Abstract
Pyropia yezoensis is an important economic macroalga widely cultivated in the East Asia countries of China, Korea, and Japan. The ATP-binding cassette (ABC) transporter gene family is one of the largest transporter families in all forms of life involved in various biological processes. The characteristics of ABC transporter genes in P. yezoensis (PyABC) and their functions in stress resistance, however, remain largely unknown. In this study, PyABCs were identified and characterized their expression patterns under low-temperature stress. A total of 48 PyABCs transporters were identified and divided into eight subfamilies, which are mostly predicted as membrane-binding proteins. The cis-elements of phytohormone and low-temperature response were distinguished in promoter sequences of PyABCs. Transcriptome analysis showed that PyABCs are involved in response to low-temperature stress. Among them, 12 PyABCs were significantly up-regulated after 24 h of exposure to low temperature (2 °C). Further quantitative RT-PCR analysis corroborated the highest expression happened at 24 for detected genes of PyABCC8, PyABCF3, and PyABCI1, extraordinarily for PyABCF3, and followed by decreased expression at 48 h. The expression of PyABCI1 was generally low in all tested strains. Whereas, in a strain of P. yezoensis with lower tolerance to low temperature, the expression was observed higher in PyABCC1, PyABCC8, and remarkably high in PyABCF3. This study provided valuable information on ABC gene families in P. yezoensis and their functional characteristics, especially on low-temperature resistance, and would help to understand the adaptive mechanisms of P. yezoensis to adverse environments.
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Introduction
ATP-binding cassette (ABC) transporters are one of the largest protein families discovered so far among living organisms. They are involved in a variety of biological processes (Liu 2019). Most of their members are membrane-binding proteins located in multiple subcellular structures such as plasma membrane, chloroplast, mitochondria, vacuoles, and peroxisome. They can use the energy released by ATP hydrolysis to transport a variety of compounds across membranes and regulate a variety of important physiological activities in organisms (Henikoff et al. 1997; Theodoulou and Kerr 2015).
There are eight subfamilies of ABC family found in plants with seven of these, namely ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, and ABCG also found in other organisms, and one of plant-only subfamily ABCI (Hung et al. 1998; Theodoulou 2000; Rea 2007; Verrier et al. 2008; Shao et al. 2013). Basically, ABC transporters contain two domains of the nucleotide binding domain (NBD) and the transmembrane domain (TMD). Subfamilies of ABCA to ABCD possess a forward TMD–NBD structure, while subfamily ABCG encodes a reverse NBD–TMD organization. The ABCE and ABCF subfamilies have two NBDs with no TMD, and ABCI has only a single NBD or accessory domains.
Most ABC transporters are membrane-binding proteins responsible for the transmembrane transport of various materials, such as hormones, secondary metabolites, heavy metal ions, and other substances (Davidson et al. 2008; Theodoulou and Kerr 2015; Liu 2019). Plant ABC transporters are involved in regulating plant growth and development as well as in biological and abiotic stress and heavy metal tolerances and in other biological processes helping plants to survive under harsh environmental conditions (Theodoulou 2000; Shao et al. 2013; Lane et al. 2016). Compared with animals and microorganisms, plant ABC transporters have a large number of members, among which there are more than 120 ABC transporter family members in Arabidopsis thaliana and Oryza sativa (rice). This was considered to be a result of plant evolution to cope with the complex living environment and to adapt to a variety of metabolic and signal transmission pathways in vivo (Higgins 1992; Sanchez-Fernandez et al. 2001; Garcia et al. 2004; Wang et al. 2017). Algae, a group of much older yet simpler organisms, were revealed to have fewer unique members of ABC transporters than the vascular land plants (Lane et al. 2016). For instance, in Chlamydomonas reinhardtii, an algal model species, only 75 ABC transporters were well identified (Li et al. 2022b).
