Genome-wide identification and molecular characterization of the MAPK family members in sand pear (Pyrus pyrifolia)

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However, the rapid production of ethylene after harvest significantly shortens its shelf life, becoming a major limiting factor for enhancing its commercial value. Mitogen-activated protein kinase (MAPK), a highly conserved family of transferases in eukaryote. Although the importance of this family has been extensively studied in other plants, the precise composition and functional mechanisms of MAPK members in sand pear remain elusive. Results: This study conducted an in-depth analysis of four PpMAPK genes identified in the transcriptome of the ‘Whangkeumbae’( Pyrus pyrifolia ) and 22 PpMAPKs in the Pyrus pyrifolia genome, demonstrating a high degree of concordance between the transcriptomic and genomic data. Specifically, the transcriptomic PpMAPK3-L (GenBank accession number: PP992971), PpMAPK7-L(GenBank accession number: PP992972), PpMAPK10-L (GenBank accession number: PP992973), and PpMAPK16-L (GenBank accession number: PP992974) exhibited sequence homology values of 99.19%, 100%, 94.51%, and 95.75%, respectively, with their corresponding genomic counterparts (EVM0007944.1, EVM0004426.1, EVM0027166.1, EVM0023771, EVM0028755.1, EVM0015862.1). These findings indicate that the integrated analysis of transcriptomic and genomic data provides critical genetic insights into the MAPK genes in sand pear, culminating in the identification of a total of 25 PpMAPK genes in this species. Further phylogenetic analysis classified these genes into four subfamilies (A, B, C, and D), with subfamilies A and B each comprising six members, subfamily C with four members, and subfamily D with nine members. The potential functional differences among the gene members of each subfamily provide valuable clues for future research into MAPK signaling pathways. Additionally, interaction analysis revealed a significant interaction between PpMAPK3-L and PpbZIP2, which coordinatively regulate the senescence traits of fruits in sand pear through their joint influence during the senescence process. Conclusion: The results of this study suggest that PpMAPK3-L , PpMAPK7-L , PpMAPK10-L , and PpMAPK16-L are likely to play pivotal roles in the maturation and senescence of sand pear fruit. Specifically, the interaction between PpMAPK3-L and PpbZIP2 could play a key role in the regulation of fruit senescence, indicating that the MAPK signaling pathway may modulate the fruit's physiological state through interactions with transcription factors. This finding offers significant insights for further investigation into the functions of MAPK genes in the maturation and senescence of sand pear fruit and provides a new direction for investigating biotechnological approaches for delaying senescence and prolonging shelf life. Pyrus pyrifolia MAPK Molecular characterization BZIP Sand pear senescence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Senescence is the final stage of fruit development [1]. This process causes a series of irreversible events, such as fruit softening, respiratory bursts, enhanced ethylene production, cell wall modifications, and color changes [2]. According to the difference in ethylene produced during ripening and senescence, fruits are divided into non-climacteric and climacteric. Those fruits with low ethylene production and no significant ethylene peak during ripening and senescence were non-climacteric fruits. In the process of ripening and senescence, ethylene increased rapidly in the climacteric fruit [3]. Sand pear ( Pyrus pyrifolia ) is a typical climacteric fruit, it has a short shelf life and rapid senescence after harvesting, which limits the development of its industry. Therefore, delaying the aging of sand pear fruit and extending the shelf life of sand pear is the key problem facing the sand pear industry. Salicylic acid (SA), also known as 2-hydroxybenzoic acid, is a phenolic compound widely distributed in the plant kingdom. As a key signaling molecule and plant hormone, SA plays an essential role in various physiological processes in plants [4]. It is involved in fundamental physiological functions such as seed germination, photosynthesis, transpiration, stomatal regulation, thermogenesis, cell growth, and ion absorption. Moreover, SA plays a crucial role in the plant immune system, regulating both local and systemic defense mechanisms [5]. In recent years, SA has gained significant attention in the postharvest management of horticultural crops due to its remarkable effects in inhibiting senescence, delaying maturation, and enhancing resistance to various biotic and abiotic stresses [6]. Studies have shown that SA can significantly extend the shelf life of crops, reduce the incidence of diseases, and improve crop performance under adverse environmental conditions, highlighting its great potential in modern agriculture. Mitogen-activated protein kinase (MAPK), as a specific serine/threonine protein kinase, is one of the largest transferases in eukaryotes [7]. Because this kinase is associated with a group of proteins that are phosphorylated by tyrosine residues under the action of mitogen, it is named mitogen-activated protein kinase. MAPK belongs to a multi-gene family. So far, MAPK family members have been identified in a variety of plants. Such as Arabidopsis thaliana [8], maize [9], corn [10], bananas [11], apples [12], tomatoes [13], and cucumbers [14]. The MAPK signaling pathway plays a crucial role in a variety of biological processes. MAPK can catalyze the phosphorylation of substrate proteins by transferring a phosphate group from ATP to the amino acid residues of the substrate proteins. MAPK cascade systems are widely present in eukaryotic organisms and play an important role in regulating cell growth and development and responding to external environmental stimuli [15]. It has been shown that MabZIP21 can interact with MaMPK6-3. The interaction between MaMPK6-3 and MabZIP21 enhanced the transcriptional activation of MabZIP21. Ser-436 and Thr-318 are the major phosphorylation sites of MabZIP21 by MaMPK6-3 [16]. In rice, MAPK5 can interact with OsWRKY72 to regulate the growth, development, and defense response of rice [17]. In tomato, the yeast two-hybridization (Y2H) experiment showed that SlASR4 protein interacts with SlMAPK3 protein, representing a class of proteins with conserved ABA/WDS domains and forming protein complexes capable of responding to various stresses [18]. In Arabidopsis thaliana , the phosphokinase MAPK6 phosphorylates ACS6 and increases its protein stability, thereby increasing ethylene production [19]. Currently, most research on MAPK is focused on stress resistance and morphogenesis. As an important protein kinase, MAPK must play an important regulatory role in the process of fruit senescence. However, there are very few research reports on this aspect. Therefore, how MAPK regulates fruit senescence and the specific signal transduction mechanisms for regulation are the hot and key issues in fruit tree research. To reveal the relationship between MAPK signal and fruit shelf life, it is necessary to deeply analyze the unique role of MAPK signal in the sand pear fruit senescence process. Transcription factors (TFs) play a crucial role in plant physiological processes, acting as key regulators in plant growth, development, and responses to environmental changes [20]. Among all transcription factor families, the basic leucine zipper (bZIP) family is one of the largest and most diverse. The name of the bZIP transcription factor is derived from its unique structural domain-the bZIP domain, which typically consists of 60 to 80 amino acids and contains two functional regions: a basic region and a leucine zipper region [21]. In bZIP proteins, the basic region consists of approximately 16 amino acid residues and contains a highly conserved motif, N-x7-R/K-x9, which primarily functions in nuclear localization and DNA binding. In contrast, the leucine zipper region is composed of multiple heptad repeat motifs of amino acids, usually dominated by leucine (Leu), but may also include other large hydrophobic amino acids such as isoleucine (Ile), valine (Val), phenylalanine (Phe), or methionine (Met). Although the amino acid sequence in this region is more variable and not as highly conserved as the basic region, it plays a crucial role in protein dimerization. Specifically, the leucine zipper region is involved not only in the homodimerization of bZIP transcription factors but also in the formation of heterodimers with other transcription factors [22]. The bZIP gene family plays an indispensable role in various biological processes in plants. Extensive research has shown that bZIP transcription factors regulate plant growth, development, and responses to environmental stress. BZIP TFs control several aspects of plant development, particularly important growth events such as fruit ripening, and plant responses to environmental stress [23, 24]. For example, in peaches, the expression of PpbZIP genes is higher at the early stages of fruit maturation and gradually decreases as ripening progresses [25]. In lychees, several bZIP genes, including LcbZIP17 , LcbZIP4 , LcbZIP5/7/21 , LcbZIP2/19/28 , LcbZIP9/32/33/44/53 , and LcbZIP24/29/40/41 , are primarily expressed during the postharvest phase, participating in the regulation of fruit postharvest growth and maturation [26]. Additionally, the bZIP11 gene in Arabidopsis thaliana influences root development by regulating the connection between low-energy signals and auxin-mediated primary root growth [27]. In maize, overexpression of the ZmbZIP4 gene leads to increased lateral root numbers, elongation of primary roots, and a stronger root system [28]. In tomatoes, SlbZIP33 ( SlAREB1 ) not only participates in stress-induced responses but also regulates the expression of genes involved in critical metabolic pathways during fruit ripening, playing a key role in metabolic programming [29]. This study conducted an in-depth analysis of the transcriptomic and genomic data of the ‘Whangkeumbae’ ( Pyrus pyrifolia ) variety, 25 PpMAPK genes were identified in sand pear. These include four genes identified from the transcriptome and 22 genes discovered from the genome. To further explore the evolutionary relationships of these genes, a phylogenetic analysis was performed, revealing that these genes can be categorized into four major subfamilies (A, B, C, and D). Specifically, subfamilies A and B each contain six genes, subfamily C consists of four genes, and subfamily D, the largest, includes nine genes. These genes may have undergone varying degrees of functional differentiation during evolution, with potential functional differences among the subfamily members offering key insights into the mechanisms of the MAPK signaling pathway. Additionally, based on protein-protein interaction network analysis, the study identified that PpMAPK3-L interacts with the transcription factor PpbZIP2. This interaction may collaboratively regulate the fruit senescence process in sand pear during ripening, providing new perspectives on the molecular regulatory mechanisms of MAPK signaling during fruit maturation and senescence. These findings also present potential research directions for breeding sand pear varieties that delay fruit senescence. Results Identification and phylogenetic analysis of PpMAPK in sand pear This study identified four PpMAPK from the transcriptome data [30] of sand pear, which were designated as PpMAPK3-L, PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L. By comparing protein sequences in the genomic database [31], and removing duplicates and incomplete sequences, a total of 22 PpMAPK protein sequences were successfully identified in the sand pear genome. Among these genomic datasets, the sequence of PpMAPK3-L in the transcriptome exhibited a high similarity of 99.19% to that of EVM0007944 in the genome database. PpMAPK7-L was identical to EVM0004426, with a similarity of 100%. The sequence similarity between PpMAPK10-L and EVM0027166 was 94.51%, while PpMAPK16-L shared a similarity of 95.75% with EVM0023771. In summary, by integrating transcriptome and genomic data, a total of 25 PpMAPK were identified in the sand pear. Based on this, the study further constructed a phylogenetic tree of the PpMAPK genes to reveal the evolutionary relationships between these genes. According to the classification criteria for the MAPK protein family in Arabidopsis thaliana . Based on the homology with Arabidopsis thaliana MAPK proteins and their amino acid sequence characteristics, the MAPK proteins were classified into four subgroups according to the classification standards for the MAPK protein family in Arabidopsis thaliana . (Fig. 1). The classification results indicate that the MAPK family members of Arabidopsis thaliana and sand pear are distributed across all subgroups. In subgroups A, B, and C, the MAPK proteins are of the TEY type, while those in subgroup D are of the TDY type. Specifically, subgroup A includes three Arabidopsis MAPK members and six sand pear MAPK members; subgroup B consists of four Arabidopsis MAPK members and six sand pear MAPK members; subgroup C comprises four Arabidopsis MAPK members and four sand pear MAPK members; subgroup D has the largest number of MAPK members, with nine MAPK members in both Arabidopsis and sand pear. The analysis of these data provides a clearer understanding of the classification and evolutionary characteristics of the sand pear MAPK family. Phylogenetics, conserved motifs, domains , and gene structures of 22 PpMAPKs Based on the obtained MAPK protein sequences, this study constructed a phylogenetic tree of the PpMAPKs and systematically analyzed their conserved motifs, conserved domains, and gene structures (Fig. 2). The analysis revealed that all PpMAPK protein sequences contain at least eight motifs. The shared motifs across each subfamily include motif 1, motif 2, motif 4, motif 5, motif 6, and motif 9. Notably, motif 6 is present in all protein sequences, indicating that this motif is highly conserved within the MAPK family. As a conserved motif of MAPKs, motif 6 is characterized by the sequence T(D/E)YVV(A)TRWYRAPEL, where the TxY part corresponds to the "T-loop" structure. This structural feature not only reflects the structural consistency of MAPK proteins but also suggests that PpMAPK possesses highly conserved functional characteristics. Motif 9 represents the CD domain, which plays an essential functional role in all MAPK proteins (Fig. 2B). To further explore the conserved domains of PpMAPK, this study employed the CD-search tool from the National Center for Biotechnology Information (NCBI) database for in-depth analysis. The results indicated that all 22 PpMAPK proteins in sand pear contain the MAPK domain, highlighting the high functional conservation of these proteins. PpMAPK proteins in subgroups A, B, and C share the same conserved domain (STKc_TEY_MAPK), whereas PpMAPK proteins in subgroup D possess a distinct conserved domain (STKc_TDY_MAPK). These domain differences reveal the functional diversity between the subgroups (Fig. 2C). Further gene structure analysis showed a clear pattern in the number and distribution of exons and introns in PpMAPK genes. In particular, genes within the same evolutionary branch exhibited high similarity in exon and intron numbers and their distribution. All PpMAPK genes contain both introns and exons, with the PpMAPKs of the TDY type having relatively more exons, ranging from 9 to 11, with most genes containing 9 or 10 exons. The genes EVM0014865 , EVM0016074 , and EVM00180 have the highest number of exons, reaching 11. In contrast, the PpMAPKs of the TEY type typically have fewer exons, usually ranging from 2 to 6, with the genes EVM0020253 , EVM0008427 , and EVM0015322 having the fewest exons, with only 2. Additionally, 9 PpMAPK genes contain UTR regions, specifically PpMAPK7-L , EVM0001504 , PpMAPK3-L , EVM0014865 , EVM0033302 , EVM0000507 , EVM0016652 , EVM0027754 , and EVM0009261 . These results suggest that the PpMAPK genes have undergone structural and functional diversification during their evolution (Fig. 2D). P hysicochemical properties and c hromosomal localization of Pp MAPK s Through a systematic analysis of the physicochemical properties and related characteristics of 22 PpMAPK proteins, we observed significant differences in the number of amino acid residues in sand pear MAPK proteins (Table 1). Specifically, EVM0018010 has the largest number of amino acid residues, reaching 635, while EVM0001504 contains only 367, highlighting a significant difference between the two. Furthermore, the relative molecular mass of these proteins ranges from 42,064.88 Daltons (Da) to 71,683.59 Da, further indicating the structural diversity of PpMAPK proteins. In the isoelectric point (pI) analysis, the majority of PpMAPK proteins exhibited acidic characteristics, with 16 proteins having pI values lower than 7, indicating they are acidic proteins, while 6 proteins exhibited pI values above 7, indicating they are basic proteins. Notably, EVM0001504 had the lowest pI value of 5.06, making it the most acidic, while EVM0027166 and EVM0020832 both had pI values of 9.15, showing strong basic characteristics. Regarding protein instability, the instability index of PpMAPK proteins ranged from 31.11 to 48.19. Specifically, EVM0015322 exhibited the lowest instability index at 31.11, while EVM0000507 had the highest index at 48.19. The aliphatic amino acid index analysis revealed that PpMAPK16-L had the lowest index at 76.13, while EVM0020253 had the highest at 97.49. Hydrophilicity analysis showed that the GRAVY values of all PpMAPK family members were negative, further confirming that they are hydrophilic proteins. Subcellular localization prediction results indicated that these proteins are all localized in the nucleus, suggesting that they may be involved in signal transduction and regulatory processes within the nucleus. Table 1 The characterizations of PpMAPKs in sand pear. Gene name Gene ID Accession no. Amino acids No. Mw(Da) pI Instability index Aliphatic index GRAVY Subcellular localization EVM0008427 GWHPBAOS037580 372 42582.21 5.96 34.86 95.70 -0.227 Nucleus EVM0015322 GWHPBAOS017334 372 42605.31 6.38 31.11 96.21 -0.234 Nucleus MAPK7-L (GenBank accession number: PP992972) EVM0004426（100%) GWHPBAOS023153 379 43546.59 8.30 36.76 95.46 -0.203 Nucleus EVM0020253 GWHPBAOS015187 378 43357.27 7.26 37.45 97.49 -0.176 Nucleus EVM0033302 GWHPBAOS034299 373 42945.09 6.03 46.15 89.65 -0.369 Nucleus EVM0000507 GWHPBAOS001398 373 42962.96 6.02 48.19 89.12 -0.365 Nucleus EVM0016652 GWHPBAOS032599 378 43139.22 6.17 44.93 88.52 -0.335 Nucleus EVM0027707 GWHPBAOS012688 379 43147.21 5.97 43.26 89.31 -0.299 Nucleus EVM0026334 GWHPBAOS040261 370 42064.88 5.87 41.72 88.86 -0.305 Nucleus EVM0001504 GWHPBAOS005430 367 42183.22 5.06 39.39 95.91 -0.257 Nucleus EVM0038345 GWHPBAOS025676 370 42557.05 5.79 41.29 94.38 -0.259 Nucleus MAPK3-L (GenBank accession number: PP992971) EVM0007944 (99.19%) GWHPBAOS005442 370 42626.93 5.62 39.72 92.54 -0.32 Nucleus EVM0027754 GWHPBAOS024101 403 46322.92 5.90 41.59 85.21 -0.361 Nucleus EVM0009261 GWHPBAOS024083 403 46293.93 5.83 41.60 85.46 -0.346 Nucleus EVM0023260 GWHPBAOS016125 406 46384.87 5.66 42.74 86.75 -0.313 Nucleus EVM0016074 GWHPBAOS011680 621 70605.48 6.54 37.90 77.28 -0.648 Nucleus EVM0018010 GWHPBAOS031618 635 71683.59 6.36 38.07 76.65 -0.603 Nucleus EVM0014865 GWHPBAOS011287 583 66525.65 6.59 45.10 81.63 -0.494 Nucleus MAPK16-L (GenBank accession number: PP992974) EVM0023771 (95.75%) GWHPBAOS009972 564 63919.95 8.73 36.68 76.13 -0.502 Nucleus MAPK10-L (GenBank accession number: PP992973) EVM0027166 (94.51%) GWHPBAOS038791 618 70715.07 9.15 34.31 80.19 -0.486 Nucleus EVM0040595 GWHPBAOS024513 608 68982.2 9.04 38.51 80.53 -0.458 Nucleus EVM0020832 GWHPBAOS006687 598 67812.97 9.15 38.37 79.93 -0.438 Nucleus In conclusion, the PpMAPK proteins from sand pear exhibit significant