Genome-wide identification and expression analysis of the OSC gene family in Platycodon grandiflorus

Identification of OSC genes in Platycodon grandiflorus

The genome sequences of Platycodon grandiflorus downloaded from China National center for Bioinformation (https://www.cncb.ac.cn/), Accession number: GWHARYT00000000. The published protein sequence of OSC family members of Arabidopsis thaliana, Panax ginseng, Taraxacum mongolicum were downloaded from the National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/). Subsequently, OSC protein sequences from different species (Arabidopsis thaliana, Panax ginseng, Taraxacum mongolicum) were used as templates for sequence alignment in the genome of Platycodon grandiflorus, candidate OSC family genes were generated from the intersection of sequence alignment by ToolKit Biologists Tools (TBtools) software (https://github.com/CJ-Chen/TBtools). The identified PgOSC gene sequence information were listed in Table S2. The molecular formula, molecular weight, and isoelectric point were from Expasy (https://web.expasy.org/protparam/).

Phylogenetic analysis and classification of PgOSC genes in Platycodon grandiflorus

The OSC family protein sequences of A. thaliana (six), S. barbata (five), Platycodon grandifloras (15), T. coreanum (five), Panax ginseng (12) were downloaded from National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) and listed in Table S3. The multiple amino acid sequence alignment of OSC proteins was performed, and the phylogenetic tree was constructed by neighbor joining (NJ) method using ClustalW method implemented in Molecular Evolutionary Genetic Analysis software (MEGA 7). The generated tree was displayed using the iTOL website (https://itol.embl.de/upload.cgi).

Collinearity analysis and chromosomal mapping of the PgOSC family genes

The annotation information and the whole genome protein sequences of A. thaliana, Panax ginseng and T. mongolicum obtained from National Center for Biotechnology Information database (http://www.ncbi.nlm.nih.gov/). The collinearity relationships of the OSC genes between Platycodon grandiflorus and Rabidopsis thaliana, T. mongolicum and Panax ginseng genomes were determined and visualized by using the Multiple Collinearity Scan toolkit (MCScanX) of TBtools software with default parameters. The physical positions of PgOSC genes on the chromosomes of the Platycodon grandiflorus genome were determined and visualized using the TBtools software according to the genome annotation file.

Gene structure, motif analysis and cis-elements analysis

The whole genome sequences and CDS sequences of PgOSC genes were downloaded and subsequently utilized for in-depth gene structure analysis using the TBtools software. The motif analysis of the PgOSC family proteins was conducted utilizing the MEME website (http://meme-suite.org/tools/meme), while the domain analysis of the same protein family was performed through the Batch CD-Search tool available at (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi). The promoter sequences, encompassing 2000 base pairs upstream of the ATG start codon, were extracted from the PgOSC genes and subsequently analyzed for cis-elements using the Plant CARE platform (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The types and quantities of these cis-elements were listed in Table S4.

Protein three-dimensional structure prediction of PgOSC proteins

The three-dimensional structures of PgOSC proteins were precisely modeled utilizing the SWISS-MODEL interactive workspace software (https://swissmodel.expasy.org/interactive). To ensure the accuracy and rationality of the homologous modeling outcomes, the SAVESv6.0 platform (https://saves.mbi.ucla.edu/) was employed for a comprehensive assessment of the modeling results.

Plant treatment and quantitative RT-PCR

One-year-old Platycodon grandiflorus plants were utilized to assess the expression levels of the PgOSC genes across various tissues, encompassing the stem, leaf, root, and beard root. To investigate the expression patterns of the PgOSC genes under various stress conditions, one-year-old potted Platycodon grandiflorus plants were subjected to salt stress (100 mM NaCl), heat stress (37 °C for 2 h), and drought stress (2 weeks). Subsequently, the expression level of the PgOSC genes were analyzed to assess its response to these stress environments. Total RNA was extracted from Platycodon grandiflorus roots using RNAiso Plus (Takara, Kusatsu, Japan). The extracted RNA was ultimately dissolved in 30µL of DEPC-treated water. Subsequently, 1.5 µg of total RNA was utilized for cDNA synthesis, and dilutions of this cDNA were employed in real-time RT-PCR experiments. The expression levels of the tested genes were normalized to PgACTIN (a constitutively expressed gene). The gene expression level was calculated by the formula (E_control)${}_{\mathrm{control}}^{\mathrm{CT}}$/(E_Target)${}_{\mathrm{Target}}^{\mathrm{CT}}$ (Livak & Schmittgen, 2001).

