Genome-wide identification and analysis of YTH gene family and its response to MeJA and salt treatment in Panax ginseng C. A. Meyer

Genome-wide identification and analysis of YTH gene family and its response to MeJA and salt treatment in Panax ginseng C. A. 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A. Meyer Ting Yang, Yiming Sun, Wanqing Yang, Yadong Zhuang, Tengfei Qin, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7523802/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted 13 You are reading this latest preprint version Abstract YTH domain-containing RNA-binding proteins function as m 6 A readers that specifically bind to m 6 A-modified RNAs. YTH domain-containing proteins participate in various biological processes, such as hormone signaling pathways, regulation of stress responses, RNA stability, and cellular differentiation. Despite these important roles, the characteristics and functions of YTH family genes in ginseng ( Panax ginseng ), a traditional medicinal herb, particularly regarding their response to MeJA treatment and salt stress on a genome-wide scale, have not yet been studied. In this study, 18 YTH genes were identified based on telomere-to-telomere reference genome of ginseng. These PgYTH genes were grouped into four subgroups by phylogenetic analysis. Moreover, the chromosomal distribution, synteny analysis, gene structures and cis -elements of PgYTH genes, and the motifs of YTH proteins were analyzed. Expression profiling results indicated that the PgYTH genes were tissue-specific and spatiotemporally-specific in 14 different tissues of 4-year-old ginseng, in ginseng roots of four different ages, and among 42 different cultivars of 4-year-old ginseng roots. The expression of the majority of PgYTH genes was downregulated in response to MeJA, an elicitor of the ginsenoside biosynthesis pathway. The expression of PgYTH8-12 was upregulated under salt treatment. Additionally, PgYTH12 was localized to the endoplasmic reticulum. Overall, these results lay the groundwork for future functional investigations of PgYTH genes, advancing our understanding of their role in the regulation of the ginsenoside biosynthesis pathway and stress resistance in ginseng. YTH Panax ginseng m6A RNA methylation gene family Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Ginseng ( Panax ginseng C. A. Mey.) is one of the most valuable traditional Chinese medicinal materials in China, often referred to as the "King of Herbs," and it possesses significant medicinal value. Similar to changes in DNA epigenetic modifications, typical components of RNA, such as adenine (A), uracil (U), guanine (G), and cytosine (C), also undergo a series of chemical modifications. These modifications can occur either during transcription or after transcription has been completed, and they persist throughout the entire lifespan of most RNA molecules [ 1 ]. RNA modifications are crucial for the characteristics and functions of RNA, affecting stability, translation, splicing, and transport, which in turn increase the functional versatility and diversity of RNA molecules [ 2 ]. To date, over 170 different RNA modifications have been identified [ 3 ]. In RNA molecules, adenosine (A) is methylated by methyltransferases, where the hydrogen atom at the sixth nitrogen position (N6) is replaced by a methyl group (CH3). The changes in m 6 A methylation are primarily regulated by various factors, including m 6 A methyltransferases, demethylases, and m 6 A recognition proteins. m 6 A modifications have been detected in several plants, including Arabidopsis and rice [ 4 ]. Additionally, proteins associated with m 6 A modifications have been progressively identified in plants. In Arabidopsis, the m 6 A methyltransferase complex consists of mRNA adenosine methyltransferase A (MTA) [ 5 ], MTB, FKBP12 interacting protein 37 (FIP37) [ 6 ], VIRILIZER (VIR) [ 7 ], and HAKAI [ 8 ], while m 6 A demethylation is regulated by ALKBH9B and ALKBH10B [ 9 ]. Research indicates that m 6 A modifications play significant regulatory roles in processes such as fruit ripening, abiotic stress responses, and secondary metabolite synthesis in plants. m 6 A methylation plays an essential regulatory role in the processes of fruit ripening and quality metabolism in kiwifruit. Overexpression of AcALKBH10 greatly elevates the soluble sugar content in the fruit while markedly reducing the accumulation of acids, whereas silencing AcALKBH10 exhibits the opposite trend [ 10 ]. In apples, sorbitol significantly affects the m 6 A methylation of mRNAs by controlling the expression of two m 6 A methyltransferases, MdVIR1 and MdVIR2. This RNA modification is essential for the resistance against Alternaria alternata, a pathogen controlled by sorbitol [ 11 ]. Additionally, m 6 A-mediated regulatory mechanisms coordinate the accumulation of terpenoid metabolites and the formation of tea aroma during the sunlight wilting stage of tea leaves [ 12 ]. m 6 A exerts its regulatory functions primarily by recruiting specific m6A-binding proteins known as "readers." In mammals, various m 6 A-binding proteins have been identified, with the most extensively studied being the family of proteins containing the YT521-B homology (YTH) domain [ 13 ], which is categorized into YTHDC1, YTHDC2, and YTHDF1-3 subfamilies. These proteins recognize the m 6 A modification through a conserved YTH domain characterized by a "hydrophobic pocket" formed by two or three aromatic residues necessary for binding to the m 6 A site [ 14 ]. YTHDF1, YTHDF2, YTHDF3, and YTHDC2 are all cytoplasmic m 6 A readers that function by binding to mature mRNAs containing m 6 A. In contrast, YTHDC1 is localized in the nucleus and selectively binds pre-mRNA containing m 6 A [ 15 ]. The m 6 A readers play a broad regulatory role in RNA transcription-related processes, including alternative splicing, mRNA stability, and translation. Thirteen YTH proteins have been identified in Arabidopsis , which are classified into two evolutionary clades: YTHDF and YTHDC [ 16 ]. Eleven genes (AtECT1-11) belong to the YTHDF clade, while AtCPSF30 and AtECT12 (AtDC1) belong to the YTHDC clade [ 17 ]. In recent years, members of the YTH family have been successively identified in plants such as rice [ 18 ], tomato [ 19 ], and wheat [ 20 ] through bioinformatics approaches. However, The YTH proteins in ginseng have not yet been identified. The functional role of m 6 A reader YTH proteins in response to hormone and abiotic stress in Panax ginseng has not yet been reported. In this study, a total of 18 PgYTH genes were identified in ginseng and classified into four subgroups, with detailed analyses of their chromosomal distribution and collinearity, gene structures, cis -acting elements, and protein motifs. We also examined the expression profiles of these PgYTH genes. The PgYTH gene family was found to respond to MeJA and salt treatments. These findings provide a foundation for further investigation into the roles of YTH genes in response to hormone and abiotic stress. 2. Methods 2.1 Identification and characterization of physiochemical properties of YTH genes in ginseng The gene annotation and telomere-to-telomere reference genome files of ginseng from Jilin Province, China, were retrieved based on the data availability in the previous study [ 21 ]. Protein sequences of YTH genes from Arabidopsis and rice were sourced from the TAIR database ( https://www.arabidopsis.org/ ) [ 22 ] and Rice Genome Annotation Project ( http://rice.plantbiology.msu.edu/index.shtml ), respectively. Candidate YTH gene sequences were aligned against the protein sequences of ginseng using the blastp algorithm. The presence of the YTH domain in the candidate genes was verified using the NCBI Conserved Domain Database (CDD). Confirmation of the identified YTH genes was further conducted using the Hmmsearch function [ 23 ]. The physicochemical properties, including relative molecular mass (MW) and theoretical isoelectric point (PI), were calculated utilizing the ExPASy proteomics server [ 24 ]. Additionally, the chromosomal locations of the YTH genes were extracted from the annotation files and visualized using TBtools [ 25 ]. 2.2 Evolutionary, gene structure, and conserved motif analysis of Pg YTH s The identified YTH protein sequences from Jilin ginseng, Arabidopsis , and rice were aligned using the MUSCLE algorithm implemented in MEGA-X. Phylogenetic tree construction was performed using the neighbor-joining (NJ) method in MEGA-X, employing the Poisson model with 1,000 bootstrap replicates. The resulting phylogenetic tree was visualized and enhanced using Evolview. Collinearity analysis of YTH genes in ginseng was conducted using TBtools. Conserved motifs within the ginseng YTH protein sequences were identified and analyzed using the MEME program. The gene structures were visually displayed using TBtools. 2.3 Chromosomal distribution and synteny analysis of Pg YTHs Ginseng reference genome annotation files from Jilin Province, China, were used to determine the chromosomal distribution of YTH genes [ 21 ]. The chromosomal distribution of ginseng YTH genes was drafted from top to bottom by TBtools according to gene positions in the genome annotation. 2.4 Regulatory elements analysis of Pg YTHs The 2000 bp region upstream of the coding sequence (CDS) of YTH genes was extracted with TBtools and designated as the promoter region. Putative cis -regulatory elements within these promoter regions were identified using PlantCARE [ 26 ]. The distribution and organization of these elements were then visualized with TBtools. 2.5 Subcellular Localization of PgYTH12 The full-length coding sequence of PgYTH12 was amplified using specific primers PgYTH12-GFP-F (5’-ACTATTTACAATTACGGATCatggctaccgttgctcctcgg-3’) and PgYTH12-GFP-R (5’-TCCTCGCCCTTGCTCACCATagatcctcctccagatcctcctc-3’), and subsequently cloned into the vector pBWA(V)H2STMVΩ-3xflag-ccdB-egfp. The resulting recombinant plasmid, pBWA(V)H2STMVΩ-PgYTH12-GFP, was introduced into Agrobacterium tumefaciens strain GV3101, which was then used to infiltrate fully expanded leaves of Nicotiana benthamiana . Expression of GFP in the infiltrated cells was visualized using a confocal laser scanning microscope (Nikon C2-ER) with argon laser excitation at 488 nm. 2.6 Expression profiles analysis of PgYTHs To investigate the expression patterns of YTH genes in ginseng, RNA-Seq datasets were retrieved from NCBI (accession number PRJNA302556). These datasets include samples from 14 different tissues of 4-year-old ginseng, ginseng roots at four different growth stages, and roots of 42 different cultivated varieties of 4-year-old ginseng (Table S1 ) were collected from Jilin, China [ 27 ]. Transcript assembly and expression quantification were conducted through a pipeline involving Hisat2, StringTie, Kallisto. 2.7 The PgYTH genes response under MeJA treatment in ginseng To explore the potential involvement of the PgYTH gene family in hormone response, ginseng adventitious roots were treated with MeJA. The adventitious roots (1 g) of ginseng were inoculated in 250 mL triangular flasks containing 150 mL of liquid B5 medium and incubated in a shaker at 22℃, 110 rpm for 21 days. On the 22nd day, 200 mM MeJA was added to the culture flasks, and this time point was designated as 0 h of treatment. Samples were collected at 0, 6, 12, 24, 48, and 72 hours after treatment, with three biological replicates for each time point. The 0 h samples served as the untreated control group. All collected samples were immediately frozen in liquid nitrogen and stored at -80°C for further analysis. Adventitious roots subjected to MeJA treatment were induced from Jilin ginseng plants (variety named “Fuxing No. 2”) originating in Jilin Province, China. All ginseng plant materials were stored in Medical Laboratory Testing Technology and Analytical Laboratory, Beihua University. 