diversity in their physicochemical properties, which may be closely related to the differentiation of their functions within plant cells, providing a possible physiological basis for their roles in various biological processes. To further explore the distribution of PpMAPK gene family members in the genome, we conducted chromosome mapping and visualization analysis using TBtools software (Fig. 3). The analysis revealed that the PpMAPK genes are randomly distributed across 11 chromosomes of the sand pear genome, located on chromosomes 1, 2, 3, 6, 7, 8, 9, 11, 13, 14, and 15. However, the gene EVM0026334 could not be successfully mapped to any chromosome. The distribution of PpMAPK genes on each chromosome showed significant variation, with the highest number of genes found on chromosomes 2, 11, and 15, each containing three genes. On other chromosomes, the number of PpMAPK genes was relatively low, particularly on chromosomes 1, 7, 8, and 9, which each contained only one PpMAPK gene: EVM0000507 , EVM0033302 , EVM0008427 , and PpMAPK10-L , respectively. These results indicate an uneven distribution of PpMAPK genes in the sand pear genome, with the majority of genes located on specific chromosomes. This provides important genomic insights for further studying the functions and evolution of PpMAPK genes. Cis-element analysis of PpMAPK gene promoters To investigate the functional roles of the PpMAPK gene family in greater depth, we extracted the 2 kb upstream sequences of 22 PpMAPK genes in sand pear and conducted a systematic analysis of their hormone-responsive cis-regulatory elements (Fig. 4). Notably, no hormone response elements were identified in the upstream sequence of the EVM0020832 gene. In contrast, the upstream sequences of the remaining 21 PpMAPK genes contain five distinct types of hormone-responsive cis-regulatory elements, namely abscisic acid, auxin, gibberellin, jasmonic acid, and SA response elements. Among these five hormone response elements, the abscisic acid response element was the most common. Except for EVM0020253 , EVM0023260 , EVM0018010 , and EVM0014865 , nearly all PpMAPK genes contain this element. The auxin response element, on the other hand, was relatively rare and was found in only seven genes, including EVM0015322 , PpMAPK7-L , EVM0020253 , EVM0016652 , EVM0009261 , EVM0040595 , and EVM0027754 . The gibberellin response element, however, was more widely distributed, being present in the promoter sequences of 11 genes: EVM0008427 , EVM0020253 , EVM0016652 , EVM0027707 , EVM0001504 , PpMAPK3-L , EVM000926 , EVM0023260 , EVM0014865 , PpMAPK10-L , and EVM0027754 . The Methyl Jasmonate response element also showed a relatively broad distribution, with only a few genes lacking this cis-regulatory element: EVM0033302 , EVM0027707 , EVM0026334 , EVM0001504 , and EVM0018010 . The SA response element, however, was more limited in distribution, being present in only seven genes: EVM0008427 , PpMAPK7-L , EVM0000507 , EVM0027707 , EVM0038345 , EVM0023260 , and EVM0018010 . In conclusion, the presence of various hormone-responsive cis-regulatory elements in the PpMAPK gene family of sand pear suggests that these genes may play important roles in plant hormone signaling pathways. The different hormone response elements likely reflect the functional differences of these genes in various physiological processes. Expression patterns of PpMAPKs in sand pear ( Pyrus pyrifolia ) Specific quantitative primers for PpMAPK3-L , PpMAPK7-L , PpMAPK10-L , and PpMAPK16-L genes were designed based on transcriptome data, with their primers provided in Additional file 1 (Table S1). Using these primers, detailed expression pattern analyses of the four PpMAPK genes were conducted across different tissue types (Fig. 5). The results revealed significant tissue-specific expression of the PpMAPK genes. Specifically, PpMAPK3-L exhibited the highest expression in petals and the lowest in leaves (Fig. 5A). PpMAPK7-L showed the lowest expression in leaves but peaked in shoot tips (Fig. 5B). PpMAPK10-L was predominantly expressed in anthers, with minimal expression in leaves (Fig. 5C). Similarly, PpMAPK16-L exhibited the weakest expression in leaves but showed the strongest expression in shoots (Fig. 5D). Furthermore, all four PpMAPK genes were expressed to varying degrees in fruit tissues, suggesting their potential involvement in fruit development. To further explore the roles of PpMAPK genes in fruit development, their expression patterns were dynamically tracked across different stages of fruit development (Fig. 6). The results indicated that these genes were expressed post-harvest, each displaying distinct expression peaks. Specifically, PpMAPK3-L and PpMAPK10-L reached their highest expression levels 25 days after harvest (DAH), while PpMAPK7-L peaked at 30 DAH. In contrast, PpMAPK16-L exhibited its highest expression as early as 5 DAH. Regulatory analysis of PpMAPK genes by SA and ETH To explore the potential roles of PpMAPK genes in the process of fruit senescence, pears were treated post-harvest with different concentrations of SA: 0.002, 0.02, 0.2, and 2 mM (Fig. 7A, B, C, D). The expression of the four PpMAPK genes was then monitored. The experimental results showed that at a concentration of 0.002 mM SA, no significant differences in the expression levels of the four PpMAPK genes were observed compared to the control group. Under the 0.02 mM SA treatment, the expression level of PpMAPK10-L significantly increased, suggesting that it may play an important regulatory role under this condition. In contrast, the expression changes of other PpMAPK genes were relatively small. When the SA concentration was increased to 0.2 mM, only PpMAPK3-L exhibited significantly higher expression compared to the control group, suggesting that this concentration of SA may stimulate the expression of PpMAPK3-L , while the expression levels of the other three genes ( PpMAPK7-L , PpMAPK10-L , and PpMAPK16-L ) showed no significant differences from the control, indicating that these genes might be less sensitive or less responsive to this concentration of SA. At 2 mM SA, the expression of PpMAPK3-L was significantly lower than that of the control group, suggesting that this high concentration of SA may inhibit the expression of PpMAPK3-L . In contrast, PpMAPK7-L exhibited significantly higher expression, indicating that this concentration of SA may upregulate PpMAPK7-L expression through some mechanism. However, the expression of PpMAPK10-L and PpMAPK16-L did not differ significantly from the control group, suggesting that these two genes might be less responsive or unaffected by the 2 mM SA treatment. To further elucidate the role of PpMAPK genes in fruit senescence and based on previous experimental designs, the flesh of post-harvest ‘Whangkeumbae’ pear was treated with 1 µ/L ethephon (ETH), and the expression of PpMAPK genes was analyzed at 3, 6, 12, 24, 36, and 48 h after treatment (Fig. 7E, F, G, H). The results showed that the expression of PpMAPK3-L was significantly lower at 3, 6, 36, and 48 h post-treatment compared to the control group, suggesting that ETH may inhibit the expression of this gene through some mechanism. The expression of PpMAPK7-L was significantly higher at 12 h post-treatment and also showed a significant increase at 36 h, indicating that ETH may activate the expression of this gene after prolonged treatment. The expression of PpMAPK10-L was significantly higher at 3, 24, and 48 h post-treatment, suggesting that ETH may participate in the fruit senescence process by up-regulating the expression of this gene at these time points. Finally, the expression of PpMAPK16-L was significantly increased at all time points (3, 12, 24, 36, and 48 h) after ETH treatment, indicating that ETH treatment may enhance the role of PpMAPK16-L in fruit senescence. Interaction between PpMAPK3-L and PpbZIP2 Based on the aforementioned experimental results, the four PpMAPK genes each play distinct roles in fruit senescence. To further investigate the potential interaction mechanisms of these MAPKs during fruit senescence, we employed the online tool STRING to predict their potential interacting proteins. The prediction results suggested that PpMAPK3-L may interact with PpbZIP2 (Gene ID: EVM0004317.1), while PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L may interact with PpbZIP3 (Additional file 2: Fig. S1). To further confirm these predictions, we established a Y2H system to analyze the interactions between PpMAPK and PpbZIP. PpMAPK3-L, PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L were individually cloned into the pGBKT7 (BD) vector to generate the recombinant vectors PpMAPK-BD. Concurrently, PpbZIP2 and PpbZIP3 were cloned into the pGADT7 (AD) vector to yield the recombinant vectors PpbZIP-AD. Initially, self-activation assays were conducted to verify that the constructed vectors did not possess the ability to spontaneously activate transcription. The experimental results demonstrated that after co-transforming the four PpMAPK-BD and PpbZIP-AD vectors, cells were able to grow normally on synthetic dropout medium/-tryptophan/-leucine (SD/-Leu/-Trp) medium, confirming the successful introduction of these genes into AH109 competent cells. Further tests revealed no growth on SD/-Trp/-Leu/-histones/-adenine/X-alpha gal (SD/-Trp/-Leu/-His/-Ade/X-α-gal) medium, confirming the absence of self-activation in the PpMAPK genes (Additional file 3: Fig. S2). Next, after co-transforming the four PpMAPK-BD vectors with the two PpbZIP-AD vectors into AH109 cells, the results revealed normal growth on SD/-Leu/-Trp medium, confirming the successful introduction of PpMAPK and PpbZIP into the competent cells. Among these experiments, only PpMAPK3-L-BD and PpbZIP2-AD exhibited significant growth on SD/-Leu/-Trp/-Ade/-His medium, further confirming the existence of an interaction between PpMAPK3-L and PpbZIP2. This study offers compelling evidence for understanding the molecular mechanisms of PpMAPK and PpbZIP proteins in