ABC transporters are widely involved in plant stress resistance, including exposure to drought, low or high temperature, heavy metals, high salt, and other stresses (Theodoulou 2000; Rea 2007; Verrier et al. 2008; Shao et al. 2013). A large number of studies have been conducted on the stress response mode of ABC transporters, and the function of some members has been identified in model organisms such as A.thaliana and O. sativa (Higgins 1992; Garcia et al. 2004). Some members of the ABC transporter family have been shown to participate in plant response to low-temperature stress. This response may be related to their substance transport functions and their promoter elements that work especially through hormone signal transduction to mobilize the expression of ABC transporter genes to maintain homeostasis in plants (Marinoia et al. 2002; Nagy et al. 2009; Kuromori et al. 2010; Xu et al. 2012; Wang et al. 2017; Niu et al. 2021). In algae, expression patterns of ABC transporter family in stress response have not been studied except in C. reinhardtii Li et al. 2022b). Based on transcriptomic analysis, green algae, red algae, and bryophytes were found to have significantly more ABCF subfamily members that function in antibiotic and stress resistance (Lane et al. 2016).
Pyropia yezoensis is an important economic macroalga that grows naturally in the intertidal zone and is widely cultivated in coastal areas in the Western North Pacific (Blouin et al. 2011; Bito et al. 2017). Thalli of P. yezoensis grow naturally and are artificially cultured during cold seasons from late autumn through winter, till the following early spring. Their ability to tolerate cold temperatures appears to be a key factor affecting their growth and yield. Understanding the cold tolerance mechanism of P. yezoensis is therefore critical and forms an important basis for its successful genetic breeding and strain improvement under the background of global climate change.
Based on our previous transcriptome data of P. yezoensis under low-temperature stress, we found an enrichment of ABC transporter genes involved in environmental information processing (Ma et al. 2024). In the present study, these ABC transporter genes were further identified and analyzed based on their genomic information and expression profile using transcriptome and fluorescence quantitative real-time PCR (qRT-PCR) to explore the possible function of these ABC transporters during algal response to low temperature.
Materials and Methods
Genome-Wide Identification and Characterization of ABC Family
High-quality Pyropia yezoensis reference genome assemblies and protein sequences were obtained from our laboratory (Wang et al. 2020). The Hidden Markov Model (PF00005) of ABC_tran secondary structure of ABC transporter was downloaded from the Pfam database (http://pfam-legacy.xfam.org). The HMMER 3.0 for Windows was used to search for ABC in P. yezoensis genome (Finn et al. 2011). The protein sequences of ABC transporter in Arabidopsis thaliana were downloaded from TAIR database (https://www.arabidopsis.org/index.jsp), and its homologous sequences in P. yezoensis were searched by Blast 2.13.0 program. To ensure credibility, the E-value of two searching methods was set at 10−5. The common genes screened by the two methods were identified as candidate genes for ABC transporter genes of P. yezoensis (PyABC). Conserved domain prediction was carried out in NCBI Batch CD-Search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and SMART (https://smart.embl.de).
The number of amino acids, molecular weights, theoretical isoelectric points (pI), instability indexes, aliphatic index, and grand averages of hydropathicity of all predicted PyABC transporters were calculated using ExPASy (https://web.expasy.org/protparam) (Artimo et al. 2012). Subcellular protein localization was predicted using WoLF PSORT, Yloc, Cell-PLoc 2.0, BUSCA, and MULocDeep (Yu et al. 2006; Briesemeister et al. 2010; Chou et al. 2010; Lin and Hu 2013; Savojardo et al. 2018).
Classification and Phylogenetic Analysis
The ABC protein sequences of A. thaliana, Chlamydomonas reinhardtii, and P. yezoensis were aligned using the MEGA 11-MUSCLE program with default parameters. ML tree was inferred by IQ-TREE 2.1.3 (Minh et al. 2020), and the best-fit substitution model was automatically selected using ModelFinder implemented in IQ-TREE (Kalyaanamoorthy et al. 2017). Branch support was calculated using ultrafast bootstrap approximation with 1000 replicates (Hoang et al. 2018). The phylogenetic tree was visualized by Evolview (http://www.evolgenius.info/evolview/#/treeview).
Gene Structure, Conserved Motifs, and Conserved Domain Analysis
TBtools and NCBI Batch CD-Search (https://www.ncbi.nlm.nih.gov) were applied to analyze and visualize gene structure and conserved domains of PyABC transporters (Marchler-Bauer et al. 2015; Chen et al. 2020). The conserved motifs of PyABC transporters were identified using MEME (https://meme-suite.org/meme/tools/meme) (Bailey et al. 2015). Ten motifs were set as the maximum number, others using default parameters.
