spinach was markedly inhibited by NaCl treatment, and the sensitivity to NaCl changed in light and dark environments\u003csup\u003e33\u003c/sup\u003e. These hormone response elements are mostly associated with the plant's response to environmental stress. Heavy metals hurt the growth, enzyme activity, and photosynthetic activities of microalgae, particularly on PSⅠ and PSⅡ. This impact is dose-dependent, demonstrating a clear dose-response relationship\u003csup\u003e34\u003c/sup\u003e. The 33 \u003cem\u003eToLhc\u003c/em\u003e genes were subjected to qRT-PCR validation. The results indicate that under different concentrations of Cr\u003csup\u003e6+\u003c/sup\u003e stress, the expression levels of \u003cem\u003eToLhcb5\u003c/em\u003e/\u003cem\u003e2.6\u003c/em\u003e/\u003cem\u003e2.3\u003c/em\u003e/\u003cem\u003e2.4\u003c/em\u003e/\u003cem\u003e2.5\u003c/em\u003e and \u003cem\u003eToLhca1.1\u003c/em\u003e/\u003cem\u003e2.1\u003c/em\u003e/\u003cem\u003e2.3\u003c/em\u003e/\u003cem\u003e2.2\u003c/em\u003e/\u003cem\u003e1.4\u003c/em\u003e/\u003cem\u003e1.2\u003c/em\u003e/\u003cem\u003e1.3\u003c/em\u003e were highest under no treatment (0 mg/L), and then the expression levels showed a dose-response relationship. However, the gene expression levels of \u003cem\u003eToLhcb5\u003c/em\u003e/\u003cem\u003e2.6\u003c/em\u003e/\u003cem\u003e2.3\u003c/em\u003e/\u003cem\u003e2.4\u003c/em\u003e/\u003cem\u003e2.5\u003c/em\u003e and \u003cem\u003eToLhca1.1\u003c/em\u003e/\u003cem\u003e2.1\u003c/em\u003e/\u003cem\u003e2.3\u003c/em\u003e/\u003cem\u003e2.2\u003c/em\u003e/\u003cem\u003e1.4\u003c/em\u003e/\u003cem\u003e1.2\u003c/em\u003e/\u003cem\u003e1.3\u003c/em\u003e reached the lowest point at 3 mg/L. This may be related to the defense mechanism of \u003cem\u003eT. obliquus\u003c/em\u003e, reducing gene expression under stress to resist damage. In contrast, \u003cem\u003eToLhca1\u003c/em\u003e/\u003cem\u003e5.1\u003c/em\u003e/\u003cem\u003e4.1\u003c/em\u003e showed an increase at 1.5 mg/L, possibly due to a low-dose stimulation effect, and some expression levels were still detected at 3 mg/L, indicating partial recovery of photosynthetic capacity after adaptation for a certain period. For \u003cem\u003eToLhca1\u003c/em\u003e/\u003cem\u003e5.1\u003c/em\u003e/\u003cem\u003e4.1\u003c/em\u003e, there was no significant difference between 0 mg/L and 0.1 mg/L, indicating that the impact of low concentration on \u003cem\u003eT. obliquus\u003c/em\u003e is minimal. However, as the concentration increased, the expression levels showed a decreasing trend, and \u003cem\u003eToLhca3.1\u003c/em\u003e and \u003cem\u003eToLhcb1.1\u003c/em\u003e still exhibited partial expression at 6 mg/L.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study identified a total of 33 members of the \u003cem\u003eLhc\u003c/em\u003e gene family in \u003cem\u003eT. obliquus\u003c/em\u003e, distributed across its 14 chromosomes. The \u003cem\u003eLhc\u003c/em\u003e genes of \u003cem\u003eT. obliquus\u003c/em\u003e were grouped into 5 subfamilies in the phylogenetic tree, and genes in the same subfamily displayed conserved motifs and similar gene structures. The expression of \u003cem\u003eLhc\u003c/em\u003e genes showed a dose-response relationship under Cr treatment. It has been proposed that \u003cem\u003eLhc\u003c/em\u003e genes are involved in how algae react to heavy metal stress.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e \u003cb\u003eIdentification and Sequence Analysis of\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003eGenes in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThrough the NCBI (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) database, the full-genome protein sequences of Light-Harvesting Complex \u003cem\u003e(Lhc\u003c/em\u003e) in \u003cem\u003eT. obliquus\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and \u003cem\u003eChlorella vulgaris\u003c/em\u003e were retrieved. The PF00504 (Ethylene-insensitive 2) domain was downloaded using HMMER3.0, and validation was performed through Pfam (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://Pfam.xfam.org\u003c/span\u003e\u003cspan address=\"http://Pfam.xfam.