fruit senescence and provides new insights for future functional research and regulation of fruit senescence (Fig. 8). Expression Pattern of PpbZIP2 To further investigate the interaction mechanism between PpMAPK3-L and PpbZIP2 , this study comprehensively analyzed the expression pattern of PpbZIP2 . The results revealed that PpbZIP2 is expressed in various tissues of sand pear, with the highest expression observed in the leaves and the lowest in the petals. This suggests that PpbZIP2 may play distinct physiological roles in different tissues (Fig. 9A). To explore the role of PpbZIP2 during fruit development, the expression dynamics of PpbZIP2 throughout fruit development were monitored. The analysis showed that the expression of PpbZIP2 fluctuated during fruit development, peaking at 30 DAH and reaching its lowest level of 60 days after full bloom (DAFB) (Fig. 9B). Notably, the expression of PpbZIP2 is induced by SA (Fig. 9C), while ETH significantly inhibits its expression (Fig. 9D). This finding provides crucial insights into the role of PpbZIP2 in fruit senescence and reveals potential molecular mechanisms by which SA and ETH regulate PpbZIP2 expression. Discussion In this study, four PpMAPK genes were initially identified from the transcriptome of sand pear ( Pyrus pyrifolia ) and named PpMAPK3-L , PpMAPK7-L , PpMAPK10-L , and PpMAPK16-L . Subsequently, using 20 Arabidopsis thaliana MAPK protein sequences (AtMAPK1–AtMAPK20) as query sequences [8], an additional 22 MAPK family members were identified in the sand pear genome database through BLAST comparison and domain validation. The results revealed that PpMAPK7-L shows a 100% similarity with the EVM0004426 sequence in the genome. Based on the combined transcriptomic and genomic data analysis, a total of 25 PpMAPK genes were identified in sand pear, providing a solid foundation for a comprehensive study of the PpMAPK family. Comparative analysis revealed a remarkable diversity of MAPK genes across plant species. MAPK family members were identified in various plants, including rice [32], maize [9], chickpea [33], kiwifruit [34], barley [35], Gossypium raimondii [36], bread wheat [37], Fragaria vesca [38], Fagopyrum tataricum [39], Brachypodium distachyon [40], tomato [13], tobacco [41], poplar [42], apple [12], soybean [43] with varying numbers of family members (Additional file 4: Table S2 ). This indicates that, although the MAPK signaling pathway is highly conserved in eukaryotes, the evolution and function of its family members exhibit considerable species-specific variation. This study conducted an in-depth analysis of the tissue-specific expression patterns of the PpMAPK genes. PpMAPK3-L is highly expressed in petals, PpMAPK7-L shows dominant expression in the shoots, PpMAPK10-L is mainly expressed in the anthers, while PpMAPK16-L exhibits the highest expression in the shoots. Furthermore, all four genes are expressed the least in the leaves (Fig. 5 ). As observed in other plants, the expression patterns of the MAPK genes in sand pear exhibit significant tissue specificity. For example, in buckwheat, FtMAPK1 and FtMAPK3 are highly expressed in the roots, stems, and leaves. In watermelon, distinct expression patterns of MAPK genes are observed in the roots, stems, and leaves [44]. In addition, the PpMAPK genes also exhibit distinct dynamic expression profiles during the senescence process of sand pear fruit. PpMAPK3-L and PpMAPK10-L peak at 25 DAH, PpMAPK7-L peaks at 30 DAH, while PpMAPK16-L shows the highest expression at 5 DAH (Fig. 6 ). These results suggest that these genes may be involved in the fruit senescence process through different temporal regulatory mechanisms. This study further verified the importance of the MAPK signaling pathway in hormone response, and revealed the response mechanism of four PpMAPK genes under different hormone conditions. The results showed that these genes were significantly regulated by SA and ETH, respectively (Fig. 7 ), suggesting that the PpMAPK signaling pathway may play a key role in the physiological regulation of fruit through hormone-mediated mechanisms. The expression levels of four PpMAPK genes also showed significant differences between diseased fruit and control fruit. Specifically, the expression levels of PpMAPK3-L and PpMAPK10-L in fruit were significantly up-regulated, which may be related to disease-induced defense response. In contrast, the expression of PpMAPK7-L and PpMAPK16-L in diseased fruit was significantly lower than that in control, suggesting that they may play an important role in the maintenance of healthy fruit (Additional file 5: Fig. S3 ). These differences indicate that different PpMAPK genes may play a role in the mechanism of pear fruit disease resistance. To further investigate the potential interaction mechanisms of MAPK genes in fruit senescence, this study first used the online tool STRING to predict possible interacting proteins of these MAPK genes (Additional file 2: Fig. S1 ). The predicted interactions were then validated using Y2H technology. The experimental results demonstrated an interaction between PpMAPK3-L and PpbZIP2 (Fig. 8 ). This finding is consistent with previous studies, which have shown that many MAPK members interact with transcription factors to play critical roles in various physiological processes in plants. For instance, the interaction between MPK4 and MYB1 is crucial for regulating anthocyanin accumulation [45], while the interaction between MdERF17 and MdMPK4 is important at different stages of plant growth [46]. In sand pear fruit, the expression of PpbZIP2 peaked at 30 DAH (Fig. 9 B). Further experiments revealed that SA significantly induced PpbZIP2 expression (Fig. 9 C), while ETH treatment significantly suppressed it (Fig. 9 D). These findings suggest that the expression of PpbZIP2 is regulated by various signaling molecules. Additional mechanistic studies revealed that the interaction between PpbZIP2 and PpMAPK3-L may delay fruit senescence through synergistic effects, regulating the expression of senescence-related genes, thus providing new molecular mechanisms and research directions for preserving sand pear fruit. In summary, this study revealed the potential function of the MAPK genes in fruit senescence and disease resistance regulation, and provided valuable clues for resolving the biological role of the MAPK genes in complex physiological processes of pear fruit. More importantly, these findings laid an important theoretical foundation for improving stress resistance, extending the shelf life, and improving the quality of fruit by molecular breeding in the future, and pointed out the direction for further research on the MAPK signaling pathway. Conclusion This study provides an in-depth analysis of the PpMAPKs in ‘Whangkeumbae’ ( Pyrus pyrifolia ) through the integration of transcriptomic and genomic data. A total of 25 PpMAPK genes were identified, including four from transcriptomic data ( PpMAPK3-L , PpMAPK7-L , PpMAPK10-L , and PpMAPK16-L ) and 22 from genomic data. Further homologous sequence alignment revealed that the transcriptomic PpMAPKs (PpMAPK3-L, PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L) shared high sequence identity with their corresponding genomic counterparts (EVM0007944.1, EVM0004426.1, EVM0027166.1, EVM0023771, EVM0028755.1, and EVM0015862.1), with identity values of 99.19%, 100%, 94.51%, and 95.75%, respectively. This indicates a high degree of consistency in gene identification between the transcriptomic and genomic data. These findings provide a solid data foundation for understanding the genetic characteristics of MAPK genes in Pyrus pyrifolia . Phylogenetic analysis revealed that the 25 PpMAPKs were classified into four subfamilies (A, B, C, and D). Subfamilies A and B each contained six members, subfamily C contained four members, and subfamily D comprised nine members. The potential functional differences among the gene members of each subfamily provide valuable insights for the study of MAPK signaling pathways. Further interaction analysis identified a significant interaction between PpMAPK3-L and the transcription factor PpbZIP2. Based on this finding, it is hypothesized that PpMAPK3-L and PpbZIP2 may jointly regulate fruit senescence in Pyrus pyrifolia , offering new perspectives for regulating the MAPK signaling pathway in plant senescence processes. Materials and Methods Plant materials Pear ( Pyrus pyrifolia ) fruit were selected as materials from Hebei Agricultural University. Flowers were marked at the flowering stage to assess the fruit ripening stage. Fruit were harvested at these stages: 30, 60, 90, 120, 130, 140, and 145 DAFB. The harvested flesh were quickly frozen with liquid nitrogen and stored in the refrigerator at -80℃ for later use. Similarly, shoots, stems, young leaves, petals and anthers taken from pear trees were taken back to the laboratory and placed in a -80℃ refrigerator. The ripe pear fruit of 10 DAH were immediately brought back to the laboratory. The pulp were treated with 0.002, 0.02, 0.2 and 2 mM SA for 12 h, respectively. Double-distilled water treatment as the control. The fresh meat slices of the fruit were immediately frozen in liquid nitrogen and stored at -80℃ for further analysis. Genome-wide identification of MAPK in sand pear First, all of the DNA sequences (FASTA format), cDNA sequences (FASTA format), CDS sequences (FASTA Format), the entire protein sequence (FASTA format), and the GFF3 gene annotation file were downloaded from the national genome data center (NGDC) ( https://bigd.big.ac.cn/gwh ). From the classification of protein database ( https://www.ebi.ac.uk/interpro/download/Pfam/ ) download the Hidden Markov Model (HMM). TBtools' simple HMM search tool was used to obtain all protein sequences with protein kinase domains, set the confidence to 10 − 5 [47]. Only one transcript is kept for each gene and the others are deleted. At NCBI-CDD ( https://www.ncbi.nlm.nih.gov/-cdd/ ) and SMART test conservative ( http://smart.embl.de/ ) integrity of