Analysis of Cis-Acting Regulatory Elements in PyABC Genes Promoters
The upstream 2 kbp promoter sequences of PyABC were extracted using TBtools (Chen et al. 2020) and were submitted to PlantCARE (Lescot et al. 2002). The location and number of detected cis-acting regulatory elements were displayed using TBtools.
Chromosome Localization and Collinearity Analysis
Chromosomal locations of PyABC were visualized using the Gene Location Visualize from GTF/GFF tool of TBtools. The collinearity relationship of ABC transporter between P. yezoensis and P. haitanensis was carried out by One Step MSCcanX-Super Fast tool of TBtools (Cao et al. 2020; Wang et al. 2012), and the output was imported into Dual Systeny Plot tool for visualization.
Low-Temperature Expression Pattern Analysis of PyABC Genes
Expression at Different times Under Low Temperature
Based on the transcriptome data of P. yezoensis (PY440, purified strain of our Laboratory of Algae Genetics and Breeding, Ocean University of China) under low-temperature of 2 °C at 0 h, 2 h, 6 h, 24 h, and 48 h from our previous study (Ma et al. 2024), heat map of PyABC expression under different low-temperature exposure times was made by HeatMap tool in TBtools based on FPKM values (Chen et al. 2020). PyABC genes with higher FPKM and significantly up-regulated expression (Log2 FC ≥ 1 and FDR < 0.01) were randomly selected for further verification. The qRT-PCR, using HiScript III RT SuperMix for qPCR Kit (Vazyme, China) and SYBR Green qPCR Mix (ABclonal, China), was performed on four selected PyABC genes under the same condition of low-temperature stress at different times (0, 2, 6, 24, 48 h). The 2−ΔΔCt method was applied to calculate the relative expression levels with an internal control using β-actin gene (Kong et al. 2015). The selected genes and their specific primers are shown in Table 1. Each qRT-PCR was repeated at least three times.
Expression Among Different Strains Under Low Temperature
To further investigate the expression pattern of PyABC, qRT-PCR was carried out on four genes in five strains of P. yezoensis following the same protocol described above. The performances of five strains NY57, NY79, NY84, NY319, and NY446 under 3d low temperature (2 °C) were examined by growth rate and photosynthetic efficiency compared to suitable temperature (10 °C). The daily growth rate (DGR) and photosynthetic parameters of Fv/Fm (maximum PSII quantum yield), ΦPSII (effective PSII quantum yield), and NPQ (non-photochemical quenching) were derived following our previous works (Du et al. 2022; Zhong et al. 2023; Ma et al. 2024).
Prediction of Protein Structure of Up-regulated PyABC Genes
To assist the analysis on the potential function of PyABC, protein tertiary structure and transmembrane helix of 12 significantly up-regulated genes at 24 h of low-temperature treatment were predicted using SWISS-MODEL (https://swissmodel.expasy.org) and TMHMM-2.0 (https://services.healthtech.dtu.dk/service.php?TMHMM-2.0).
Statistical Analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the t-test to compare the differences among treatments using IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA).
Results
Identification of PyABC Genes
Forty-eight ABC transporters were identified from P. yezoensis (Supplementary Table S1) by protein sequence alignment and hidden Markov model search. Physicochemical property analysis showed significant differences among the family members, with amino acid sequences ranging from 149 aa to 1823 aa and molecular weight ranging from 14.90 to 185.46 kDa. Among them, PyABCC1 has the smallest amino acid numbers (149) and molecular weight (14.9), and PyABCA2 has the largest amino acid numbers (1823) and the largest molecular weight (185.46). The pI of PyABCs ranged from 4.57 to 11.02, the instability index ranged from 26.47 to 49.41 and the aliphatic index ranged from 81.56 to 117.35. The grand averages of hydropathicity ranged from −0.492 to 0.653, implying that most PyABCs are hydrophobic proteins. The grand averages of hydropathicity of subfamilies ABCE and ABCF were less than 0, indicating a stronger hydrophilicity. The prediction of subcellular localization showed that most PyABCs were located in the plasma membrane and chloroplast, while some were located in the membrane of other organelles and cytoplasmic matrix. This predication is consistent with the property of ABC family as being basically membrane-binding protein.