2.8 The PgYTH genes response under salt treatment in ginseng To investigate the potential role of the PgYTH gene family in salt stress response, ginseng adventitious roots were subjected to NaCl treatment. Adventitious roots measuring 1 cm in length were cultured on B5 medium supplemented with varying concentrations of NaCl (0 mM, 70 mM, 80 mM, 90 mM, and 100 mM) at 25°C for 30 days. After treatment, the samples were rapidly frozen in liquid nitrogen before being stored at -80°C for subsequent gene expression analysis. The adventitious roots used for salt treatment were induced from Jilin ginseng plants (variety named “Fuxing No. 2”) sourced from Jilin Province, China. All ginseng plant materials were stored in Medical Laboratory Testing Technology and Analytical Laboratory, Beihua University. 2.9 Quantitative Real-Time PCR (qPCR) analysis Total RNA from Jilin ginseng (variety named “Fuxing No. 2”) was extracted using the TRIzol method. Subsequently, the Super RT III Kit (Biosharp Biotech) was used to reverse-transcribe the RNA into cDNA. Primers for qPCR were synthesized by Sangon Biotech (Shanghai, China), with their sequences provided in Supplementary Table S2 . The β-actin gene served as the internal reference. qPCR assays were conducted using the SYBR Premix Ex Taq™ II (Tli RNaseH Plus) kit (TaKaRa). Each experiment was performed in triplicate for every sample group, and the relative expression levels were calculated using the 2 −ΔΔCT method [ 28 ]. 2.10 Liquid–Liquid Phase Separation (LLPS) Prediction The prion-like domain (PrLD) and intrinsically disordered region (IDR) associated with LLPS in PgYTH proteins were predicted using the PLAAC tool, which employs a hidden Markov model (HMM) algorithm. 3. Result 3.1 Genome-wide identification of ginseng YTH genes In total, we identified 18 YTH genes in Panax ginseng (Table 1 ). The complete information for these genes, including the gene ID, number of amino acids (aa), molecular weight (MW), isoelectric point (pI), location and subcellular localization were analyzed. The lengths of the corresponding YTH proteins ranged from 436 aa (PgYTH7) to 720 aa (PgYTH11). The pI exhibited variability, spanning from 5.13 (PgYTH6) to 9.23 (PgYTH5). Additionally, the MW of these proteins was found to range approximately from 49.4 kDa (PgYTH7) to 79.8 kDa (PgYTH1). The subcellular localization prediction results indicated that 4 (PgYTH1, PgYTH2, PgYTH7, and PgYTH16) out of the 18 proteins were localized in the nucleus, while the remaining 14 were exclusively localized in the cytoplasm. Table 1 Characteristics of YTH genes and their encoded proteins in ginseng. Gene Name Gene ID Number of Amino Acid Molecular Weight (MW) isoelectric point (pI) Location Subcellular Localization PgYTH1 pg_2000603.t01 718 79775.36 6.12 Chr02:6144965–6159906(+) Nucleus PgYTH2 pg_5003162.t01 455 51297.58 7.6 Chr05:34521511–34528108(-) Nucleus PgYTH3 pg_5013137.t01 600 65984.59 5.19 Chr05:159669959–159681112(+) Cytoplasm PgYTH4 pg_7001001.t01 516 57747.22 5.6 Chr07:9116362–9123416(-) Cytoplasm PgYTH5 pg_8011634.t01 552 61306.36 9.23 Chr08:157638510–157645259(+) Cytoplasm PgYTH6 pg_11012144.t01 609 67004.63 5.13 Chr11:168557286–168570003(+) Cytoplasm PgYTH7 pg_12002421.t03 436 49447.37 8.03 Chr12:25766424–25773204(-) Nucleus PgYTH8 pg_12010946.t01 600 65955.74 5.14 Chr12:148177398–148189627(+) Cytoplasm PgYTH9 pg_13012092.t01-1 700 76689.87 6.69 Chr13:159000112–159003943(+) Cytoplasm PgYTH10 pg_13012092.t01-2 711 78013.78 7.94 Chr13:159006392–159011254(+) Cytoplasm PgYTH11 pg_14000049.t02 720 79627.76 7.24 Chr14:500279–505414(-) Cytoplasm PgYTH12 pg_14000050.t01 705 77160.4 6.52 Chr14:507290–511491(-) Cytoplasm PgYTH13 pg_14009897.t01 609 67009.61 5.24 Chr14:128673911–128685027(+) Cytoplasm PgYTH14 pg_15008119.t01 552 61095.98 9.08 Chr15:110326382–110333089(+) Cytoplasm PgYTH15 pg_16000974.t01 524 58769.19 6.08 Chr16:7869738–7876491(-) Cytoplasm PgYTH16 pg_17000373.t01 712 78999.64 6.16 Chr17:3182577–3197335(-) Nucleus PgYTH17 pg_21010025.t01 716 78401.42 6.3 Chr21:124088321–124093891(-) Cytoplasm PgYTH18 pg_23008814.t02 716 78322.42 6.3 Chr23:92385522–92391328(-) Cytoplasm 3.2 Phylogenetic analysis of YTH genes A phylogenetic tree was constructed with YTH protein sequences from ginseng, Arabidopsis , and rice to analyze the evolutionary relationships among YTH genes (Fig. 1 ). In our study, the YTH proteins of ginseng were classified into two families, DC and DF, based on previous studies in other plant species. The DC group contains 4 members: PgYTH1, PgYTH2, PgYTH7, and PgYTH16. The DF group was further divided into 3 subfamilies (DFA, DFB, and DFC). PgYTH9-12 were classified into subfamily DFA. PgYTH3/6/8/13 were classified into subfamily DFB. PgYTH5/14/15/17/18 were classified into subfamily DFC (Fig. 1 ). 3.3 Motifs, conserved domain and gene structure analysis An unrooted phylogenetic tree was constructed to investigate the evolutionary patterns and classification of YTH genes in ginseng. We examined the conserved motifs of the YTH proteins and the gene structures of the identified YTH genes. The MEME program was employed to analyze the organization of conserved motifs in ginseng YTH proteins, resulting in the detection of a total of 10 distinct motifs. These genes contained 4 to 9 motifs. Notably, motifs 1, 2, 3, and 5 were present in YTH genes. PgYTH9-12 contain all motifs except motif10. PgYTH3/6/8/13 contain all motifs except motif10. YTH family members that cluster within a clade share similar motif compositions (Fig. 2 A). The YTH domains in the candidate genes were verified through analysis in the NCBI CDD. The YTH domains of most YTH genes are located near the 3' end. The YTH domains of PgYTH2 and PgYTH7 are located near the 5' end. The YTH domains are in the middle of PgYTH1 and PgYTH16 (Fig. 2 B). To evaluate the consistency of exon-intron patterns among YTH genes, we performed a comprehensive gene structure analysis. The results revealed that the number of exons in the YTH gene family of ginseng ranged from 7 to 10. The analysis of exon and intron arrangements offered valuable insights into the evolutionary relationships among different members of this gene family. Notably, genes that are closely related displayed a higher degree of structural similarity, with variations primarily observed in the lengths of their introns and exons. A significant positive correlation was identified between phylogenetic relationships and exon-intron structures (Fig. 2 C). 3.4 Chromosome distribution and collinearity analysis of YTH genes Considering that ginseng is an allotetraploid plant, the previous study categorized the ginseng chromosomes into two subgenomes (subgenomes A and B) [ 21 ]. The BLASTN search was conducted to determine the chromosomal distribution of YTH genes in ginseng. The PgYTH genes were found to be roughly evenly distributed across both subgenomes of ginseng. The ginseng YTH gene is located on chromosome Chr03A-07A, Chr10A and Chr12A as well as chromosome Chr03B-07B and Chr10B. Three YTH genes (PgYTH11-13) are anchored on chromosome Chr04B. With the exception of four genes (PgYTH9-12), the chromosomal locations of the remaining genes are largely conserved between subgenome A and subgenome B (Fig. 3 A). These results imply that divergent evolutionary pressures have likely shaped the evolution of ginseng. To further investigate the phylogenetic mechanisms of the ginseng YTH gene family, we performed an intraspecific collinearity analysis of PgYTH genes. A total of 19 pairs of colinear PgYTH genes were identified (Fig. 3 B). To explore the potential selective pressure on PgYTHs , the Ka (non-synonymous) and Ks (synonymous) substitution rates, along with the Ka/Ks ratios, were analyzed. The Ka values for the PgYTH family genes ranged from 0.005 to 0.150, the Ks values for the PgYTH family genes ranged from 0.015 to 0.401, and the Ka/Ks ratios varied between 0.206 and 0.716 (Table S3 ). The results reveal that PgYTH has mainly undergone purifying selection throughout its evolution, as all PgYTH gene pairs exhibited Ka/Ks values below 1. 3.5 cis -regulatory element analysis of YTH genes Understanding the cis -regulatory elements that control YTH gene expression is crucial for elucidating their regulatory mechanisms and potential functions in ginseng. To this end, we extracted the 2000 bp region upstream of the start codon of each ginseng YTH gene as its promoter region for further analysis. A total of 469 cis -regulatory elements, representing 17 different types, were identified in the promoter regions of PgYTH genes (Fig. 4 A). Among them, light-responsive elements were the most abundant, accounting for nearly half of the total number (224) (Table S4 ). Stress-responsive regulatory elements were also identified, including those associated with anaerobic environments, MYB binding sites involved in drought stress inducibility, low-temperature stress, wound response, mixed stress conditions, and anoxia (Fig. 4 B). A total of 85 environmental stress-related cis -acting elements were identified. Over 70% of these elements were anaerobic induction elements and drought-responsive MYB binding sites, highlighting their dominant roles in stress response (Fig. 4 B). In addition, several hormone regulatory sites were identified, including those responsive to MeJA, abscisic acid (ABA), gibberellin (GA3), auxin, and salicylic acid (SA). The distribution of cis -acting elements in the promoter regions suggests that PgYTH genes may be primarily regulated by MeJA and ABA (Fig. 4 C). Furthermore, cis -acting elements related to zein, meristem, endosperm, palisade mesophyll cells and circadian were also detected. 3.6 Expression patterns of ginseng YTH genes Gene expression patterns are strongly associated with gene functions. To better understand the expression patterns of PgYTH genes in ginseng, we obtained expression data for PgYTH transcripts across 42 farm cultivars (S1–S42) (Table S1 ), 14 different tissues, and ginseng roots of four varying ages (5, 12, 18, and 25 years). The expression levels of PgYTH9-12 in ginseng roots of four different ages were significantly higher than that of other PgYTH genes (Fig. 5 A). The PgYTH genes exhibited dynamic and tissue-specific expression patterns throughout these organs, reflecting their diverse functional roles in ginseng development and metabolism. PgYTH1-8 and PgYTH13-18 exhibited generally low expression levels across all ginseng tissues. Compared to other PgYTH genes, PgYTH12 exhibited higher expression levels across various tissues, indicating its potentially broader or more significant functional role in ginseng. Moreover, PgYTH12 showed the highest expression level in the main root cortex (Fig. 5 B). Furthermore, PgYTH12 exhibited the highest expression level across 42 farm cultivars (Fig. 5 C), indicating that this gene may play an important role in the root of ginseng. 3.7 Expression analysis of PgYTH genes under MeJA treatment in ginseng MeJA was found to be involved in the regulation of ginsenoside biosynthesis. qRT-PCR was conducted to determine the expression profiles of the PgYTH genes under MeJA treatment. The qRT-PCR analysis showed a predominant downregulation trend across most members of this gene family. Among the 18 PgYTH genes examined, only PgYTH2 , PgYTH14 , PgYTH15 , and PgYTH16 showed no downregulation (Fig. 6 ), indicating that these genes may be governed by different regulatory mechanisms or have functions that are less affected by MeJA signaling. The expression levels of PgYTH3 , PgYTH4 , PgYTH10 , PgYTH12 , PgYTH13 , PgYTH17 , and PgYTH18 were significantly decreased compared to the control at all time points post-treatment, suggesting that they may serve as key components in the MeJA signaling pathway. 