Identification of the OSC Gene Family in the Platycodon grandiflorus Genome

To detect potential oxidized squalene cyclases (OSC) in Platycodon grandiflorus, we utilized two OSC protein sequences of Arabidopsis thaliana (AT5G48010, AT4G15370) as reference templates and aligned them against the protein database of Platycodon grandiflorus. As a result, we identified 17 promising candidate proteins for OSC in Platycodon grandiflorus, and consistent results were achieved when employing the OSC protein sequence of Panax ginseng or T. mongolicum as an alternative template for alignment, and two members with few amino acids were eliminated by subsequent gene structure and motif analysis. Consequently, we successfully identified 15 OSC members in Platycodon grandiflorus and provided an overview of their physicochemical properties in Table 1.

Despite a total of nine chromosomes in Platycodon grandiflorus, the PgOSC members are unevenly distributed among just five of them (Chr 1, Chr2, Chr 4, Chr 7, Chr 8). The lengths of the 15 identified PgOSC proteins to range from 611 to 847 aa, and their molecular weights range from 69.14 kDa (PgOSC15) to 96.45 kDa (PgOSC8). The isoelectric point ranged from 5.85 (PgOSC7) to 7.84 (PgOSC15), and the instability index ranged from 33.97 (PgOSC11) to 48.83 (PgOSC7), and the Aliphatic index ranged from 70.75 (PgOSC9) to 88.34 (PgOSC11). In addition, the hydrophilicity values of all PgOSC proteins are less than 0, indicating that all of them are hydrophilic proteins, and amino acid hydrophilicity of all PgOSC proteins were listed in Table S1.

Chromosomal location and collinearity analysis of OSC family genes in Platycodon grandiflorus

The distribution of PgOSC genes on nine chromosomes in Platycodon grandiflorus was predicted based on gene assembly information published previously. Not all chromosomes of Platycodon grandiflorus, PgOSC family genes are specifically distributed across only five chromosomes, including four OSC genes on chromosomes 2 and 8, five OSC genes on chromosome 4, and only one OSC gene each on chromosomes 2 and 7 (Fig. 1). Furthermore, PgOSC genes are predominantly distributed in the form of gene clusters on the chromosomes of Platycodon grandiflorus. The gene clusters on chromosomes 4 and 8 contain up to four paralogous OSC genes, whereas the two gene clusters situated on chromosome 1 comprise only two members each. The clustering distribution of PgOSC family genes implies their functional similarity, indicating that they may share common biological roles and regulatory mechanisms.

Phylogenetic relationship analysis of OSC proteins

To explore the phylogenetic relationships between the OSC proteins, a phylogenetic tree was constructed by MEGA7 software using OSC proteins sequence from A. thaliana (six), S. barbata (five), Platycodon grandifloras (15), T. coreanum (five), Panax ginseng (12), respectively. A total of 43 OSC protein sequences, originating from five diverse species, were categorized into four distinct subfamilies (Fig. 2). Four OSC proteins belonged to the subfamily I are all from A. thaliana, these may be related to the different evolutionary directions of A. thaliana and medicinal plants. Subfamily III contains the largest number of PgOSC proteins (six) and a small amount of proteins from other species. In addition, subfamily II encompasses 14 proteins, while subfamily IV comprises 16 proteins and the majority of which originate from Platycodon grandiflorus and Panax ginseng.

Collinearity analysis of OSC family members

To elucidate the collinear relationship among OSC family gene species, we conducted a collinearity analysis employing the genomes of A. thaliana, Panax ginseng, S. miltiorrhiza, and Platycodon grandiflorus. Within this analysis, the OSC family members were emphasized through the utilization of red lines. As shown in Fig. 3, There is no collinearity between the OSC genes of Platycodon grandiflorus and A. thaliana, suggesting that they may not have a common ancestral gene. However, both Panax ginseng and S. miltiorrhiza, renowned for their medicinal properties, exhibit a distinct collinear relationship with Platycodon grandiflorus, such as PgOSC15 (PGRA_01400, Chr 2) in Platycodon grandiflorus and EVM0005942.1 (Chr 5) and EVM0039524.1 (Chr 12) in Panax ginseng; PgOSC9 (PGRA_07650 Chr 4) in Platycodon grandiflorus and EVM0056770.1 (Chr 3) and EVM0062132.1 (Chr 19)in Panax ginseng. These findings suggest that the OSC family genes of medicinal plants, including Platycodon grandiflorus, Panax ginseng, and S. miltiorrhiza, are more intimately related in the evolutionary process compared to those of A. thaliana. Unfortunately, hampered by the limitations of incomplete genomic data available in public databases, we were unable to carry out an intra-species collinearity analysis of Platycodon grandiflorus.