Phylogenetic Analysis of PyABC Transporters
Based on the phylogenetic tree, 48 PyABCs were divided into eight subfamilies whose members clustered with the same subfamily of plant such as model species A. thaliana and microalga C. reinhardtii, namely 2 ABCAs, 15 ABCBs, 9 ABCCs, 2 ABCDs, 1 ABCE, 8 ABCFs, 8 ABCGs, and 3 ABCIs (Fig. 1). Other than subfamily ABCI which was relatively dispersed, the ABCs of each species basically clustered together exhibiting certain homology. Subfamilies ABCG, ABCB, and ABCC have more members, making up 33.3%, 20.5%, and 13.6% of the total number, respectively. The members of PyABC subfamilies ABCB, ABCC, and ABCG mostly clustered together respectively, whereas those of other subfamilies were separated and clustered with those of A. thaliana and C. reinhardtii.
Conserved Domain, Conserved Motif, and Gene Structure of PyABCs
There are at least two conserved motifs in the PyABC transporters (Fig. 2a). The number and location distribution of conserved domains of PyABC showed all members to contain at least one conserved ABC_tran nucleotide-binding domain (Fig. 2b). In membrane binding domain, ABC2_membrane is unique to subfamily ABCG, ABC_ membrane2 exists only in subfamily ABCD, ABC3_ membrane3 only in subfamilies ABCA and ABCI, and ABC_membrane only in subfamilies ABCB and ABCC. In addition, ABC_tran_Xtn exists only in subfamily ABCF. The subfamilies ABCA and ABCF have the AAA_21 domain, and some members of subfamilies ABCB, ABCC, and ABCI contain the AAA_29 domain. The distribution of the domain is similar in the same subfamily. The gene structure of PyABC indicated all members contain introns, and the number of exons varied from 2 to 10, among which PyABCA2 has the largest number of exons (Fig. 2c). The number and length of exons within the same subfamily are diverse.
Conserved Motifs are further illustrated in Fig. 3. Motif 4 is present in all family members with a sequence that is similar to Walker B canonical sequence (ILLDE). Most PyABCs contain at least one of Motif 2 or Motif 7, both having ABC characteristic motif (LSGGQ). Motif 1 and Motif 8 contain Walker A typical sequence (GxxGxGKST), which also exists in most PyABCs. Other conserved motifs are mainly found in subfamilies ABCB and ABCC.
Cis-acting Elements of PyABC Gene Promoter
The promoter region of PyABC contains various cis-acting elements (Fig. 4), which are most related to hormone response. All of PyABC contain numbers of methyl jasmonate and light response elements. Except for PyABCA2, all PyABCs contain abscisic acid response elements. Some members also have cis-acting elements related to MYB transcription factors, salicylic acid, and environmental stress, among which 20 family members have 1~3 low-temperature responsive elements distributed in subfamilies ABCA ~ ABCG.
Chromosomal Localization and Collinearity Analysis
As shown in Fig. 5, 48 PyABC genes were mapped to three chromosomes of P. yezoensis. The number of PyABC genes of chromosomes 1, 2, and 3 is 21, 11, and 16, respectively, with the PyABC genes evenly distributed in chromosomes 1 and 2. Two densely located regions appeared on chromosome 3, which only contained members of subfamilies ABCB, ABCC, ABCF, and ABCG. No Tandem duplication or Segmental duplication was found in all PyABC genes, and only PyABCG4 and PyABCG5 were Proximal duplicate genes on chromosomes.
Blast comparison was conducted between the genomes of P. yezoensis and P. haitanensis, and a collinearity relationship was established based on the comparison results and annotation information (Fig. 6). As shown in Fig. 7, there are 34 pairs of ABC transporter genes with collinearity relationship, accounting for about 70% of PyABC genes. There is only a one-to-one relationship between collinearity genes, indicating that the evolution of ABC genes in both Pyropia species was conservative.