3.8 Expression analysis of PgYTH genes under salt treatment in ginseng Ginseng growing in natural environments is influenced by multiple stress factors, and gene expression patterns in these specific conditions are commonly utilized to study gene functions. qRT-PCR was used to verify the response of PgYTH genes to salt stress. Ginseng adventitious roots were treated with different concentrations of salt (0, 70, 80, 90, and 100 mM NaCl) in B5 medium for 30 days. As the salt concentration increased, the growth of ginseng adventitious roots was increasingly inhibited (Fig. 7 A). Compared to the control, the expression levels of all genes except PgYTH8-12 remained largely unchanged following salt treatment (Fig. 7 B). All PgYTH genes within the DFA subfamily ( PgYTH9-12 ) demonstrated a significant upregulation trend under salt treatment, suggesting that these genes may have crucial roles in the molecular mechanisms governing salt stress tolerance. In addition, the PgYTH8 gene, which belongs to the DFB subfamily, also exhibited a significant upregulation trend under salt treatment. These results suggest that while many PgYTH genes remain stable under salt stress, a targeted subset is actively engaged, highlighting functional differentiation within the PgYTH gene family in adapting to salt stress. 3.8 Subcellular Localization of PgYTH12 To investigate the subcellular localization of PgYTH12 and its potential function in roots, we fused the coding sequence of PgYTH12 to pBWA(V)H2STMVΩ-3xflag-ccdB-egfp vector and transiently expressed it in expanded leaves of N. benthamiana . Transient expression assays revealed that GFP signals in the GFPPgYTH12-expressed N. benthamiana leaves were observed in the endoplasmic reticulum (ER) (Fig. 7 ). These results suggested that PgYTH12 may be involved in ER-associated processes, such as protein synthesis, folding, and stress responses. 3.9 Liquid–Liquid Separation of PgYTH Proteins The PrLD domain has been demonstrated to facilitate the liquid–liquid phase separation of proteins. The online tool Prion-like Amino Acid Composition (PLAAC) has proven to be an effective method for identifying prion-like domains (PrLDs) in proteins. In this study, we utilized PLAAC to predict prion domains within PgYTH proteins to assess their potential for phase transition. PgYTH proteins from different subfamilies exhibit varying numbers of highly disordered PrLDs. The DFA subfamily includes three PrLDs, indicating a strong propensity for phase separation. The DFB subfamily contains one to two PrLDs. Within the DFC subfamily, four PgYTH proteins have one PrLD each, while two proteins lack PrLDs. In the DC subfamily, two PgYTH proteins contain three PrLDs, and two proteins have none. This indicates that PgYTH proteins, especially those belonging to the DFA subfamily, might undergo phase separation similarly to YTHDF1–YTHDF3 proteins of human. 4. Discussion m 6 A RNA methylation is the most prevalent reversible modification found on eukaryotic RNAs and is one of the earliest reported chemical modifications, with its abundance varying across developmental stages and different tissues [ 29 ]. The recognition of m 6 A-modified RNAs is largely mediated by reader proteins containing the YTH domain, which features an aromatic cage that specifically binds to the GG(m 6 A)C sequence [ 30 ]. In this study, we identified 18 PgYTH genes distributed across 2 subgenomes of ginseng. This number contrasts with previous findings of 13 YTH genes in Arabidopsis [ 31 ], 12 in rice [ 18 ], 26 in apple [ 32 ], 10 in Camellia chekiangoleosa [ 33 ], 53 in alfalfa [ 34 ], and 10 in Ginkgo biloba [ 35 ]. Such variation in YTH gene family size among species suggests that the m 6 A-YTH regulatory system has undergone species-specific expansion or contraction, likely reflecting adaptations to distinct biological needs or environmental pressures. In Arabidopsis , ECT2, ECT3, and ECT4 exhibit significant redundant regulatory functions in leaf growth, organogenesis, and trichome branching. Additionally, ECT2/ECT3/ECT4 collaborate by directly recruiting poly(A) binding proteins, which enhances the stability of target genes and regulates ABA responses [ 36 ]. In recent years, bioinformatics approaches have identified members of the YTH family in rice, tomato, apple, and other plants. OsYTH3 , OsYTH5 , and OsYTH10 are m 6 A readers in rice that are highly expressed across various tissues, showing significant protein homology with AtECT2. The three genes OsYTH3/5/10 regulate rice plant height through functional redundancy. Further transcriptomic analysis indicates that the reduced height of yth03/05/10 triple mutant plants may result from disrupted pathways for terpenoid and brassinosteroid synthesis [ 18 ]. This highlights a shared mechanism by which YTH proteins regulate growth-related processes via m 6 A-mediated RNA recognition. In tomato, SlYTH2 is highly homologous to Arabidopsis ECT2, ECT3, and ECT4. SlYTH2 possesses the ability to recognize m 6 A and promotes the formation of protein-RNA condensates through liquid-liquid phase separation, thus enabling precise regulation of molecules related to fruit aroma quality [ 37 ]. In our study, PgYTH9-12 were classified into subfamily DFA together with AtECT2/3/4 and OsYTH3/5/10 . This conserved classification indicates that PgYTH9-12 could contribute to key developmental and physiological processes, possibly including growth regulation and secondary metabolite production. Gene expression patterns are closely related to gene functions. In C. chekiangoleosa , Solanum lycopersicum, and strawberry, the Y TH genes, belonging to the YTHDFA subfamily, show significantly higher expression compared to other genes in different tissues [ 33 , 38 , 39 ]. In Salvia miltiorrhiza , the SmYTH3 gene, which is classified into the YTHDFA subfamily, exhibited significantly higher expression levels in roots compared to other tissues [ 40 ]. In this study, PgYTH9–12 , which were also classified into the YTHDFA subfamily, showed significantly higher expression in roots of varying ages, suggesting that genes in the YTHDFA subfamily may be particularly important for root development and function throughout the plant's lifespan. Among the PgYTH family, PgYTH12 , stood out due to its consistently elevated expression across diverse tissues, the main root cortex, and a wide range of farm cultivars. This broad and robust expression pattern points to a potentially central role for PgYTH12 in coordinating key physiological and developmental processes in ginseng. Ginsenoside, a secondary metabolite of ginseng, is a triterpenoid saponin exhibit significant therapeutic effects in areas such as anti-tumor activity, anti-aging, immune enhancement, and neuroprotection [ 41 ]. Recent studies have shown that saponin synthesis is not only directly regulated by transcription factors such as MYB and WRKY [ 42 , 43 ], but also influenced by epigenetic modifications (such as DNA methylation and histone methylation,) that dynamically regulate chromatin structure and gene transcription activity. These modifications may serve as critical regulatory nodes in the synthesis of secondary metabolites [ 44 ]. Understanding the regulatory mechanisms underlying ginsenoside biosynthesis is of great significance for improving the medicinal value of ginseng. MeJA is widely used as an inducer in research on the ginsenoside synthesis pathway because of its effective and consistent induction properties [ 45 – 47 ]. In our study, analysis of the promoter regions of PgYTH genes revealed the presence of MeJA-responsive elements, suggesting that these genes may be directly regulated by jasmonate signaling. Correspondingly, expression profiling demonstrated that PgYTH9-12 were significantly downregulated following MeJA treatment. This finding strongly indicates that members of the PgYTH family are subject to fine hormonal regulation by MeJA. The pattern of downregulation of PgYTH genes in response to MeJA mirrors observations in S. miltiorrhiza , where most SmYTH1–SmYTH6 genes exhibited high sensitivity to MeJA, with predominantly decreased expression. In S. miltiorrhiza , this downregulation correlated with increased accumulation of phenolic acids, bioactive compounds of medicinal significance. [ 40 ]. It is speculated that PgYTH gene family may mediate the post-transcriptional regulation of downstream hormone signaling genes by binding to the m 6 A modification on mRNA, thereby affecting the accumulation of medicinal components in ginseng. The YTH domain-containing gene family has emerged as a key regulator in plant responses to various abiotic stresses, particularly salt and drought stress, across a diverse range of plant species, including Arabidopsis , apple, Setaria italica , cotton, and C. chekiangoleosa . AtECT8 functions as a sensor for salt stress, promoting mRNA degradation that is essential for preserving transcriptome balance and improving salt stress tolerance in Arabidopsis [ 48 ]. The absence of ECT8 leads to impaired sequestration of m 6 A -modified PYRABACTIN RESISTANCE 1-LIKE 7 (PYL7) within stress granules, allowing increased translation of PYL7 transcripts. This results in the overactivation of ABA-responsive genes and the development of ABA-hypersensitive traits, such as enhanced drought tolerance [ 49 ]. In Arabidopsis , AtECT12 was identified to modulate the stability of m 6 A-marked RNA transcripts, thereby promoting Arabidopsis tolerance to salt or drought stress [ 50 ]. The m 6 A reader SiYTH1 improves drought tolerance in S. italica by modulating the stability of messenger RNAs associated with stomatal closure and reactive oxygen species scavenging [ 51 ]. Functional validation demonstrated that silencing GhYTH8 decreased drought tolerance in the upland cotton TM-1 line [ 52 ]. C. chekiangoleosa CchYTH genes expression levels vary in response to drought stress [ 33 ]. These observations highlight an evolutionarily conserved role for YTH domain-containing proteins in modulating the response to abiotic stresses through post-transcriptional regulation mechanisms, especially via interaction with m 6 A-modified RNAs. In this study, the expression levels of PgYTH9-12 with DFA subfamily were significantly induced by salt stress, supporting the pivotal role of YTH genes in modulating stress tolerance mechanisms in plants. 5. Conclusion In this study, a comprehensive analysis of the ginseng YTH gene family was conducted, encompassing identification, phylogenetic relationships, gene structure, chromosomal location, expression patterns. This research offers a foundation for future metabolic regulation of the ginsenoside biosynthesis pathway. Furthermore, this research sheds new light on the intricate mechanism of m 6 A reader-mediated salt tolerance and lays a foundation for enhancing salt tolerance in ginseng breeding. Declarations Ethics approval and consent to participate Clinical trial: Not applicable Consent for publication Not applicable. Availability of data and materials The original contributions of this study are contained within the article/Supplementary Material. 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Supplementary Files TableS1.Plantmaterialsusedforthisstudy..xls TableS2.PrimersusedforqRTPCR..xlsx TableS3.cisactingelementsofPgYTHs.xlsx TableS4.TheKaKsratiosandestimateddivergencetimesforduplicatepairsofPgYTHs..xlsx Cite Share Download PDF Status: Published Journal Publication published 26 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 10 Nov, 2025 Reviews received at journal 05 Nov, 2025 Reviews received at journal 20 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers agreed at journal 18 Oct, 2025 Reviews received at journal 11 Oct, 2025 Reviewers agreed at journal 21 Sep, 2025 Reviewers agreed at journal 19 Sep, 2025 Reviewers invited by journal 18 Sep, 2025 Editor assigned by journal 18 Sep, 2025 Editor invited by journal 18 Sep, 2025 Submission checks completed at journal 17 Sep, 2025 First submitted to journal 17 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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13:36:17","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":157892,"visible":true,"origin":"","legend":"","description":"","filename":"35dbd62bee664f8480261ffe9e3557431structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/3e498f8aabeae58b5095b3bf.xml"},{"id":92510096,"identity":"a0b08f77-b421-4e91-a0db-cc0c1ae8dbb4","added_by":"auto","created_at":"2025-09-30 13:20:17","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":172522,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/1c9e105c747b0bc80173324a.html"},{"id":92510066,"identity":"28b79c7a-0ac0-477b-942b-d335729c3eaf","added_by":"auto","created_at":"2025-09-30 13:20:17","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":870852,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003eYTH\u003c/em\u003e gene family in ginseng, \u003cem\u003eArabidopsis\u003c/em\u003e, and rice. The NJ tree was constructed with a bootstrap value of 1,000. The phylogenetic tree classified \u003cem\u003eYTH\u003c/em\u003e genes into 4 groups. Pg, ginseng; At, \u003cem\u003eArabidopsis\u003c/em\u003e; Os, rice.