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and InterProScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/InterProScan\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/InterProScan\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e35\u003c/sup\u003e. he protein sequences of \u003cem\u003eToLhc\u003c/em\u003e without the PF00504 (Ethylene-insensitive 2) domain, as well as those with redundancy, incompleteness, and annotations were removed\u003csup\u003e36\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003ePhylogenetic Analysis of\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003eGenes in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eToLhc\u003c/em\u003e genes were retrieved from \u003cem\u003eT. obliquus\u003c/em\u003e, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and \u003cem\u003eChlorella vulgaris\u003c/em\u003e. Multiple sequence alignment (MSA) was performed using ClustalW2, aligning the \u003cem\u003eToLhc\u003c/em\u003e protein sequences from \u003cem\u003eT. obliquus\u003c/em\u003e with those from \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eC. vulgaris\u003c/em\u003e\u003csup\u003e37\u003c/sup\u003e, The ITOL website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/login.cgi\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/login.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to visualize and annotate the phylogenetic tree, which was created using the neighbor-joining method. This allowed for the grouping and naming of \u003cem\u003eLhc\u003c/em\u003e genes according to their evolutionary relationships\u003csup\u003e38\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosomal localization analysis of the\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe GFF3 annotation file of the reference genome of \u003cem\u003eT. obliquus\u003c/em\u003e was downloaded from the NCBI database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). From the GFF3 file, gene structure annotations for members of the \u003cem\u003eToLhc\u003c/em\u003e family were extracted. The chromosomal distribution map was created using the start and end position information of these genes on the chromosomes with the MapInspect tool, followed by refinement and adjustments for visual clarity\u003csup\u003e39\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003eMotifs and Gene Structure in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eBased on the genome annotation information of \u003cem\u003eT. obliquus\u003c/em\u003e, the gene structure analysis diagram was drawn using TBtools. Next, the \u003cem\u003eLhc\u003c/em\u003e protein sequences were uploaded to MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://memesuite.org/index.html\u003c/span\u003e\u003cspan address=\"http://memesuite.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e40\u003c/sup\u003e and to find their conserved motifs, Smart Motif (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used\u003csup\u003e41\u003c/sup\u003e. The motif analysis was set with a motif quantity of 10 and a maximum width of 50. TBtools was employed to draw the Motif analysis diagram, achieving the visualization of conserved motif sequences\u003csup\u003e42\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eProtein Characteristics and Tertiary Structure Prediction of\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003eGenes in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe protein characteristics of \u003cem\u003eToLhc\u003c/em\u003e were analyzed using the protein analysis tool ExPASy Server10 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prosite.expasy.ory/PS50011\u003c/span\u003e\u003cspan address=\"https://prosite.expasy.ory/PS50011\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These properties include stability, average hydrophobicity (GRAVY), amino acid count, isoelectric point (pI), and relative molecular weight (MW). Furthermore, the relevant data were arranged and the subcellular localization of \u003cem\u003eLhc\u003c/em\u003e genes was predicted using the online tool Plant-mPLoc (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/cgibin/PlantPLoc.cgi\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/cgibin/PlantPLoc.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e43\u003c/sup\u003e. Homology modeling of \u003cem\u003eToLhc\u003c/em\u003e was performed using SWISS-MODEL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.swissmodel.expasy.org/\u003c/span\u003e\u003cspan address=\"https://www.swissmodel.