Expression Pattern of PyABC Under Low Temperature
Expression of PyABC at Different Durations of Low-Temperature Exposure
The PyABCs retrieved from transcriptome data of samples exposed to low-temperature stress for different durations was analyzed for their expression pattern at different times, taking FPKM ≥ 1 as the threshold value (Fig. 7). Among a total of 48 PyABCs, six genes were unexpressed, including four members of subfamily ABCB, one member of subfamily ABCC and one member of subfamily ABCF. Among those expressed PyABCs, the most increased expression was detected under low temperature. Clustering analysis showed that these 48 PyABC genes could be divided into five groups: Group I showed decreased or no expression; Group II showed the expression level was basically unchanged in the first 6 h, and increased after 24 h; Group III, the expression level firstly decreased within 6 h and increased after 24 h; Group IV, the expression level increased within 6 h, decreased after 24 h, and then fluctuated slightly; Group V, the overall expression level increased within 48 h, with some genes showing slight fluctuation. The genes which expressed significant differentially were defined by Log2 FC ≥ 1 and FDR < 0.01. There were 6, 9, 12, and 13 genes up-regulated significantly at 2 h, 6 h, 24 h, and 48 h, respectively, and 3, 4, 2, and 1 genes down-regulated significantly respectively with compared to before treatment (Fig. 7). The similar expression patterns were presented between 2 and 6 h and between 24 and 48 h.
The expression of the four selected PyABCs exhibited a similar trend but at a relatively different level over the treatment times (Fig. 8). Increased expression response to low-temperature treatments of 2, 6, and 24 h was detected in all these PyABCs examined. However, the response decreased under 48 h treatment. During 2 h to 24 h, the expression of PyABCC1 was relatively higher at 2 and 6 h (p < 0.05); whereas for the other three genes, the highest level of expression was exhibited at 24 h and especially for PyABCF3 which reached an extraordinary highest level of expression (p < 0.05) (Fig. 8).
Expression of PyABCs in Different Strains of P. yezoensis Under Low-Temperature Treatments
The qRT-PCR results of the four selected PyABCs examined in five strains of P. yezoensis showed different degrees of increased gene expression levels for individuals under the 24 h low-temperature treatment (Fig. 9). The expression levels of PyABCC1 and PyABCF3 under low temperature in five tested strains were more than twice of that of the control (10 °C, p < 0.05). This is especially so for PyABCF3 which reached the highest level in strain NY319. On the other hand, the level of PyABCC11 expression was generally low in all these five strains except for NY319 and NY446 which exhibited a relatively increase under low temperature. The strain NY319 had higher expression in three genes of PyABCC1, PyABCC8, and PyABCF3 than other strains, extremely in PyABCF3.
The performance of five strains to low-temperature stress was exhibited on growth rate and photosynthetic activities compared with suitable temperature (Fig. 10). Among them, the strains NY 319 presented extremely higher sensitivity to low temperature than other strains, with remarkably lower growth rate, Fv/Fm, ΦPSII, and NPQ under low temperature compared to suitable temperature, which also severer than other four strains.
Prediction of Protein Structure for Differently Expressed PyABCs
Transcriptome analysis showed an increase in the expression levels of most PyABC genes under 24 h of low-temperature stress with the levels of increase being significantly different. The protein tertiary and transmembrane helical structures of the 12 up-regulated PyABC genes of 24 h were therefore predicted. Results of the protein tertiary structure prediction showed that most PyABC transporters belonged to subfamily ABCC, and all of them contained different amounts of α helices that make up the structure of transport proteins in PyABCB4, PyABCB15, PyABCC3, PyABCC5, and PyABCC7 (Fig. 11). These genes contain different number of transmembrane helices (TMH). However, no TMHs were predicted for PyABCC1, PyABCF3, and PyABCI1 genes (Table 2).
Discussion
Characteristics of PyABC Gene
Although ABC gene is recognized as one of the largest transporter families in all forms of life, few studies have been carried out to evaluate its basic bioinformation and functions in algae. In our previous transcriptome analysis on P. yezoensis response to low-temperature stress, ABC transporters were annotated by KEGG pathway enrichment of differentially expressed genes. To further explore their characteristics and functions, ABC genes in P. yezoensis (PyABC) were identified and analyzed in the present study. A total of 48 PyABC transporters were identified in P. yezoensis, which is less than the 120 genes identified in A. thaliana and O. sativa. However, these transporter genes in P. yezoensis belong to eight subfamilies which are also found in higher plants (Theodoulou 2000; Rea 2007; Kang et al. 2011).