\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/954d41411e7145f7991c7ddf.jpg"},{"id":92511594,"identity":"b188426e-34c3-4cd2-9e49-6f0e24c9a4b2","added_by":"auto","created_at":"2025-09-30 13:36:17","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1352099,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree, protein motifs, conserved domain, and gene structure of \u003cem\u003eYTH\u003c/em\u003e genes in ginseng. (A) Phylogenetic tree and motif distribution of \u003cem\u003eYTH\u003c/em\u003e genes in ginseng. Each colored box represents a specific motif. (B) Conserved domain in ginseng YTH proteins. (C) Exon-intron structure of ginseng YTH genes. Green boxes denote untranslated regions (UTRs), yellow boxes indicate exons, and black lines represent introns.\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/3e5dc23e1c0ad3026251f906.jpg"},{"id":92511595,"identity":"31eedfe2-bc2a-439a-8a67-596ae892f899","added_by":"auto","created_at":"2025-09-30 13:36:17","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2373471,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal locations and collinearity analysis of the PgYTH genes. (A) Chromosomal distribution of PgYTH genes across both A and B subgenomes. (B) Covariance analysis of ginseng YTH gene family members in ginseng chromosomes.\u003c/p\u003e","description":"","filename":"image3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/f8c367fafd3eb04fbe15687a.jpg"},{"id":92511597,"identity":"5ccd81da-fa80-4e3c-9f78-ecaa9518bbeb","added_by":"auto","created_at":"2025-09-30 13:36:17","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1354753,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ecis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-regulatory elements of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePgYTH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes.\u003c/strong\u003e (A) Distribution of \u003cem\u003ecis\u003c/em\u003e-regulatory elements within the promoter regions of \u003cem\u003ePgYTH\u003c/em\u003e genes. Gray lines depict the gene sequences. Each cis-element is illustrated as a distinct colored square with annotations positioned to the right. (B) Distribution of different types of stress responsive \u003cem\u003ecis\u003c/em\u003e-elements. \u0026nbsp;(C) Distribution of different types of Hormone responsive \u003cem\u003ecis\u003c/em\u003e-elements.\u003c/p\u003e","description":"","filename":"image4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/31c86a9a15c2fda082c99376.jpg"},{"id":92511245,"identity":"fb6a43b3-ac2f-4afb-bffe-efc5c2dd7aac","added_by":"auto","created_at":"2025-09-30 13:28:17","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6357366,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eYTH\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes in ginseng. \u003c/strong\u003e(A) Expression of \u003cem\u003ePgYTH \u003c/em\u003egenes in ginseng roots at four different ages (5, 12, 18, and 25 years). (B) Expression patterns of \u003cem\u003ePgYTHs\u003c/em\u003e in 14 different tissues of 4-year-old ginseng. (C) Expression patterns of \u003cem\u003ePgYTHs\u003c/em\u003e in in the 42 farm cultivars of 4-year-old ginseng roots.\u003c/p\u003e","description":"","filename":"image5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/bb400a4795053669e8d654c0.jpg"},{"id":92510073,"identity":"1ba1fb5b-c774-401c-91c6-9f2d1cb06a83","added_by":"auto","created_at":"2025-09-30 13:20:17","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2660888,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression of the PgYTH genes under MeJA treatment in ginseng.\u003c/p\u003e","description":"","filename":"image6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/cf0b55971ae9af6ef2a33915.jpg"},{"id":92511244,"identity":"9205c20e-103d-486d-a0c5-9cb0d4056af6","added_by":"auto","created_at":"2025-09-30 13:28:17","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4930755,"visible":true,"origin":"","legend":"\u003cp\u003eThe responses of the \u003cem\u003ePgYTH\u003c/em\u003e genes to salt stress. (A) Ginseng adventitious roots cultured with different concentrations of NaCl for 30 days on B5 medium. (B) The relative expressions of the \u003cem\u003ePgYTH \u003c/em\u003egenes in the salt-stressed ginseng roots. “*” indicates P ≤ 0.05; “**” indicates P ≤ 0.01.\u003c/p\u003e","description":"","filename":"image7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/bfa6c79e723db66d08f5e9fc.jpg"},{"id":92511241,"identity":"108af372-b93b-41a5-9637-8050cae91609","added_by":"auto","created_at":"2025-09-30 13:28:17","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":115371,"visible":true,"origin":"","legend":"\u003cp\u003eSubcellular analysis of PgYTH12 protein. The PgYTH12::GFP and GFP alone were transiently expressed via agroinfiltration using \u003cem\u003eN. benthamiana\u003c/em\u003e leaves. Green fluorescence was visualized using confocal laser microscopy (left), and the same cells were examined by transmission microscopy. The two images were then overlaid (right). Scale bar =20 μm.\u003c/p\u003e","description":"","filename":"image8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/9e5687f4c959cc4a0384a56e.jpeg"},{"id":92511599,"identity":"9dac90e8-0f1d-4ea6-9bfb-340e65ccccb9","added_by":"auto","created_at":"2025-09-30 13:36:17","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":5743680,"visible":true,"origin":"","legend":"\u003cp\u003ePrLD and disordered region predictions of PgYTH proteins by PLAAC.\u003cstrong\u003e \u003c/strong\u003eThe black line indicates the background signal, while the red line represents the predicted region of the prion structure. When the red line rises above the baseline, it indicates the presence of a prion structure at that position, suggesting a strong likelihood of phase transition.\u003c/p\u003e","description":"","filename":"image9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/d76132799fb4705a83a57313.jpg"},{"id":99172871,"identity":"6ebde285-6547-45ff-9135-257677ecc1d1","added_by":"auto","created_at":"2025-12-29 16:11:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":27047602,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/5b856fc4-45a2-4814-bbcf-52ec7b5b07ac.pdf"},{"id":92511236,"identity":"2fead737-761e-4704-9025-8d61822c7a22","added_by":"auto","created_at":"2025-09-30 13:28:17","extension":"xls","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":25600,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.Plantmaterialsusedforthisstudy..xls","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/f5f4cbd305c05c6b337a4dc8.xls"},{"id":92510062,"identity":"183c5323-7346-4e15-8da6-478cedf9ea73","added_by":"auto","created_at":"2025-09-30 13:20:17","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12650,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.PrimersusedforqRTPCR..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/c75fad356bee0c6c5cd8c8d3.xlsx"},{"id":92511596,"identity":"1eb51249-996d-4044-9a5e-c953f9314ff2","added_by":"auto","created_at":"2025-09-30 13:36:17","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10504,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.cisactingelementsofPgYTHs.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/c2568ec086831a6f69f94568.xlsx"},{"id":92511238,"identity":"ca575b6e-43f9-4b5e-a288-bc9e686ec507","added_by":"auto","created_at":"2025-09-30 13:28:17","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11988,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.TheKaKsratiosandestimateddivergencetimesforduplicatepairsofPgYTHs..xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7523802/v1/d6332fcb19cbb88fb16aae75.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification and analysis of YTH gene family and its response to MeJA and salt treatment in Panax ginseng C. A. Meyer","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eGinseng (\u003cem\u003ePanax ginseng\u003c/em\u003e C. A. Mey.) is one of the most valuable traditional Chinese medicinal materials in China, often referred to as the \"King of Herbs,\" and it possesses significant medicinal value. Similar to changes in DNA epigenetic modifications, typical components of RNA, such as adenine (A), uracil (U), guanine (G), and cytosine (C), also undergo a series of chemical modifications. These modifications can occur either during transcription or after transcription has been completed, and they persist throughout the entire lifespan of most RNA molecules [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. RNA modifications are crucial for the characteristics and functions of RNA, affecting stability, translation, splicing, and transport, which in turn increase the functional versatility and diversity of RNA molecules [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To date, over 170 different RNA modifications have been identified [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn RNA molecules, adenosine (A) is methylated by methyltransferases, where the hydrogen atom at the sixth nitrogen position (N6) is replaced by a methyl group (CH3). The changes in m\u003csup\u003e6\u003c/sup\u003eA methylation are primarily regulated by various factors, including m\u003csup\u003e6\u003c/sup\u003eA methyltransferases, demethylases, and m\u003csup\u003e6\u003c/sup\u003eA recognition proteins. m\u003csup\u003e6\u003c/sup\u003eA modifications have been detected in several plants, including Arabidopsis and rice [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Additionally, proteins associated with m\u003csup\u003e6\u003c/sup\u003eA modifications have been progressively identified in plants. In Arabidopsis, the m\u003csup\u003e6\u003c/sup\u003eA methyltransferase complex consists of mRNA adenosine methyltransferase A (MTA) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], MTB, FKBP12 interacting protein 37 (FIP37) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], VIRILIZER (VIR) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and HAKAI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], while m\u003csup\u003e6\u003c/sup\u003eA demethylation is regulated by ALKBH9B and ALKBH10B [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Research indicates that m\u003csup\u003e6\u003c/sup\u003eA modifications play significant regulatory roles in processes such as fruit ripening, abiotic stress responses, and secondary metabolite synthesis in plants. m\u003csup\u003e6\u003c/sup\u003eA methylation plays an essential regulatory role in the processes of fruit ripening and quality metabolism in kiwifruit. Overexpression of AcALKBH10 greatly elevates the soluble sugar content in the fruit while markedly reducing the accumulation of acids, whereas silencing AcALKBH10 exhibits the opposite trend [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In apples, sorbitol significantly affects the m\u003csup\u003e6\u003c/sup\u003eA methylation of mRNAs by controlling the expression of two m\u003csup\u003e6\u003c/sup\u003eA methyltransferases, MdVIR1 and MdVIR2. This RNA modification is essential for the resistance against Alternaria alternata, a pathogen controlled by sorbitol [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, m\u003csup\u003e6\u003c/sup\u003eA-mediated regulatory mechanisms coordinate the accumulation of terpenoid metabolites and the formation of tea aroma during the sunlight wilting stage of tea leaves [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e\u003cp\u003em\u003csup\u003e6\u003c/sup\u003eA exerts its regulatory functions primarily by recruiting specific m6A-binding proteins known as \"readers.