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003csup\u003e44\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of cis-acting elements in the promoter regions of the\u003c/b\u003e \u003cb\u003eToLhc\u003c/b\u003e \u003cb\u003egene family in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo analyze the cis-acting elements in the promoter regions of the \u003cem\u003eToLhc\u003c/em\u003e gene family in \u003cem\u003eT. obliquus\u003c/em\u003e, the upstream sequences (1\u0026thinsp;~\u0026thinsp;1500 bp) of the \u003cem\u003eToLhc\u003c/em\u003e family members were manually extracted from the genome sequence. These sequences were then submitted to PlantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the identification of cis-acting elements in the promoter region\u003csup\u003e45\u003c/sup\u003e. The analysis results were organized and visualized using the R software package heatmap.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromium Toxicity Effects in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eT. obliquus\u003c/em\u003e was acquired from the Institute of Aquatic Biology, Chinese Academy of Sciences, Freshwater Algae Species Bank. The supplier of potassium dichromate (K\u003csub\u003e2\u003c/sub\u003eCr\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e7\u003c/sub\u003e) was Tianjin Kemiou Chemical Reagent Co., Ltd.; the remaining reagents were analytical grade. Algae cell culture and Cr\u003csup\u003e6+\u003c/sup\u003e exposure protocol: The algae cells were initially cultivated following the protocol outlined by Wen et al\u003csup\u003e46\u003c/sup\u003e. \u003cem\u003eT. obliquus\u003c/em\u003e in the logarithmic growth phase were inoculated into 250mL conical flasks. At the beginning of the experiment, the density of the algae cells was approximately 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e cells/mL (initial OD\u003csub\u003e680nm\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.087) with a total solution volume of 200 mL. Cr\u003csup\u003e6+\u003c/sup\u003e concentrations were 0 (CK), 0.1, 1.5, 3, 4.5, and 6 mg/L, and the experimental period was 96 hours\u003csup\u003e47\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRNA extraction and RT-qPCR analysis in\u003c/b\u003e \u003cb\u003eT. obliquus\u003c/b\u003e\u003c/p\u003e \u003cp\u003eUsing the TRizol reagent (GenStar, Beijing, China), total RNA was extracted, and the DNaseI enzyme (Vazyme, Nanjing, China) was used to remove DNA. RevertAid reverse transcriptase (Vazyme, Nanjing, China) was used to reverse transcribe RNA into complementary DNA (cDNA). After that, cDNA was diluted using water free of enzymes. Using Primer 5.0 software, create gene-specific primers with tub serving as the internal reference gene\u003csup\u003e48\u003c/sup\u003e. The RT-qPCR analysis was conducted according to the manufacturer's instructions (Vazyme, Nanjing, China). The quantitative real-time polymerase chain reaction was performed using the 2 \u0026times; SYBR Premix Extaq 10 \u0026micro;L forward and reverse primers, 0.4 \u0026micro;L each. Two milliliters (\u0026micro;L) of ddH\u003csub\u003e2\u003c/sub\u003eO were used to dilute the cDNA There are three steps in the program: Step 1 involves pre-denaturation at 95\u0026deg;C for 30 s; step 2 involves denaturation at 95\u0026deg;C for 5 s; and step 3 involves primer annealing, extension, and fluorescence signal collection at 60\u0026deg;C for 30 s; cycles of step 2 to step 3 were repeated 40 times. The relative expression level was calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method, and GraphPad Prism 8.0 software was used for differential significance analysis\u003csup\u003e49\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not include human or animal subjects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatement on guidelines\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental studies and experimental materials involved in this research are in full compliance with relevant institutional, national, and international guidelines and legislation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments (not compulsory)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this section, you can acknowledge any support given which is not covered by the author contribution or funding sections. This may include administrative and technical support, or donations in kind (e.g., materials used for experiments).