The physicochemical properties of PyABC transporters showed that there are high variabilities in the molecular weight size, acidity, and basicity of their family members. The prediction of fat coefficient, grand averages of hydropathicity, and subcellular localization indicated that most members are membrane-binding proteins and are relatively stable. This is of great significance to these ABC transporters as they have been shown to play important function roles of material transport and maintenance of homeostasis in vivo to cope with environmental changes (Martinoia et al. 2002; Nagy et al. 2009; Xu et al. 2012; Niu et al. 2021). ABC transporters have also been found to be involved in chlorophyll synthesis and thylakoid membrane formation, which is important for the maintenance of chloroplast normal structure that can therefore affect photosynthesis (Zeng et al. 2017). In the present study, several PyABCs were found to be located in chloroplasts, indicating that they may have important effects on chloroplast formation and photosynthesis in the algal species as well.
Based on analysis of the conserved motifs and conserved domains of PyABC transporters, it was found that some members have special domain combinations. For example, “TMD-NBD-TMD” domain combinations existed in subfamilies ABCB, ABCC, and ABCG. Similar combination pattern was also found in tomato, maize, and other plant species, suggesting that they may be the structural basis of specific physiological functions in the evolutionary process (Kang et al. 2011; Pang et al. 2013; Amoak et al. 2018). There are also some ABCs that do not contain transmembrane domains, such as PyABCE which is the only member of subfamily ABCE. It is without membrane-binding domains and contains only two ATP-binding domains. The protein sequence of PyABCE is basically consistent with that of the yeast RNAaseL inhibitor, which may participate in the RNAi process for translation regulation (Bisbal et al. 1995).
Gene expression under stress is often regulated by upstream regulatory factors. Cis-acting elements, as the sites interacting with regulatory factors, are extremely important for the regulation of gene transcription initiation (Yamaguchi-Shinozaki and Shinozaki 2005). In the present study, prediction results of cis-acting elements of PyABC gene promoters suggest that nearly half of PyABC members were found containing various amounts of low-temperature responsive elements CCCAAA-motif. In transcriptome analysis, it was found that PyABC members with CCCAaA-motif had different expressions and participated in low-temperature response. However, the presence or absence of corresponding promoter elements could not be the only basis for determining whether they participated in such response. Li et al. (2022a) found that in the potato StCRKs gene family, some genes lacking low-temperature response elements could also respond to low-temperature stress signals. In the present study, not all differentially expressed PyABC genes were predicted having low-temperature response elements. Nonetheless, they basically contained methyl jasmonate response elements, light response elements, and abscisic acid response elements. In recent years, some cold stress-related studies revealed that various regulatory mechanisms such as transcription, epigenetic and post-translational modification of hormone signals, light signals, and biological rhythms can all be involved in plant response to low-temperature stress (Fowler and Thomashow 2002; Ding et al. 2019). Therefore, the differential expression of some PyABC genes under low-temperature stress might be due to the interaction between cis-acting elements related to hormone signal regulation in promoters and their corresponding hormones.
In terrestrial ABC transporters, there are more members of subfamilies ABCB, ABCC, and ABCG, where tandem repetition and chromosome fragment replication play a role in family member expansion (Hwang et al. 2016). In the present study, the above three subfamilies accounted similarly for the largest proportions of ABC transporters in P. yezoensis, but no obvious expansion of the above subfamily genes was found in gene chromosome localization and collinearity analysis. Following the phylogenetic relationship of ABC in A. thaliana, C. reinhardtii, and P. yezoensis, it could be found that subfamilies ABCB, ABCC, and ABCG showed aggregation of members of the same species but not with the other species, indicating that their sequences were highly similar within the species, but different with those of the other species. Compared with higher plants, algae have more members belonging to subfamily ABCF. In the present study, 8 ABCFs were identified in macroalga P. yezoensis, which are more than those 5 ABCFs of A. thaliana. Same as in the microalga C. reinhardtii, 11 ABCFs were found. It suggested that ABCF members may play an important role in algae adaptation to aquatic environments (Lane et al. 2016; Murina et al. 2019; Li et al. 2022b). ABC transporter subfamily ABCI not only plays a function role of material transport in plants, but also acts as a component of a variety of protein complexes (Ming et al. 2004; Rayapuram et al. 2007; Verrier et al. 2008). In the present study, members of the subfamily ABCI are divided into multiple branches in the phylogenetic tree, indicating that their genetic sequences and structures are changeable, corresponding to their functional diversity.