\" In mammals, various m\u003csup\u003e6\u003c/sup\u003eA-binding proteins have been identified, with the most extensively studied being the family of proteins containing the YT521-B homology (YTH) domain [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], which is categorized into YTHDC1, YTHDC2, and YTHDF1-3 subfamilies. These proteins recognize the m\u003csup\u003e6\u003c/sup\u003eA modification through a conserved YTH domain characterized by a \"hydrophobic pocket\" formed by two or three aromatic residues necessary for binding to the m\u003csup\u003e6\u003c/sup\u003eA site [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. YTHDF1, YTHDF2, YTHDF3, and YTHDC2 are all cytoplasmic m\u003csup\u003e6\u003c/sup\u003eA readers that function by binding to mature mRNAs containing m\u003csup\u003e6\u003c/sup\u003eA. In contrast, YTHDC1 is localized in the nucleus and selectively binds pre-mRNA containing m\u003csup\u003e6\u003c/sup\u003eA [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The m\u003csup\u003e6\u003c/sup\u003eA readers play a broad regulatory role in RNA transcription-related processes, including alternative splicing, mRNA stability, and translation. Thirteen YTH proteins have been identified in \u003cem\u003eArabidopsis\u003c/em\u003e, which are classified into two evolutionary clades: YTHDF and YTHDC [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Eleven genes (AtECT1-11) belong to the YTHDF clade, while AtCPSF30 and AtECT12 (AtDC1) belong to the YTHDC clade [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In recent years, members of the YTH family have been successively identified in plants such as rice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], tomato [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and wheat [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] through bioinformatics approaches. However, The YTH proteins in ginseng have not yet been identified. The functional role of m\u003csup\u003e6\u003c/sup\u003eA reader YTH proteins in response to hormone and abiotic stress in Panax ginseng has not yet been reported.\u003c/p\u003e\u003cp\u003eIn this study, a total of 18 \u003cem\u003ePgYTH\u003c/em\u003e genes were identified in ginseng and classified into four subgroups, with detailed analyses of their chromosomal distribution and collinearity, gene structures, \u003cem\u003ecis\u003c/em\u003e-acting elements, and protein motifs. We also examined the expression profiles of these \u003cem\u003ePgYTH\u003c/em\u003e genes. The \u003cem\u003ePgYTH\u003c/em\u003e gene family was found to respond to MeJA and salt treatments. These findings provide a foundation for further investigation into the roles of \u003cem\u003eYTH\u003c/em\u003e genes in response to hormone and abiotic stress.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Identification and characterization of physiochemical properties of \u003cem\u003eYTH\u003c/em\u003e genes in ginseng\u003c/h2\u003e\u003cp\u003eThe gene annotation and telomere-to-telomere reference genome files of ginseng from Jilin Province, China, were retrieved based on the data availability in the previous study [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Protein sequences of \u003cem\u003eYTH\u003c/em\u003e genes from \u003cem\u003eArabidopsis\u003c/em\u003e and rice were sourced from the TAIR database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and Rice Genome Annotation Project (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu/index.shtml\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu/index.shtml\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively. Candidate YTH gene sequences were aligned against the protein sequences of ginseng using the blastp algorithm. The presence of the YTH domain in the candidate genes was verified using the NCBI Conserved Domain Database (CDD). Confirmation of the identified \u003cem\u003eYTH\u003c/em\u003e genes was further conducted using the Hmmsearch function [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The physicochemical properties, including relative molecular mass (MW) and theoretical isoelectric point (PI), were calculated utilizing the ExPASy proteomics server [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Additionally, the chromosomal locations of the \u003cem\u003eYTH\u003c/em\u003e genes were extracted from the annotation files and visualized using TBtools [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Evolutionary, gene structure, and conserved motif analysis of Pg\u003cem\u003eYTH\u003c/em\u003es\u003c/h2\u003e\u003cp\u003eThe identified YTH protein sequences from Jilin ginseng, \u003cem\u003eArabidopsis\u003c/em\u003e, and rice were aligned using the MUSCLE algorithm implemented in MEGA-X. Phylogenetic tree construction was performed using the neighbor-joining (NJ) method in MEGA-X, employing the Poisson model with 1,000 bootstrap replicates. The resulting phylogenetic tree was visualized and enhanced using Evolview. Collinearity analysis of \u003cem\u003eYTH\u003c/em\u003e genes in ginseng was conducted using TBtools. Conserved motifs within the ginseng YTH protein sequences were identified and analyzed using the MEME program. The gene structures were visually displayed using TBtools.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Chromosomal distribution and synteny analysis of Pg\u003cem\u003eYTHs\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eGinseng reference genome annotation files from Jilin Province, China, were used to determine the chromosomal distribution of \u003cem\u003eYTH\u003c/em\u003e genes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The chromosomal distribution of ginseng \u003cem\u003eYTH\u003c/em\u003e genes was drafted from top to bottom by TBtools according to gene positions in the genome annotation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Regulatory elements analysis of Pg\u003cem\u003eYTHs\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe 2000 bp region upstream of the coding sequence (CDS) of YTH genes was extracted with TBtools and designated as the promoter region. Putative \u003cem\u003ecis\u003c/em\u003e-regulatory elements within these promoter regions were identified using PlantCARE [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The distribution and organization of these elements were then visualized with TBtools.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Subcellular Localization of PgYTH12\u003c/h2\u003e\u003cp\u003eThe full-length coding sequence of \u003cem\u003ePgYTH12\u003c/em\u003e was amplified using specific primers PgYTH12-GFP-F (5\u0026rsquo;-ACTATTTACAATTACGGATCatggctaccgttgctcctcgg-3\u0026rsquo;) and PgYTH12-GFP-R (5\u0026rsquo;-TCCTCGCCCTTGCTCACCATagatcctcctccagatcctcctc-3\u0026rsquo;), and subsequently cloned into the vector pBWA(V)H2STMVΩ-3xflag-ccdB-egfp. The resulting recombinant plasmid, pBWA(V)H2STMVΩ-PgYTH12-GFP, was introduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101, which was then used to infiltrate fully expanded leaves of \u003cem\u003eNicotiana benthamiana\u003c/em\u003e. Expression of GFP in the infiltrated cells was visualized using a confocal laser scanning microscope (Nikon C2-ER) with argon laser excitation at 488 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Expression profiles analysis of \u003cem\u003ePgYTHs\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eTo investigate the expression patterns of \u003cem\u003eYTH\u003c/em\u003e genes in ginseng, RNA-Seq datasets were retrieved from NCBI (accession number PRJNA302556). These datasets include samples from 14 different tissues of 4-year-old ginseng, ginseng roots at four different growth stages, and roots of 42 different cultivated varieties of 4-year-old ginseng (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) were collected from Jilin, China [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Transcript assembly and expression quantification were conducted through a pipeline involving Hisat2, StringTie, Kallisto.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 The \u003cem\u003ePgYTH\u003c/em\u003e genes response under MeJA treatment in ginseng\u003c/h2\u003e\u003cp\u003eTo explore the potential involvement of the \u003cem\u003ePgYTH\u003c/em\u003e gene family in hormone response, ginseng adventitious roots were treated with MeJA. The adventitious roots (1 g) of ginseng were inoculated in 250 mL triangular flasks containing 150 mL of liquid B5 medium and incubated in a shaker at 22℃, 110 rpm for 21 days. On the 22nd day, 200 mM MeJA was added to the culture flasks, and this time point was designated as 0 h of treatment. Samples were collected at 0, 6, 12, 24, 48, and 72 hours after treatment, with three biological replicates for each time point. The 0 h samples served as the untreated control group. All collected samples were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C for further analysis. Adventitious roots subjected to MeJA treatment were induced from Jilin ginseng plants (variety named \u0026ldquo;Fuxing No. 2\u0026rdquo;) originating in Jilin Province, China. All ginseng plant materials were stored in Medical Laboratory Testing Technology and Analytical Laboratory, Beihua University.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 The \u003cem\u003ePgYTH\u003c/em\u003e genes response under salt treatment in ginseng\u003c/h2\u003e\u003cp\u003eTo investigate the potential role of the \u003cem\u003ePgYTH\u003c/em\u003e gene family in salt stress response, ginseng adventitious roots were subjected to NaCl treatment. Adventitious roots measuring 1 cm in length were cultured on B5 medium supplemented with varying concentrations of NaCl (0 mM, 70 mM, 80 mM, 90 mM, and 100 mM) at 25\u0026deg;C for 30 days. After treatment, the samples were rapidly frozen in liquid nitrogen before being stored at -80\u0026deg;C for subsequent gene expression analysis. The adventitious roots used for salt treatment were induced from Jilin ginseng plants (variety named \u0026ldquo;Fuxing No. 2\u0026rdquo;) sourced from Jilin Province, China. All ginseng plant materials were stored in Medical Laboratory Testing Technology and Analytical Laboratory, Beihua University.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Quantitative Real-Time PCR (qPCR) analysis\u003c/h2\u003e\u003cp\u003eTotal RNA from Jilin ginseng (variety named \u0026ldquo;Fuxing No. 2\u0026rdquo;) was extracted using the TRIzol method. Subsequently, the Super RT III Kit (Biosharp Biotech) was used to reverse-transcribe the RNA into cDNA. Primers for qPCR were synthesized by Sangon Biotech (Shanghai, China), with their sequences provided in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The β-actin gene served as the internal reference. qPCR assays were conducted using the SYBR Premix Ex Taq\u0026trade; II (Tli RNaseH Plus) kit (TaKaRa). Each experiment was performed in triplicate for every sample group, and the relative expression levels were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10 Liquid\u0026ndash;Liquid Phase Separation (LLPS) Prediction\u003c/h2\u003e\u003cp\u003eThe prion-like domain (PrLD) and intrinsically disordered region (IDR) associated with LLPS in PgYTH proteins were predicted using the PLAAC tool, which employs a hidden Markov model (HMM) algorithm.