the domain. Construction and beautification of phylogenetic tree A total of 22 MAPK family members were identified in sand pear, and 4 MAPK members were identified in transcriptome. The sequence similarity between PpMAPK7-L and EVM0004426.1 was 100%. NCBI blast was used to find the protein sequences of apple, tomato, tobacco and Arabidopsis that were closely related to the four MAPKs. The protein sequences of all MAPK members were sorted into FASTA format, and the FASTA file was put into MEGA7.0 software [48]. Multiple sequences were compared by ClustalW, the phylogenetic tree was constructed by neighbor-joining (NJ) [49]. The Artificial Intelligence (AI) software was used to add branching colors and grouping information to the constructed evolutionary tree. Identification of physical and chemical properties of MAPK family members TBtools was used to extract CDS sequences, protein sequences and promoter sequences of MAPK family members for subsequent analysis. On Expasy website ( https://web.expasy.org/protparam/ ) protein hydrophobicity, molecular weight and pI were predicted. The online site Plant-mPLoc ( http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ ) was used to predict the location of PpMAPKs. STRING ( https://string-db.org/ ) was used to predict interacting proteins of PpMAPKs. Analysis of motifs, conserved domains and gene structure of PpMAPKs The evolutionary relationship of PpMAPK was analyzed and identified by using MEGA 7.0 software. The PpMAPK protein sequences were submitted to NCBI-CDD and the online MEME tool ( https://meme-suite.org/meme/index.html ) for domain and motif identification. The genomic annotation file of Pyrus pyrifolia was obtained from NGDC and the gene structure was analyzed. The CDS sequences were compared with the corresponding genomic DNA sequences using the Gene Structure Display Server (GSDS) tool ( http://gsds.cbi.pku.edu.cn/ ) to analyze the gene structure. The phylogenetic tree, conserved motifs, domains and gene structure diagrams of PpMAPK were constructed by using TBtools software. Chromosome localization and promoter cis-acting elements of PpMAPK family TBtools software was used to analyze the pear genome annotation file, determine the chromosome location information of each PpMAPK sequence, and draw the corresponding chromosome location map. Place the sequence obtained in TBtools at the first 2000 bp of the start codon ATG in the Plant CARE website ( https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Hormone-related elements were screened among all cis-acting elements. The number of each cis-acting element was counted, the Heatmap tool of TBtools was used to classify and visually analyze promoters of PpMAPK family members. Map the Location of MAPK family members on chromosomes via using the Gene Location Visualize from GTF/GFF tool in TBtools. Artificial intelligence was used to beautify pictures. Y2H assay Complete coding DNA sequences (CDS) of PpMAPK3-L , PpMAPK7-L , PpMAPK10-L , and PpMAPK16-L were obtained based on BLAST analysis of the sand pear transcriptome database [30]. The recombinant plasmid PpMAPK3-L -pGBKT7, PpMAPK7-L -pGBKT7, PpMAPK10-L -pGBKT7, and PpMAPK16-L -pGBKT7 were successfully constructed by connecting PpMAPK3-L, PpMAPK7-L, PpMAPK10-L and PpMAPK16-L with the decoy vector pGBKT7, respectively. The CDS sequences of PpbZIP2 and PpbZIP3 were connected to the prey vector to construct PpbZIP2 -pGADT7 and PpbZIP3 -pGADT7. The constructed PpMAPK3-L -pGBKT7, PpMAPK7-L -pGBKT7, PpMAPK10-L -pGBKT7, and PpMAPK16-L -pGBKT7 plasmids were fused with PpbZIP2 -pGADT7, PpbZIP3-pGADT7, respectively. The fusion plasmid was co-transferred to yeast AH109, and the transformed bacterial solution was coated on the yeast medium Trp and Leu. After incubation at 30℃ for 3 days, positive colonies were selected and strewed on SD/-Trp/-Leu and SD/-Trp/-Leu/-Ade/X-α-gal plates, respectively. Protein-protein interactions were observed after incubation at 30℃ for 3 days. Prey vectors and bait vectors without target sequences were used as negative control. RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) According to the manufacturer instructions, use RNAprep Pure Plant Plus Kit (Tian Gen, Beijing, China) Kit to extract total RNA. Using FastQuant RT Kit (with gDNase) (Tian Gen, Beijing, China) Kit will RNA transcription for cDNA. Magic SYBR mixture (CoWin Biosciences, China) was used for the qRT-PCR reaction. The fluorescence quantitative PCR instrument employed in this study was the Mastercycler ep realplex 4 (Eppendorf AG, Hamburg, Germany). The internal references utilised in this study were the PpUBI (GenBank number: AF195224) gene. Primers for RT-qPCR analysis are listed in Additional file 1 (Table S1 ). Data analysis The data represent the mean ± SD of at least three replicates. Univariate analysis of variance (ANOVA)/Duncan's multiple range test were used for the overall significance of the differences. All data were analyzed in SPSS v27.0 (IBM Corp., Armonk, NY, USA). Abbreviations MAPK: Mitogen-activated protein kinase; SA: Salicylic acid; Y2H: yeast two-hybridization; TFs: Transcription factors; bZIP: basic leucine zipper; Leu: leucine; Ile: isoleucine; Val: valine; Phe: phenylalanine; Met: methionine; NCBI: National Center for Biotechnology Information; Da: Dalton; pI: isoelectric points; DAH: days after harvest; ETH: ethephon; BD: pGBKT7; AD: pGADT7; SD/-Trp/-Leu: synthetic dropout medium/-tryptophan/-leucine; SD/-Trp/-Leu/-His/-Ade/X-α-gal: SD/-Trp/-Leu/-histones/-adenine/X-alpha gal; DAFB: days after full bloom; NGDC: national genome data center; HMM: Hidden Markov Model; NJ: neighbor-joining; GSDS: Gene Structure Display Server; AI: Artificial Intelligence; CDS: Complete coding DNA sequences; qRT-PCR: quantitative real-time polymerase chain reaction; ANOVA: Analysis of variance Declarations Supplementary Information The online version contains supplementary material availabie at Acknowledgments The authors extend their gratitude to Professor Chen Liang (Chinese Academy of Sciences) for his valuable assistance with providing the yeast two-hybrid vectors. Authors ’ contribution The work presented here was a collaborative effort among all the authors. SHY formulated the research topic. SHY, XY, and WHY analyzed data and interpreted the results. SHY and XY wrote the manuscript. SHY, XY and WHY revised the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the National Natural Science Foundation of China (32272654), the Natural Science Foundation of Hebei Province, China (C2023204016), the Hebei Province Introduced Overseas-Scholar Fund, China (C20220361), and the Hebei Province Outstanding Youth Fund, China (2016, 2019). Availability of data and materials The genome sequence information contained in this study were obtained from following websites. Pyrus pyrifolia cultivar ‘Cuiguan’ genome file from the National Genomics Date Center (NGDC): https://ngdc.cncb.ac.cn/. Arabidopsis protein sequences from the Arabidopsis information resource (TAIR) database: https://www.arabidopsis.org/. Ethics approval and consent to participate The ‘Whangkeumbae’ was used in the current study was obtained from the experimental farm of Hebei Agricultural University, China. All the required permissions have been obtained, and all the plant materials during the current study were provided free of charge and maintained following the international guidelines. Consent for publication Not applicable. Competing interests The authors declare that they have no conflicts of interest. References Liang CZ, Zheng GY, Li WZ, Wang YQ, Hu B, Wang HR, et al. MT delays leaf senescence and enhances salt stress tolerance in rice. J Pineal Res. 2015;59:91–101. Gapper NE, Mcquinn RP, Giovannoni JJ. Molecular and genetic regulation of fruit ripening. Plant Mol Biol. 2013;82:575–591. Adams-Phillips L, Barry C, Giovannoni J. Signal transduction systems regulating fruit ripening. Trends Plant Sci. 2004;9:331–338. Raskin I. Salicylate, a new plant harmone. Plant Physiol. 1992;99:799–803. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6141919","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":424314919,"identity":"28b4fa21-6c99-4161-8e00-ea175bf1bc20","order_by":0,"name":"Yue Xu","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Xu","suffix":""},{"id":424314920,"identity":"36191744-176d-46dc-b8a9-5c62148c06f7","order_by":1,"name":"Huiying Wang","email":"","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Huiying","middleName":"","lastName":"Wang","suffix":""},{"id":424314921,"identity":"a159279e-0db9-43a0-a5e7-7a5ef6155d07","order_by":2,"name":"Haiyan Shi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACCRBhwJDAwMB8gOGBAWla2BIYEojXwgDSwgPWSBjIz+4x+/Cj4E4ev0TOxw8JBYcTtzMwP3x0A48WgztnjGf2GDwrlpyRu1kiweBw4s4GNmPjHHxaJHKMgU46nLjhdu4GsJYNB3jYpPFpkZ+RY8z4B6wl5/EPorQw3MgxZobYksNGnC0GN9KKmWUMDhdLzn9mZpFgkG684TABv8jPSN7M+ObP4Tx+nsOPb3z4Yy274Xjzw8d4HcbAgRJ9zcBUgFc5CLA/QObVEVQ/CkbBKBgFIw8AAP12Ug4MenVPAAAAAElFTkSuQmCC","orcid":"","institution":"Hebei Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Haiyan","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-03-03 02:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6141919/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6141919/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-11672-0","type":"published","date":"2025-05-15T15:57:40+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":77982459,"identity":"707764ee-7c87-438d-a3ec-9747a40f6400","added_by":"auto","created_at":"2025-03-07 13:08:03","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":667672,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree and classification of 25 members of the MAPK family of the sand pear genome. ClustalW method was applied to compare the amino acid sequence of 25 MAPK proteins, bootstrap analysis was performed on 1000 repeating sequences by NJ method, and the phylogenetic tree was constructed.