Expression Pattern of PyABC Under Low Temperature
Transcriptome analysis revealed that ABC transporters showed different response modes under low-temperature stress, and most members showed an overall up-regulated expression trend within 48 h of low-temperature treatment. It is worth noting that only a few genes showed significant differences in their expression levels within 6 h of low-temperature stress, but a large number of members showed up-regulated expression at 24 h of low-temperature stress. This indicates that the PyABC transporters take a certain time to respond. This may be due to the time delay in the regulation of genes by related response pathways in vivo (Sargood et al. 2022). Especially for the ABCF subfamily members which are known to participate in translation regulation for antibiotic and stress resistance (Lane et al. 2016; Li et al. 2022b), with PyABCF3 exhibiting an extraordinarily highest expression at 24h than any of the other genes examined in the present study. In Arabidopsis, ABCF3 gene were discovered its functions in response under abiotic stresses (Faus et al. 2021). It implies the important potential role of PyABCF subfamily in the response of P. yezoensis to harsh intertidal environments. The tertiary structure and transmembrane helical prediction of PyABC genes that exhibited significant up-regulated expression under 24 h low-temperature exposure showed that some members, such as PyABCB4, PyABCB15, PyABCC3, PyABCC5, and PyABCC7, could form the typical structure of membrane binding proteins that corresponds to their material transport function. While non-helical members like PyABCC1, PyABCF3, and PyABCI1, which contain only ATP-binding domains, might take up the function in translation regulation in detoxification or in forming protein polymers (Theodoulou 2015; Lane et al. 2016; Liu 2019).
The qRT-PCR results showed the expression levels of four selected genes at different stress time under low temperature compared to suitable temperature, which strongly supports the transcriptomic revelation. However, the expressions of four genes were different among strains. In strain NY319, the expression levels of PyABCC1, PyABCC8, and PyABCF3 genes were significantly higher than those in the other strains, especially for PyABCF3. On growth and photosynthesis, the strain NY319 also exhibited the highest sensitivity to low temperature than other strains. It indicated high expression of PyABCC1, PyABCC8, and especially PyABCF3 usually happens in strains with lower tolerance to low temperature. It also implied that the PyABCF3 gene plays important functional roles in the response of P. yezoensis to low-temperature stress.
Conclusions
This study explored the PyABC gene and its role in low-temperature tolerance in the red alga P. yezoensis, providing a reference for understanding its response mechanism to environmental stress. A total of 48 putative ABC transporter genes in this alga were identified, and divided into eight subfamilies. Physicochemical properties and subcellular localization prediction indicated that most of these members tend to form membrane-binding proteins. In the prediction of the promoter sequence of PyABC, cis-acting elements including hormone response elements and low-temperature response elements were found. Transcriptome analysis showed that ABC transporters were involved in the response to low-temperature stress. Most of the transporters showed an up-regulated expression trend after 24 h of low-temperature stress, and 12 genes were significantly up-regulated compared with those before treatment. The expression of PyABCI1 was generally low in all tested strains. The genes of PyABCC1, PyABCC8, and especially PyABCF3 expressed higher in strain NY319 with lower tolerance to low-temperature. All these results suggest that PyABC genes play an important function in the low-temperature tolerance of the intertidal red alga P. yezoensis.
Data Availability
No datasets were generated or analysed during the current study.
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Acknowledgements
We thank Profs. Put O. Ang of Chinese University of Hong Kong and Fu-Li Liu of Ocean University of China for providing comments on the draft version of the manuscript.
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This study was financially supported by the National Key R&D Program of China (2023YFD2400101, 2022YFD2400105), Jiangsu Shuangchuang Rencai Project (JSSCRC2021432), and Jiangsu Yancheng City “Yellow Sea Pearl” Lingjun Rencai Project.
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Lin Tian: Data Curation, Writing-Original Draft, Validation Lan Wu: Data Curation, Writing-Original Draft, Investigation Xue-Feng Zhong: Resource, Methodology, Formal analysis Lin-Hong Ma: Investigation, Software, Visualization Guo-Ying Du: Conceptualization, Writing-Review & Editing, Project administration, Supervision All authors reviewed the manuscript.
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Tian, L., Wu, L., Zhong, XF. et al. Genome-Wide Characterization of ABC Transporter Genes and Expression Profiles in Red Macroalga Pyropia yezoensis Expose to Low-Temperature. Mar Biotechnol (2024). https://doi.org/10.1007/s10126-024-10355-3
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DOI: https://doi.org/10.1007/s10126-024-10355-3