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions statem\u003c/strong\u003e\u003cstrong\u003eent\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHailing Tan and Yonghua Ma conceived and designed the experiments;\u0026nbsp;Hailing Tan, Gaolei\u0026nbsp;Cai, Hui Tan and Yuhang Yin performed the experiment; Hailing Tan conducted the bioinformatics analysis and wrote the manuscript; Jun Yang, Fengxia Tan\u0026nbsp;and Yi Chai revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest. The funders had no role in the design\u0026nbsp;of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or\u0026nbsp;in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was partially supported by the Key Lab of Freshwater Biodiversity Conservation Ministry of Agriculture and Rural Affairs of China, Yangtze River Fisheries Research Institute(2024LFBC1113).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWang, S., Li, Q., Huang, S., Zhao, W., \u0026amp; Zheng, Z. Single and combined effects of microplastics and lead on the freshwater algae Microcystis aeruginosa. 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Postharvest Biology and Technology 136, 90\u0026ndash;98, DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.postharvbio.2017.10.013\u003c/span\u003e\u003cspan address=\"10.1016/j.postharvbio.2017.10.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (2018).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4429243/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4429243/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe process of photosynthesis depends heavily on the light-harvesting chlorophyll \u003cem\u003ea\u003c/em\u003e/\u003cem\u003eb\u003c/em\u003e-binding proteins (\u003cem\u003eLhc\u003c/em\u003e). However, to date, there has been a lack of systematic understanding of the \u003cem\u003eLhc\u003c/em\u003e gene family members in \u003cem\u003eT. obliquus\u003c/em\u003e. This study conducted a systematic identification and analysis of the \u003cem\u003eLhc\u003c/em\u003e family genes in \u003cem\u003eT. obliquus\u003c/em\u003e using bioinformatics. The findings show that 33 \u003cem\u003eToLhc\u003c/em\u003e genes in total, dispersed unevenly over 14 chromosomes, were found in \u003cem\u003eT. obliquus\u003c/em\u003e. Most \u003cem\u003eToLhc\u003c/em\u003e genes encode stable proteins, with the majority predicted to localize in the chloroplast. The most prevalent cis-acting elements were those linked to both biotic and abiotic stress responses, according to analysis.RT-qPCR analysis showed that all \u003cem\u003eToLhc\u003c/em\u003e genes were down-regulated under 6 mg/L Cr\u003csup\u003e6+\u003c/sup\u003e conditions, except for \u003cem\u003eToLhca1\u003c/em\u003e/\u003cem\u003e5.3\u003c/em\u003e and \u003cem\u003eToLhcb1.1\u003c/em\u003e, which maintained expression levels. This study systematically identified and characterized members of the \u003cem\u003eToLhc\u003c/em\u003e gene family in the green algae \u003cem\u003eT. obliquus\u003c/em\u003e. Additionally, it offered an initial comprehension of the expression patterns of 33 genes under Cr\u003csup\u003e6+\u003c/sup\u003e heavy metal stress. The aim was to assess and predict the ecological risk of heavy metal Cr\u003csup\u003e6+\u003c/sup\u003e pollution to aquatic organisms, and to offer a theoretical framework for assessing how Cr\u003csup\u003e6+\u003c/sup\u003e affects algae.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identification, Characterization and Expression Analysis of the Lhc Gene Family in Tetradesmus obliquus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-31 11:18:25","doi":"10.21203/rs.3.rs-4429243/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b34ae14a-a4ad-44cf-808d-ff857b43e268","owner":[],"postedDate":"May 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":32561365,"name":"Biological sciences/Molecular biology"},{"id":32561366,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2024-09-10T08:15:02+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-31 11:18:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4429243","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4429243","identity":"rs-4429243","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}