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/9c212348a4025b10cf74887c.png"},{"id":77982174,"identity":"a3f841d1-8146-4fc8-9490-9b4478c602b0","added_by":"auto","created_at":"2025-03-07 13:00:03","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":302588,"visible":true,"origin":"","legend":"\u003cp\u003eEvolutionary relationship, conserved protein motif, domain and gene structure of PpMAPKs. \u003cstrong\u003eA\u003c/strong\u003e The phylogenetic tree was constructed based on protein sequences using the NJ method by MEGA7.0. \u003cstrong\u003eB-E\u003c/strong\u003e Motif compositions, domains and Exon/intron structures of the PpMAPKs.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/63ce3fb6f16245cf1795546c.png"},{"id":77982458,"identity":"f82ae59a-19d5-4c4a-a1a0-a071553edc7f","added_by":"auto","created_at":"2025-03-07 13:08:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221953,"visible":true,"origin":"","legend":"\u003cp\u003eChromosome mapping of \u003cem\u003ePpMAPKs\u003c/em\u003e in sand pear.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/ae0ac6c14e5805e5e5b7eb44.png"},{"id":77983397,"identity":"cd29e23f-060e-45c5-b025-2f91285480c4","added_by":"auto","created_at":"2025-03-07 13:16:03","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":391158,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of cis-acting elements in the promoter region of \u003cem\u003ePpMAPK\u003c/em\u003e genes. \u003cstrong\u003eA\u003c/strong\u003e Types of hormone-responsive cis-acting elements contained in \u003cem\u003ePpMAPKs\u003c/em\u003e. \u003cstrong\u003eB\u003c/strong\u003eThe numbers of cis-acting components that \u003cem\u003ePpMAPKs\u003c/em\u003e contain.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/315cb4ef83d733151ad65562.png"},{"id":77982461,"identity":"268f573d-f4cf-4322-ae38-ad74e0c5f0d0","added_by":"auto","created_at":"2025-03-07 13:08:03","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59469,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L, PpMAPK10-L\u003c/em\u003e and \u003cem\u003ePpMAPK16-L\u003c/em\u003ein ‘Whangkeumbae’ tissue.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/098c0136e1374f96212ad1d6.png"},{"id":77982177,"identity":"333d350e-8ef7-4dee-b3b0-7f927e1ca670","added_by":"auto","created_at":"2025-03-07 13:00:03","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":69813,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e and \u003cem\u003ePpMAPK16-L\u003c/em\u003ein ‘Whangkeumbae’during fruit development.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/b556ded5469f5a3b1661d53c.png"},{"id":77983398,"identity":"80b06295-2a8f-401d-9b41-61a1fc382be0","added_by":"auto","created_at":"2025-03-07 13:16:03","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":92944,"visible":true,"origin":"","legend":"\u003cp\u003eSA and ETH regulate the expression of \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e and \u003cem\u003ePpMAPK16-L.\u003c/em\u003e\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/1e4bc5aa44f4b1c6ddea11a1.png"},{"id":77982186,"identity":"4ef7f63f-9b27-47d0-a83b-298083f18131","added_by":"auto","created_at":"2025-03-07 13:00:03","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":675693,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-protein interactions between PpMAPKs and PpbZIPs.\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/d53224240337afb086d3fa07.png"},{"id":77982190,"identity":"7449eeac-97b2-4c24-9573-0b663d275780","added_by":"auto","created_at":"2025-03-07 13:00:03","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":96250,"visible":true,"origin":"","legend":"\u003cp\u003eExpression pattern of \u003cem\u003ePpBZIP2\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003e The expression of \u003cem\u003ePpbZIP2\u003c/em\u003e in various tissues of the ‘Whangkeumbae’. \u003cstrong\u003eB\u003c/strong\u003e Changes in the expression of \u003cem\u003ePpbZIP2\u003c/em\u003e during fruit development in ‘Whangkeumbae’. \u003cstrong\u003eC\u003c/strong\u003eThe regulatory effect of SA on the expression of \u003cem\u003ePpbZIP2\u003c/em\u003e. \u003cstrong\u003eD \u003c/strong\u003eETH on the expression of \u003cem\u003ePpbZIP2\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Fig.9.png","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/d01e083a114486bba27da2c9.png"},{"id":83067814,"identity":"89c10ea5-4021-46c3-b31a-669b98061114","added_by":"auto","created_at":"2025-05-19 16:06:35","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3409172,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/bb33fc35-7d60-4853-b707-2f2bf6feb3fe.pdf"},{"id":77982457,"identity":"c97ef58d-9b36-48b7-b29c-760a3ec2d16f","added_by":"auto","created_at":"2025-03-07 13:08:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16224,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/b28f7a04c66eababcc735a78.docx"},{"id":77982182,"identity":"b5ab4698-d05a-4421-afcb-16a7494bccd4","added_by":"auto","created_at":"2025-03-07 13:00:03","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":395528,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/e82bb1de647990ab7d52b7df.docx"},{"id":77982467,"identity":"d73200cc-f000-4edb-a377-5a72e3cf52ee","added_by":"auto","created_at":"2025-03-07 13:08:03","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":652127,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/5113c491a08f4188222e37d6.docx"},{"id":77983399,"identity":"d211bffd-e909-48d4-bda8-65552d4a89c5","added_by":"auto","created_at":"2025-03-07 13:16:03","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":12694,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile4.docx","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/e73004869a24bf75579a383c.docx"},{"id":77982192,"identity":"b7e9df84-dc13-456b-8840-4a562a0c9980","added_by":"auto","created_at":"2025-03-07 13:00:03","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":126031,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile5.docx","url":"https://assets-eu.researchsquare.com/files/rs-6141919/v1/1239e6813e76a32e1461ed38.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification and molecular characterization of the MAPK family members in sand pear (Pyrus pyrifolia)","fulltext":[{"header":"Background","content":"\u003cp\u003eSenescence is the final stage of fruit development [1]. This process causes a series of irreversible events, such as fruit softening, respiratory bursts, enhanced ethylene production, cell wall modifications, and color changes [2]. According to the difference in ethylene produced during ripening and senescence, fruits are divided into non-climacteric and climacteric. Those fruits with low ethylene production and no significant ethylene peak during ripening and senescence were non-climacteric fruits. In the process of ripening and senescence, ethylene increased rapidly in the climacteric fruit [3]. Sand pear (\u003cem\u003ePyrus pyrifolia\u003c/em\u003e) is a typical climacteric fruit, it has a short shelf life and rapid senescence after harvesting, which limits the development of its industry. Therefore, delaying the aging of sand pear fruit and extending the shelf life of sand pear is the key problem facing the sand pear industry.\u003c/p\u003e \u003cp\u003eSalicylic acid (SA), also known as 2-hydroxybenzoic acid, is a phenolic compound widely distributed in the plant kingdom. As a key signaling molecule and plant hormone, SA plays an essential role in various physiological processes in plants [4]. It is involved in fundamental physiological functions such as seed germination, photosynthesis, transpiration, stomatal regulation, thermogenesis, cell growth, and ion absorption. Moreover, SA plays a crucial role in the plant immune system, regulating both local and systemic defense mechanisms [5]. In recent years, SA has gained significant attention in the postharvest management of horticultural crops due to its remarkable effects in inhibiting senescence, delaying maturation, and enhancing resistance to various biotic and abiotic stresses [6]. Studies have shown that SA can significantly extend the shelf life of crops, reduce the incidence of diseases, and improve crop performance under adverse environmental conditions, highlighting its great potential in modern agriculture.\u003c/p\u003e \u003cp\u003eMitogen-activated protein kinase (MAPK), as a specific serine/threonine protein kinase, is one of the largest transferases in eukaryotes [7]. Because this kinase is associated with a group of proteins that are phosphorylated by tyrosine residues under the action of mitogen, it is named mitogen-activated protein kinase. MAPK belongs to a multi-gene family. So far, MAPK family members have been identified in a variety of plants. Such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [8], maize [9], corn [10], bananas [11], apples [12], tomatoes [13], and cucumbers [14].\u003c/p\u003e \u003cp\u003eThe MAPK signaling pathway plays a crucial role in a variety of biological processes. MAPK can catalyze the phosphorylation of substrate proteins by transferring a phosphate group from ATP to the amino acid residues of the substrate proteins. MAPK cascade systems are widely present in eukaryotic organisms and play an important role in regulating cell growth and development and responding to external environmental stimuli [15]. It has been shown that MabZIP21 can interact with MaMPK6-3. The interaction between MaMPK6-3 and MabZIP21 enhanced the transcriptional activation of MabZIP21. Ser-436 and Thr-318 are the major phosphorylation sites of MabZIP21 by MaMPK6-3 [16]. In rice, MAPK5 can interact with OsWRKY72 to regulate the growth, development, and defense response of rice [17]. In tomato, the yeast two-hybridization (Y2H) experiment showed that SlASR4 protein interacts with SlMAPK3 protein, representing a class of proteins with conserved ABA/WDS domains and forming protein complexes capable of responding to various stresses [18]. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the phosphokinase MAPK6 phosphorylates ACS6 and increases its protein stability, thereby increasing ethylene production [19]. Currently, most research on MAPK is focused on stress resistance and morphogenesis. As an important protein kinase, MAPK must play an important regulatory role in the process of fruit senescence. However, there are very few research reports on this aspect. Therefore, how MAPK regulates fruit senescence and the specific signal transduction mechanisms for regulation are the hot and key issues in fruit tree research. To reveal the relationship between MAPK signal and fruit shelf life, it is necessary to deeply analyze the unique role of MAPK signal in the sand pear fruit senescence process.\u003c/p\u003e \u003cp\u003eTranscription factors (TFs) play a crucial role in plant physiological processes, acting as key regulators in plant growth, development, and responses to environmental changes [20]. Among all transcription factor families, the basic leucine zipper (bZIP) family is one of the largest and most diverse. The name of the bZIP transcription factor is derived from its unique structural domain-the bZIP domain, which typically consists of 60 to 80 amino acids and contains two functional regions: a basic region and a leucine zipper region [21].\u003c/p\u003e \u003cp\u003eIn bZIP proteins, the basic region consists of approximately 16 amino acid residues and contains a highly conserved motif, N-x7-R/K-x9, which primarily functions in nuclear localization and DNA binding. In contrast, the leucine zipper region is composed of multiple heptad repeat motifs of amino acids, usually dominated by leucine (Leu), but may also include other large hydrophobic amino acids such as isoleucine (Ile), valine (Val), phenylalanine (Phe), or methionine (Met). Although the amino acid sequence in this region is more variable and not as highly conserved as the basic region, it plays a crucial role in protein dimerization. Specifically, the leucine zipper region is involved not only in the homodimerization of bZIP transcription factors but also in the formation of heterodimers with other transcription factors [22].\u003c/p\u003e \u003cp\u003eThe \u003cem\u003ebZIP\u003c/em\u003e gene family plays an indispensable role in various biological processes in plants. Extensive research has shown that bZIP transcription factors regulate plant growth, development, and responses to environmental stress. BZIP TFs control several aspects of plant development, particularly important growth events such as fruit ripening, and plant responses to environmental stress [23, 24]. For example, in peaches, the expression of \u003cem\u003ePpbZIP\u003c/em\u003e genes is higher at the early stages of fruit maturation and gradually decreases as ripening progresses [25]. In lychees, several \u003cem\u003ebZIP\u003c/em\u003e genes, including \u003cem\u003eLcbZIP17\u003c/em\u003e, \u003cem\u003eLcbZIP4\u003c/em\u003e, \u003cem\u003eLcbZIP5/7/21\u003c/em\u003e, \u003cem\u003eLcbZIP2/19/28\u003c/em\u003e, \u003cem\u003eLcbZIP9/32/33/44/53\u003c/em\u003e, and \u003cem\u003eLcbZIP24/29/40/41\u003c/em\u003e, are primarily expressed during the postharvest phase, participating in the regulation of fruit postharvest growth and maturation [26]. Additionally, the \u003cem\u003ebZIP11\u003c/em\u003e gene in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e influences root development by regulating the connection between low-energy signals and auxin-mediated primary root growth [27]. In maize, overexpression of the \u003cem\u003eZmbZIP4\u003c/em\u003e gene leads to increased lateral root numbers, elongation of primary roots, and a stronger root system [28]. In tomatoes, \u003cem\u003eSlbZIP33\u003c/em\u003e (\u003cem\u003eSlAREB1\u003c/em\u003e) not only participates in stress-induced responses but also regulates the expression of genes involved in critical metabolic pathways during fruit ripening, playing a key role in metabolic programming [29].\u003c/p\u003e \u003cp\u003eThis study conducted an in-depth analysis of the transcriptomic and genomic data of the \u0026lsquo;Whangkeumbae\u0026rsquo; (\u003cem\u003ePyrus pyrifolia\u003c/em\u003e) variety, 25 \u003cem\u003ePpMAPK\u003c/em\u003e genes were identified in sand pear. These include four genes identified from the transcriptome and 22 genes discovered from the genome. To further explore the evolutionary relationships of these genes, a phylogenetic analysis was performed, revealing that these genes can be categorized into four major subfamilies (A, B, C, and D). Specifically, subfamilies A and B each contain six genes, subfamily C consists of four genes, and subfamily D, the largest, includes nine genes. These genes may have undergone varying degrees of functional differentiation during evolution, with potential functional differences among the subfamily members offering key insights into the mechanisms of the MAPK signaling pathway. Additionally, based on protein-protein interaction network analysis, the study identified that PpMAPK3-L interacts with the transcription factor PpbZIP2. This interaction may collaboratively regulate the fruit senescence process in sand pear during ripening, providing new perspectives on the molecular regulatory mechanisms of MAPK signaling during fruit maturation and senescence. These findings also present potential research directions for breeding sand pear varieties that delay fruit senescence.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification and phylogenetic analysis of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePpMAPK\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;in sand pear\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study identified four PpMAPK from the transcriptome data [30] of sand pear, which were designated as PpMAPK3-L, PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L. By comparing protein sequences in the genomic database [31], and removing duplicates and incomplete sequences, a total of 22 PpMAPK protein sequences were successfully identified in the sand pear genome. Among these genomic datasets, the sequence of PpMAPK3-L in the transcriptome exhibited a high similarity of 99.19% to that of EVM0007944 in the genome database. PpMAPK7-L was identical to EVM0004426, with a similarity of 100%. The sequence similarity between PpMAPK10-L and EVM0027166 was 94.51%, while PpMAPK16-L shared a similarity of 95.75% with EVM0023771. In summary, by integrating transcriptome and genomic data, a total of 25 PpMAPK were identified in the sand pear.\u003c/p\u003e\n\u003cp\u003eBased on this, the study further constructed a phylogenetic tree of the \u003cem\u003ePpMAPK\u003c/em\u003e genes to reveal the evolutionary relationships between these genes. According to the classification criteria for the MAPK protein family in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Based on the homology with \u003cem\u003eArabidopsis thaliana\u003c/em\u003e MAPK proteins and their amino acid sequence characteristics, the MAPK proteins were classified into four subgroups according to the classification standards for the MAPK protein family in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. (Fig. 1). The classification results indicate that the MAPK family members of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and sand pear are distributed across all subgroups. In subgroups A, B, and C, the MAPK proteins are of the TEY type, while those in subgroup D are of the TDY type. Specifically, subgroup A includes three \u003cem\u003eArabidopsis\u003c/em\u003e MAPK members and six sand pear MAPK members; subgroup B consists of four Arabidopsis MAPK members and six sand pear MAPK members; subgroup C comprises four Arabidopsis MAPK members and four sand pear MAPK members; subgroup D has the largest number of MAPK members, with nine MAPK members in both Arabidopsis and sand pear. The analysis of these data provides a clearer understanding of the classification and evolutionary characteristics of the sand pear MAPK family.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhylogenetics, conserved motifs, domains\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;and gene structures of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e22 PpMAPKs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the obtained MAPK protein sequences, this study constructed a phylogenetic tree of the PpMAPKs and systematically analyzed their conserved motifs, conserved domains, and gene structures (Fig. 2). The analysis revealed that all PpMAPK protein sequences contain at least eight motifs. The shared motifs across each subfamily include motif 1, motif 2, motif 4, motif 5, motif 6, and motif 9. Notably, motif 6 is present in all protein sequences, indicating that this motif is highly conserved within the MAPK family. As a conserved motif of MAPKs, motif 6 is characterized by the sequence T(D/E)YVV(A)TRWYRAPEL, where the TxY part corresponds to the \u0026quot;T-loop\u0026quot; structure. This structural feature not only reflects the structural consistency of MAPK proteins but also suggests that PpMAPK possesses highly conserved functional characteristics. Motif 9 represents the CD domain, which plays an essential functional role in all MAPK proteins (Fig. 2B).\u003c/p\u003e\n\u003cp\u003eTo further explore the conserved domains of PpMAPK, this study employed the CD-search tool from the National Center for Biotechnology Information (NCBI) database for in-depth analysis. The results indicated that all 22 PpMAPK proteins in sand pear contain the MAPK domain, highlighting the high functional conservation of these proteins. PpMAPK proteins in subgroups A, B, and C share the same conserved domain (STKc_TEY_MAPK), whereas PpMAPK proteins in subgroup D possess a distinct conserved domain (STKc_TDY_MAPK). These domain differences reveal the functional diversity between the subgroups (Fig. 2C).