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Genome-wide identification of ginseng \u003cem\u003eYTH\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eIn total, we identified 18 \u003cem\u003eYTH\u003c/em\u003e genes in \u003cem\u003ePanax ginseng\u003c/em\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The complete information for these genes, including the gene ID, number of amino acids (aa), molecular weight (MW), isoelectric point (pI), location and subcellular localization were analyzed. The lengths of the corresponding YTH proteins ranged from 436 aa (PgYTH7) to 720 aa (PgYTH11). The pI exhibited variability, spanning from 5.13 (PgYTH6) to 9.23 (PgYTH5). Additionally, the MW of these proteins was found to range approximately from 49.4 kDa (PgYTH7) to 79.8 kDa (PgYTH1). The subcellular localization prediction results indicated that 4 (PgYTH1, PgYTH2, PgYTH7, and PgYTH16) out of the 18 proteins were localized in the nucleus, while the remaining 14 were exclusively localized in the cytoplasm.\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\u003eCharacteristics of \u003cem\u003eYTH\u003c/em\u003e genes and their encoded proteins in ginseng.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"7\"\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=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene Name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eGene ID\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNumber of Amino Acid\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMolecular Weight (MW)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eisoelectric point (pI)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eLocation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eSubcellular Localization\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_2000603.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e718\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e79775.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr02:6144965\u0026ndash;6159906(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_5003162.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e455\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e51297.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr05:34521511\u0026ndash;34528108(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_5013137.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e65984.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr05:159669959\u0026ndash;159681112(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_7001001.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e516\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e57747.22\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr07:9116362\u0026ndash;9123416(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_8011634.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e552\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e61306.36\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.23\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr08:157638510\u0026ndash;157645259(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_11012144.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e609\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e67004.63\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr11:168557286\u0026ndash;168570003(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_12002421.t03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e436\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e49447.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr12:25766424\u0026ndash;25773204(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_12010946.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e600\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e65955.74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr12:148177398\u0026ndash;148189627(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_13012092.t01-1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e700\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e76689.87\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr13:159000112\u0026ndash;159003943(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_13012092.t01-2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e711\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e78013.78\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.94\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr13:159006392\u0026ndash;159011254(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_14000049.t02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e720\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e79627.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e7.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr14:500279\u0026ndash;505414(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_14000050.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e705\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e77160.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr14:507290\u0026ndash;511491(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_14009897.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e609\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e67009.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr14:128673911\u0026ndash;128685027(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_15008119.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e552\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e61095.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e9.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr15:110326382\u0026ndash;110333089(+)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH15\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_16000974.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e524\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e58769.19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.08\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr16:7869738\u0026ndash;7876491(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_17000373.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e712\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e78999.64\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr17:3182577\u0026ndash;3197335(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eNucleus\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_21010025.t01\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e716\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e78401.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr21:124088321\u0026ndash;124093891(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePgYTH18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003epg_23008814.t02\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e716\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e78322.42\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e6.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eChr23:92385522\u0026ndash;92391328(-)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eCytoplasm\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Phylogenetic analysis of \u003cem\u003eYTH\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eA phylogenetic tree was constructed with YTH protein sequences from ginseng, \u003cem\u003eArabidopsis\u003c/em\u003e, and rice to analyze the evolutionary relationships among \u003cem\u003eYTH\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). In our study, the YTH proteins of ginseng were classified into two families, DC and DF, based on previous studies in other plant species. The DC group contains 4 members: PgYTH1, PgYTH2, PgYTH7, and PgYTH16. The DF group was further divided into 3 subfamilies (DFA, DFB, and DFC). PgYTH9-12 were classified into subfamily DFA. PgYTH3/6/8/13 were classified into subfamily DFB. PgYTH5/14/15/17/18 were classified into subfamily DFC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Motifs, conserved domain and gene structure analysis\u003c/h2\u003e\u003cp\u003eAn unrooted phylogenetic tree was constructed to investigate the evolutionary patterns and classification of \u003cem\u003eYTH\u003c/em\u003e genes in ginseng. We examined the conserved motifs of the YTH proteins and the gene structures of the identified \u003cem\u003eYTH\u003c/em\u003e genes. The MEME program was employed to analyze the organization of conserved motifs in ginseng YTH proteins, resulting in the detection of a total of 10 distinct motifs. These genes contained 4 to 9 motifs. Notably, motifs 1, 2, 3, and 5 were present in \u003cem\u003eYTH\u003c/em\u003e genes. PgYTH9-12 contain all motifs except motif10. \u003cem\u003ePgYTH3/6/8/13\u003c/em\u003e contain all motifs except motif10. \u003cem\u003eYTH\u003c/em\u003e family members that cluster within a clade share similar motif compositions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e\u003cp\u003eThe YTH domains in the candidate genes were verified through analysis in the NCBI CDD. The YTH domains of most YTH genes are located near the 3' end. The YTH domains of PgYTH2 and PgYTH7 are located near the 5' end. The YTH domains are in the middle of PgYTH1 and PgYTH16 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eTo evaluate the consistency of exon-intron patterns among \u003cem\u003eYTH\u003c/em\u003e genes, we performed a comprehensive gene structure analysis. The results revealed that the number of exons in the \u003cem\u003eYTH\u003c/em\u003e gene family of ginseng ranged from 7 to 10. The analysis of exon and intron arrangements offered valuable insights into the evolutionary relationships among different members of this gene family. Notably, genes that are closely related displayed a higher degree of structural similarity, with variations primarily observed in the lengths of their introns and exons. A significant positive correlation was identified between phylogenetic relationships and exon-intron structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Chromosome distribution and collinearity analysis of \u003cem\u003eYTH\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eConsidering that ginseng is an allotetraploid plant, the previous study categorized the ginseng chromosomes into two subgenomes (subgenomes A and B) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The BLASTN search was conducted to determine the chromosomal distribution of \u003cem\u003eYTH\u003c/em\u003e genes in ginseng. The PgYTH genes were found to be roughly evenly distributed across both subgenomes of ginseng. The ginseng \u003cem\u003eYTH\u003c/em\u003e gene is located on chromosome Chr03A-07A, Chr10A and Chr12A as well as chromosome Chr03B-07B and Chr10B. Three \u003cem\u003eYTH\u003c/em\u003e genes (PgYTH11-13) are anchored on chromosome Chr04B. With the exception of four genes (PgYTH9-12), the chromosomal locations of the remaining genes are largely conserved between subgenome A and subgenome B (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). These results imply that divergent evolutionary pressures have likely shaped the evolution of ginseng.\u003c/p\u003e\u003cp\u003eTo further investigate the phylogenetic mechanisms of the ginseng \u003cem\u003eYTH\u003c/em\u003e gene family, we performed an intraspecific collinearity analysis of \u003cem\u003ePgYTH\u003c/em\u003e genes. A total of 19 pairs of colinear \u003cem\u003ePgYTH\u003c/em\u003e genes were identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). To explore the potential selective pressure on \u003cem\u003ePgYTHs\u003c/em\u003e, the Ka (non-synonymous) and Ks (synonymous) substitution rates, along with the Ka/Ks ratios, were analyzed. The Ka values for the \u003cem\u003ePgYTH\u003c/em\u003e family genes ranged from 0.005 to 0.150, the Ks values for the \u003cem\u003ePgYTH\u003c/em\u003e family genes ranged from 0.015 to 0.401, and the Ka/Ks ratios varied between 0.206 and 0.716 (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The results reveal that \u003cem\u003ePgYTH\u003c/em\u003e has mainly undergone purifying selection throughout its evolution, as all \u003cem\u003ePgYTH\u003c/em\u003e gene pairs exhibited Ka/Ks values below 1.