Analyses of gene structures and conserved motifs of PgOSC genes

In order to reveal the structural characteristics of PgOSC family genes, we analyzed the exon–intron structures and the conserved motifs of 15 PgOSC genes using the MEME online tool and TBtools software. As shown in Fig. 4A, All 15 PgOSC proteins shared seven common motifs (motif 1 to motif 7), Motif 8 is present in all PgOSC gene family, with the exception of pgosc15. The exon-intron structures of PgOSC genes were analyzed by the CDS and genome sequences. As shown in Fig. 4B, The OSC family genes of Platycodon grandiflorus typically exhibit intricate gene structures, characterized by diverse combinations of numerous exons and introns. The length of PgOSC family genes surpasses 10 kb, excluding PgOSC2, PgOSC7, and PgOSC10. The extended intron structure accounts for the augmented gene length and implied that these genes may undergo more extensive differentiation and accumulate longer-term variations, thereby elevating the recombination frequency during OSC gene replication. This phenomenon is conducive to the evolution of organisms. However, given the intricate nature of the gene structure, it is imperative to take into account the quality of the database. This necessitates the corroboration of sequencing results from diverse sources to ensure the reliability of the data.

Prediction of the three-dimensional structures of PgOSC proteins

In order to further study the characteristics of tertiary structure of OSC protein in Platycodon grandiflorus, we conducted homology modeling analysis using the SWISS-MODE online tool. As shown in Fig. 5, The fundamental structure of OSC protein comprises a small β-fold block that is enclosed within two dense clusters of α-helices and the irregular curl shuttling through it seem to maintain the stability of the entire structure. Within the PgOSC family, significant variations in the number of central β-sheets exist among different members, potentially influencing the functional properties of these proteins. Furthermore, the stereoscopic structure of PgOSC12 and PgOSC13 distinctly differs from other members in Platycodon grandiflorus, consisting of a unique complex that integrates four fundamental PgOSC structures. Determining whether this distinctive architecture indeed bestows PgOSC12 and PgOSC13 with superior catalytic efficiency necessitates meticulous experimental validation.

Prediction of subcellular localization of PgOSC family proteins

To obtain the predictive information of subcellular localization of PgOSC family proteins in Platycodon grandiflorus, we utilized the protein sequence of PgOSC to predict its subcellular localization using the WOLF PSORT website. The subcellular localization prediction of PgOSC family members are shown by heatmap. As shown in Fig. 6, Through clustering analysis, it becomes evident that PgOSC proteins exhibit three distinct patterns of subcellular distribution within cells. One pattern involves localization in mitochondria and peroxisomes, encompassing PgOSC1, PgOSC4, PgOSC13. Another pattern is found in the cytoplasm and nucleus, with high credibility in prediction results, such as PgOSC2, PgOSC5, PgOSC7 PgOSC11, PgOSC12, PgOSC15. Lastly, the remaining PgOSC family members are distributed across chloroplasts, vacuoles, Golgi apparatus, and other organelles.

Analysis of cis-acting elements of PgOSC promoter

Cis-elements are important for gene transcription, to acquire information of the Cis-elements on PgOSC gene promoters , a 2000 bp DNA sequence upstream of each gene translation start site extracted from the Platycodon grandiflorus genome database were used to make a prediction through the PlantCARE website (Fig. 7). A moderate level of adversity can promote the accumulation of secondary metabolites. We found that the PgOSC family contains a rich set of cis-acting elements that respond to drought, low temperature, and hypoxia, which are beneficial for the accumulation of triterpenoids. In addition, hormone response elements involved in auxins, abscisic acid, gibberellins, and jasmonic acid indicate that hormones remain important factors in regulating the growth and development of Platycodon grandiflorus and the accumulation of secondary metabolites. Details of cis-acting elements in the promoter region of PgOSC genes shown in Table S4.

Expression pattern analysis of PgOSC genes

To ascertain the tissue expression pattern of PgOSCs, we employed qRT-PCR to assess the expression levels of PgOSC family genes across leaf, steam, root, and root beard of Platycodon grandiflorus. As expected, the expression level of PgOSC family genes was higher in the roots of Platycodon grandiflorus than in its stem, leaf and root beard tissues. There was no significant difference in the expression of OSC family genes between stems and leaves (except PgOSC8) (Fig. 8) . These results suggest that The root of Platycodon grandiflorus may function as the primary site for triterpenoids synthesis, which aligns with the utilization of the root specifically for medicinal purposes, rather than other tissues of the plant.

Moderate stress conditions can stimulate the accumulation of secondary metabolites in plants.To clarify the relationship between the synthesis of triterpenoids in Platycodon grandiflorus and stress conditions, we subjected one-year-old plants to various types of stress treatments, including heat, drought, and salt. Subsequently, we used qRT-PCR to detect changes in the expression of PgOSC genes. Excitingly, salt stress emerged as a prominent factor, exhibiting a robust induction effect on most genes of the PgOSC family (excluding PgOSC3, PgOSC8, PgOSC15) (Fig. 9). This result suggests that salt stress effectively promotes the accumulation of triterpenoids in Platycodon grandiflorus. In addition, drought stress specifically induced the expression of PgOSC14 and PgOSC15.