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.5 \u003cem\u003ecis\u003c/em\u003e-regulatory element analysis of \u003cem\u003eYTH\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eUnderstanding the \u003cem\u003ecis\u003c/em\u003e-regulatory elements that control \u003cem\u003eYTH\u003c/em\u003e gene expression is crucial for elucidating their regulatory mechanisms and potential functions in ginseng. To this end, we extracted the 2000 bp region upstream of the start codon of each ginseng YTH gene as its promoter region for further analysis. A total of 469 \u003cem\u003ecis\u003c/em\u003e-regulatory elements, representing 17 different types, were identified in the promoter regions of \u003cem\u003ePgYTH\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Among them, light-responsive elements were the most abundant, accounting for nearly half of the total number (224) (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Stress-responsive regulatory elements were also identified, including those associated with anaerobic environments, MYB binding sites involved in drought stress inducibility, low-temperature stress, wound response, mixed stress conditions, and anoxia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A total of 85 environmental stress-related \u003cem\u003ecis\u003c/em\u003e-acting elements were identified. Over 70% of these elements were anaerobic induction elements and drought-responsive MYB binding sites, highlighting their dominant roles in stress response (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, several hormone regulatory sites were identified, including those responsive to MeJA, abscisic acid (ABA), gibberellin (GA3), auxin, and salicylic acid (SA). The distribution of \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoter regions suggests that \u003cem\u003ePgYTH\u003c/em\u003e genes may be primarily regulated by MeJA and ABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Furthermore, \u003cem\u003ecis\u003c/em\u003e-acting elements related to zein, meristem, endosperm, palisade mesophyll cells and circadian were also detected.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Expression patterns of ginseng \u003cem\u003eYTH\u003c/em\u003e genes\u003c/h2\u003e\u003cp\u003eGene expression patterns are strongly associated with gene functions. To better understand the expression patterns of \u003cem\u003ePgYTH\u003c/em\u003e genes in ginseng, we obtained expression data for \u003cem\u003ePgYTH\u003c/em\u003e transcripts across 42 farm cultivars (S1\u0026ndash;S42) (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), 14 different tissues, and ginseng roots of four varying ages (5, 12, 18, and 25 years). The expression levels of PgYTH9-12 in ginseng roots of four different ages were significantly higher than that of other \u003cem\u003ePgYTH\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The \u003cem\u003ePgYTH\u003c/em\u003e genes exhibited dynamic and tissue-specific expression patterns throughout these organs, reflecting their diverse functional roles in ginseng development and metabolism. \u003cem\u003ePgYTH1-8\u003c/em\u003e and \u003cem\u003ePgYTH13-18\u003c/em\u003e exhibited generally low expression levels across all ginseng tissues. Compared to other \u003cem\u003ePgYTH\u003c/em\u003e genes, \u003cem\u003ePgYTH12\u003c/em\u003e exhibited higher expression levels across various tissues, indicating its potentially broader or more significant functional role in ginseng. Moreover, \u003cem\u003ePgYTH12\u003c/em\u003e showed the highest expression level in the main root cortex (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, \u003cem\u003ePgYTH12\u003c/em\u003e exhibited the highest expression level across 42 farm cultivars (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), indicating that this gene may play an important role in the root of ginseng.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.7 Expression analysis of\u003c/b\u003e \u003cb\u003ePgYTH\u003c/b\u003e \u003cb\u003egenes under MeJA treatment in ginseng\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eMeJA was found to be involved in the regulation of ginsenoside biosynthesis. qRT-PCR was conducted to determine the expression profiles of the \u003cem\u003ePgYTH\u003c/em\u003e genes under MeJA treatment. The qRT-PCR analysis showed a predominant downregulation trend across most members of this gene family. Among the 18 \u003cem\u003ePgYTH\u003c/em\u003e genes examined, only \u003cem\u003ePgYTH2\u003c/em\u003e, \u003cem\u003ePgYTH14\u003c/em\u003e, \u003cem\u003ePgYTH15\u003c/em\u003e, and \u003cem\u003ePgYTH16\u003c/em\u003e showed no downregulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e), indicating that these genes may be governed by different regulatory mechanisms or have functions that are less affected by MeJA signaling. The expression levels of \u003cem\u003ePgYTH3\u003c/em\u003e, \u003cem\u003ePgYTH4\u003c/em\u003e, \u003cem\u003ePgYTH10\u003c/em\u003e, \u003cem\u003ePgYTH12\u003c/em\u003e, \u003cem\u003ePgYTH13\u003c/em\u003e, \u003cem\u003ePgYTH17\u003c/em\u003e, and \u003cem\u003ePgYTH18\u003c/em\u003e were significantly decreased compared to the control at all time points post-treatment, suggesting that they may serve as key components in the MeJA signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e\u003cb\u003e3.8 Expression analysis of\u003c/b\u003e \u003cb\u003ePgYTH\u003c/b\u003e \u003cb\u003egenes under salt treatment in ginseng\u003c/b\u003e\u003c/h2\u003e\u003cp\u003eGinseng growing in natural environments is influenced by multiple stress factors, and gene expression patterns in these specific conditions are commonly utilized to study gene functions. qRT-PCR was used to verify the response of \u003cem\u003ePgYTH\u003c/em\u003e genes to salt stress. Ginseng adventitious roots were treated with different concentrations of salt (0, 70, 80, 90, and 100 mM NaCl) in B5 medium for 30 days. As the salt concentration increased, the growth of ginseng adventitious roots was increasingly inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Compared to the control, the expression levels of all genes except PgYTH8-12 remained largely unchanged following salt treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). All \u003cem\u003ePgYTH\u003c/em\u003e genes within the DFA subfamily (\u003cem\u003ePgYTH9-12\u003c/em\u003e) demonstrated a significant upregulation trend under salt treatment, suggesting that these genes may have crucial roles in the molecular mechanisms governing salt stress tolerance. In addition, the \u003cem\u003ePgYTH8\u003c/em\u003e gene, which belongs to the DFB subfamily, also exhibited a significant upregulation trend under salt treatment. These results suggest that while many \u003cem\u003ePgYTH\u003c/em\u003e genes remain stable under salt stress, a targeted subset is actively engaged, highlighting functional differentiation within the \u003cem\u003ePgYTH\u003c/em\u003e gene family in adapting to salt stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.8 Subcellular Localization of PgYTH12\u003c/h2\u003e\u003cp\u003eTo investigate the subcellular localization of PgYTH12 and its potential function in roots, we fused the coding sequence of \u003cem\u003ePgYTH12\u003c/em\u003e to pBWA(V)H2STMVΩ-3xflag-ccdB-egfp vector and transiently expressed it in expanded leaves of \u003cem\u003eN. benthamiana\u003c/em\u003e. Transient expression assays revealed that GFP signals in the GFPPgYTH12-expressed \u003cem\u003eN. benthamiana\u003c/em\u003e leaves were observed in the endoplasmic reticulum (ER) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). These results suggested that PgYTH12 may be involved in ER-associated processes, such as protein synthesis, folding, and stress responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.9 Liquid\u0026ndash;Liquid Separation of PgYTH Proteins\u003c/h2\u003e\u003cp\u003eThe PrLD domain has been demonstrated to facilitate the liquid\u0026ndash;liquid phase separation of proteins. The online tool Prion-like Amino Acid Composition (PLAAC) has proven to be an effective method for identifying prion-like domains (PrLDs) in proteins. In this study, we utilized PLAAC to predict prion domains within PgYTH proteins to assess their potential for phase transition. PgYTH proteins from different subfamilies exhibit varying numbers of highly disordered PrLDs. The DFA subfamily includes three PrLDs, indicating a strong propensity for phase separation. The DFB subfamily contains one to two PrLDs. Within the DFC subfamily, four PgYTH proteins have one PrLD each, while two proteins lack PrLDs. In the DC subfamily, two PgYTH proteins contain three PrLDs, and two proteins have none. This indicates that PgYTH proteins, especially those belonging to the DFA subfamily, might undergo phase separation similarly to YTHDF1\u0026ndash;YTHDF3 proteins of human.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003em\u003csup\u003e6\u003c/sup\u003eA RNA methylation is the most prevalent reversible modification found on eukaryotic RNAs and is one of the earliest reported chemical modifications, with its abundance varying across developmental stages and different tissues [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The recognition of m\u003csup\u003e6\u003c/sup\u003eA-modified RNAs is largely mediated by reader proteins containing the YTH domain, which features an aromatic cage that specifically binds to the GG(m\u003csup\u003e6\u003c/sup\u003eA)C sequence [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In this study, we identified 18 \u003cem\u003ePgYTH\u003c/em\u003e genes distributed across 2 subgenomes of ginseng. This number contrasts with previous findings of 13 \u003cem\u003eYTH\u003c/em\u003e genes in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], 12 in rice [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], 26 in apple [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], 10 in \u003cem\u003eCamellia chekiangoleosa\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], 53 in alfalfa [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and 10 in \u003cem\u003eGinkgo biloba\u003c/em\u003e [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Such variation in \u003cem\u003eYTH\u003c/em\u003e gene family size among species suggests that the m\u003csup\u003e6\u003c/sup\u003eA-YTH regulatory system has undergone species-specific expansion or contraction, likely reflecting adaptations to distinct biological needs or environmental pressures.\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, ECT2, ECT3, and ECT4 exhibit significant redundant regulatory functions in leaf growth, organogenesis, and trichome branching. Additionally, ECT2/ECT3/ECT4 collaborate by directly recruiting poly(A) binding proteins, which enhances the stability of target genes and regulates ABA responses [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In recent years, bioinformatics approaches have identified members of the YTH family in rice, tomato, apple, and other plants. \u003cem\u003eOsYTH3\u003c/em\u003e, \u003cem\u003eOsYTH5\u003c/em\u003e, and \u003cem\u003eOsYTH10\u003c/em\u003e are m\u003csup\u003e6\u003c/sup\u003eA readers in rice that are highly expressed across various tissues, showing significant protein homology with AtECT2. The three genes \u003cem\u003eOsYTH3/5/10\u003c/em\u003e regulate rice plant height through functional redundancy. Further transcriptomic analysis indicates that the reduced height of yth03/05/10 triple mutant plants may result from disrupted pathways for terpenoid and brassinosteroid synthesis [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. This highlights a shared mechanism by which YTH proteins regulate growth-related processes via m\u003csup\u003e6\u003c/sup\u003eA-mediated RNA recognition. In tomato, SlYTH2 is highly homologous to \u003cem\u003eArabidopsis\u003c/em\u003e ECT2, ECT3, and ECT4. SlYTH2 possesses the ability to recognize m\u003csup\u003e6\u003c/sup\u003eA and promotes the formation of protein-RNA condensates through liquid-liquid phase separation, thus enabling precise regulation of molecules related to fruit aroma quality [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In our study, \u003cem\u003ePgYTH9-12\u003c/em\u003e were classified into subfamily DFA together with \u003cem\u003eAtECT2/3/4\u003c/em\u003e and \u003cem\u003eOsYTH3/5/10\u003c/em\u003e. This conserved classification indicates that \u003cem\u003ePgYTH9-12\u003c/em\u003e could contribute to key developmental and physiological processes, possibly including growth regulation and secondary metabolite production.\u003c/p\u003e\u003cp\u003eGene expression patterns are closely related to gene functions. In \u003cem\u003eC. chekiangoleosa\u003c/em\u003e, Solanum lycopersicum, and strawberry, the Y\u003cem\u003eTH\u003c/em\u003e genes, belonging to the YTHDFA subfamily, show significantly higher expression compared to other genes in different tissues [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. In \u003cem\u003eSalvia miltiorrhiza\u003c/em\u003e, the SmYTH3 gene, which is classified into the YTHDFA subfamily, exhibited significantly higher expression levels in roots compared to other tissues [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In this study, \u003cem\u003ePgYTH9\u0026ndash;12\u003c/em\u003e, which were also classified into the YTHDFA subfamily, showed significantly higher expression in roots of varying ages, suggesting that genes in the YTHDFA subfamily may be particularly important for root development and function throughout the plant's lifespan. Among the \u003cem\u003ePgYTH\u003c/em\u003e family, \u003cem\u003ePgYTH12\u003c/em\u003e, stood out due to its consistently elevated expression across diverse tissues, the main root cortex, and a wide range of farm cultivars. This broad and robust expression pattern points to a potentially central role for PgYTH12 in coordinating key physiological and developmental processes in ginseng.\u003c/p\u003e\u003cp\u003eGinsenoside, a secondary metabolite of ginseng, is a triterpenoid saponin exhibit significant therapeutic effects in areas such as anti-tumor activity, anti-aging, immune enhancement, and neuroprotection [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Recent studies have shown that saponin synthesis is not only directly regulated by transcription factors such as MYB and WRKY [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], but also influenced by epigenetic modifications (such as DNA methylation and histone methylation,) that dynamically regulate chromatin structure and gene transcription activity. These modifications may serve as critical regulatory nodes in the synthesis of secondary metabolites [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Understanding the regulatory mechanisms underlying ginsenoside biosynthesis is of great significance for improving the medicinal value of ginseng. MeJA is widely used as an inducer in research on the ginsenoside synthesis pathway because of its effective and consistent induction properties [\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In our study, analysis of the promoter regions of \u003cem\u003ePgYTH\u003c/em\u003e genes revealed the presence of MeJA-responsive elements, suggesting that these genes may be directly regulated by jasmonate signaling. Correspondingly, expression profiling demonstrated that \u003cem\u003ePgYTH9-12\u003c/em\u003e were significantly downregulated following MeJA treatment. This finding strongly indicates that members of the \u003cem\u003ePgYTH\u003c/em\u003e family are subject to fine hormonal regulation by MeJA. The pattern of downregulation of \u003cem\u003ePgYTH\u003c/em\u003e genes in response to MeJA mirrors observations in \u003cem\u003eS. miltiorrhiza\u003c/em\u003e, where most \u003cem\u003eSmYTH1\u0026ndash;SmYTH6\u003c/em\u003e genes exhibited high sensitivity to MeJA, with predominantly decreased expression. In \u003cem\u003eS. miltiorrhiza\u003c/em\u003e, this downregulation correlated with increased accumulation of phenolic acids, bioactive compounds of medicinal significance. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. It is speculated that \u003cem\u003ePgYTH\u003c/em\u003e gene family may mediate the post-transcriptional regulation of downstream hormone signaling genes by binding to the m\u003csup\u003e6\u003c/sup\u003eA modification on mRNA, thereby affecting the accumulation of medicinal components in ginseng.\u003c/p\u003e\u003cp\u003eThe YTH domain-containing gene family has emerged as a key regulator in plant responses to various abiotic stresses, particularly salt and drought stress, across a diverse range of plant species, including \u003cem\u003eArabidopsis\u003c/em\u003e, apple, \u003cem\u003eSetaria italica\u003c/em\u003e, cotton, and \u003cem\u003eC. chekiangoleosa\u003c/em\u003e. AtECT8 functions as a sensor for salt stress, promoting mRNA degradation that is essential for preserving transcriptome balance and improving salt stress tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The absence of ECT8 leads to impaired sequestration of m\u003csup\u003e6\u003c/sup\u003eA -modified PYRABACTIN RESISTANCE 1-LIKE 7 (PYL7) within stress granules, allowing increased translation of PYL7 transcripts. This results in the overactivation of ABA-responsive genes and the development of ABA-hypersensitive traits, such as enhanced drought tolerance [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, AtECT12 was identified to modulate the stability of m\u003csup\u003e6\u003c/sup\u003eA-marked RNA transcripts, thereby promoting \u003cem\u003eArabidopsis\u003c/em\u003e tolerance to salt or drought stress [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The m\u003csup\u003e6\u003c/sup\u003eA reader SiYTH1 improves drought tolerance in \u003cem\u003eS. italica\u003c/em\u003e by modulating the stability of messenger RNAs associated with stomatal closure and reactive oxygen species scavenging [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Functional validation demonstrated that silencing \u003cem\u003eGhYTH8\u003c/em\u003e decreased drought tolerance in the upland cotton TM-1 line [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. \u003cem\u003eC. chekiangoleosa CchYTH\u003c/em\u003e genes expression levels vary in response to drought stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. These observations highlight an evolutionarily conserved role for YTH domain-containing proteins in modulating the response to abiotic stresses through post-transcriptional regulation mechanisms, especially via interaction with m\u003csup\u003e6\u003c/sup\u003eA-modified RNAs. In this study, the expression levels of \u003cem\u003ePgYTH9-12\u003c/em\u003e with DFA subfamily were significantly induced by salt stress, supporting the pivotal role of \u003cem\u003eYTH\u003c/em\u003e genes in modulating stress tolerance mechanisms in plants.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, a comprehensive analysis of the ginseng \u003cem\u003eYTH\u003c/em\u003e gene family was conducted, encompassing identification, phylogenetic relationships, gene structure, chromosomal location, expression patterns. This research offers a foundation for future metabolic regulation of the ginsenoside biosynthesis pathway. Furthermore, this research sheds new light on the intricate mechanism of m\u003csup\u003e6\u003c/sup\u003eA reader-mediated salt tolerance and lays a foundation for enhancing salt tolerance in ginseng breeding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eClinical trial: Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions of this study are contained within the article/Supplementary Material.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Science and Technology Development Project of Jilin Province (20240602082RC).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTY: Data curation, Formal Analysis, Investigation, Methodology, and Writing original draft. YM: Visualization, Methodology, and Writing original draft. YZ: Data curation, Formal Analysis, Investigation, Writing\u0026ndash; review \u0026amp; editing. WY: Investigation, Writing\u0026ndash; review \u0026amp; editing. TQ: Supervision, Conceptualization, and Writing and editing. WH: Funding acquisition, Project administration, Resources, Validation, Visualization, Writing\u0026ndash; review \u0026amp; editing. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSharma B, Prall W, Bhatia G, Gregory BD. The Diversity and Functions of Plant RNA Modifications: What We Know and Where We Go from Here. Annu Rev Plant Biol. 2023;74:53\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGe L, Pan F, Jia M, Pott DM, He H, Shan H, Lozano-Duran R, Wang A, Zhou X, Li F. RNA modifications in plant biotic interactions. 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ECT12, an YTH-domain protein, is a potential mRNA m(6)A reader that affects abiotic stress responses by modulating mRNA stability in Arabidopsis. Plant Physiol Biochem. 2024;206:108255.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLuo W, Tang Y, Li S, Zhang L, Liu Y, Zhang R, Diao X, Yu J. The m(6) A reader SiYTH1 enhances drought tolerance by affecting the messenger RNA stability of genes related to stomatal closure and reactive oxygen species scavenging in Setaria italica. J Integr Plant Biol. 2023;65(12):2569\u0026ndash;86.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHao W, Wang W, Xiao X, Sun J, Wu B, Zhao Y, Pei S, Fan W, Xu D, Qin T. Genome-Wide Identification and Evolutionary Analysis of Gossypium YTH Domain-Containing RNA-Binding Protein Family and the Role of GhYTH8 in Response to Drought Stress. Plants (Basel) 2023, 12(5).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"YTH, Panax ginseng, m6A RNA methylation, gene family","lastPublishedDoi":"10.21203/rs.3.rs-7523802/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7523802/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eYTH domain-containing RNA-binding proteins function as m\u003csup\u003e6\u003c/sup\u003eA readers that specifically bind to m\u003csup\u003e6\u003c/sup\u003eA-modified RNAs. YTH domain-containing proteins participate in various biological processes, such as hormone signaling pathways, regulation of stress responses, RNA stability, and cellular differentiation. Despite these important roles, the characteristics and functions of \u003cem\u003eYTH\u003c/em\u003e family genes in ginseng (\u003cem\u003ePanax ginseng\u003c/em\u003e), a traditional medicinal herb, particularly regarding their response to MeJA treatment and salt stress on a genome-wide scale, have not yet been studied. In this study, 18 \u003cem\u003eYTH\u003c/em\u003e genes were identified based on telomere-to-telomere reference genome of ginseng. These \u003cem\u003ePgYTH\u003c/em\u003e genes were grouped into four subgroups by phylogenetic analysis. Moreover, the chromosomal distribution, synteny analysis, gene structures and \u003cem\u003ecis\u003c/em\u003e-elements of \u003cem\u003ePgYTH\u003c/em\u003e genes, and the motifs of YTH proteins were analyzed. Expression profiling results indicated that the \u003cem\u003ePgYTH\u003c/em\u003e genes were tissue-specific and spatiotemporally-specific in 14 different tissues of 4-year-old ginseng, in ginseng roots of four different ages, and among 42 different cultivars of 4-year-old ginseng roots. The expression of the majority of \u003cem\u003ePgYTH\u003c/em\u003e genes was downregulated in response to MeJA, an elicitor of the ginsenoside biosynthesis pathway. The expression of \u003cem\u003ePgYTH8-12\u003c/em\u003e was upregulated under salt treatment. Additionally, PgYTH12 was localized to the endoplasmic reticulum. Overall, these results lay the groundwork for future functional investigations of \u003cem\u003ePgYTH\u003c/em\u003e genes, advancing our understanding of their role in the regulation of the ginsenoside biosynthesis pathway and stress resistance in ginseng.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification and analysis of YTH gene family and its response to MeJA and salt treatment in Panax ginseng C. A. 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