\u003c/p\u003e\n\u003cp\u003eFurther gene structure analysis showed a clear pattern in the number and distribution of exons and introns in \u003cem\u003ePpMAPK\u003c/em\u003e genes. In particular, genes within the same evolutionary branch exhibited high similarity in exon and intron numbers and their distribution. All \u003cem\u003ePpMAPK\u003c/em\u003e genes contain both introns and exons, with the \u003cem\u003ePpMAPKs\u003c/em\u003e of the TDY type having relatively more exons, ranging from 9 to 11, with most genes containing 9 or 10 exons. The genes \u003cem\u003eEVM0014865\u003c/em\u003e, \u003cem\u003eEVM0016074\u003c/em\u003e, and \u003cem\u003eEVM00180\u003c/em\u003e have the highest number of exons, reaching 11. In contrast, the \u003cem\u003ePpMAPKs\u003c/em\u003e of the TEY type typically have fewer exons, usually ranging from 2 to 6, with the genes \u003cem\u003eEVM0020253\u003c/em\u003e, \u003cem\u003eEVM0008427\u003c/em\u003e, and \u003cem\u003eEVM0015322\u003c/em\u003e having the fewest exons, with only 2. Additionally, 9 \u003cem\u003ePpMAPK\u003c/em\u003e genes contain UTR regions, specifically \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003eEVM0001504\u003c/em\u003e, \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003eEVM0014865\u003c/em\u003e, \u003cem\u003eEVM0033302\u003c/em\u003e, \u003cem\u003eEVM0000507\u003c/em\u003e, \u003cem\u003eEVM0016652\u003c/em\u003e, \u003cem\u003eEVM0027754\u003c/em\u003e, and \u003cem\u003eEVM0009261\u003c/em\u003e. These results suggest that the \u003cem\u003ePpMAPK\u0026nbsp;\u003c/em\u003egenes have undergone structural and functional diversification during their evolution (Fig. 2D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003cstrong\u003ehysicochemical properties\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eand c\u003c/strong\u003e\u003cstrong\u003ehromosomal localization of Pp\u003c/strong\u003e\u003cstrong\u003eMAPK\u003c/strong\u003e\u003cstrong\u003es\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough a systematic analysis of the physicochemical properties and related characteristics of 22 PpMAPK proteins, we observed significant differences in the number of amino acid residues in sand pear MAPK proteins (Table 1). Specifically, EVM0018010 has the largest number of amino acid residues, reaching 635, while EVM0001504 contains only 367, highlighting a significant difference between the two. Furthermore, the relative molecular mass of these proteins ranges from 42,064.88 Daltons (Da) to 71,683.59 Da, further indicating the structural diversity of PpMAPK proteins. In the isoelectric point (pI) analysis, the majority of PpMAPK proteins exhibited acidic characteristics, with 16 proteins having pI values lower than 7, indicating they are acidic proteins, while 6 proteins exhibited pI values above 7, indicating they are basic proteins. Notably, EVM0001504 had the lowest pI value of 5.06, making it the most acidic, while EVM0027166 and EVM0020832 both had pI values of 9.15, showing strong basic characteristics. Regarding protein instability, the instability index of PpMAPK proteins ranged from 31.11 to 48.19. Specifically, EVM0015322 exhibited the lowest instability index at 31.11, while EVM0000507 had the highest index at 48.19. The aliphatic amino acid index analysis revealed that PpMAPK16-L had the lowest index at 76.13, while EVM0020253 had the highest at 97.49. Hydrophilicity analysis showed that the GRAVY values of all PpMAPK family members were negative, further confirming that they are hydrophilic proteins. Subcellular localization prediction results indicated that these proteins are all localized in the nucleus, suggesting that they may be involved in signal transduction and regulatory processes within the nucleus.\u003c/p\u003e\n\u003cp\u003eTable 1 The characterizations of PpMAPKs in sand pear.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003eGene name\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eGene ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eAccession no.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003eAmino acids No.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003eMw(Da)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003epI\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003eInstability index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003eAliphatic index\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eGRAVY\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eSubcellular localization\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0008427\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS037580\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42582.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e34.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e95.70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.227\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0015322\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS017334\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e372\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42605.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e6.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e31.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e96.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.234\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003eMAPK7-L\u003c/p\u003e\n \u003cp\u003e(GenBank accession number: PP992972)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0004426（100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS023153\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e379\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e43546.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e8.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e36.76\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e95.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.203\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0020253\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS015187\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e43357.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e7.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e37.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e97.49\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.176\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0033302\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS034299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42945.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e6.03\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e46.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e89.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.369\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0000507\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS001398\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e373\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42962.96\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e6.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e48.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e89.12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.365\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0016652\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS032599\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e378\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e43139.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e6.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e44.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e88.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.335\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0027707\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS012688\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e379\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e43147.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e43.26\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e89.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.299\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0026334\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS040261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42064.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e41.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e88.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.305\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0001504\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS005430\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e367\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42183.22\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.06\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e39.39\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e95.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0038345\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS025676\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42557.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e41.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e94.38\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.259\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003eMAPK3-L (GenBank accession number: PP992971)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0007944\u003c/p\u003e\n \u003cp\u003e(99.19%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS005442\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e370\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e42626.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e39.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e92.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0027754\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS024101\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e403\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e46322.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e41.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e85.21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.361\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0009261\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS024083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e403\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e46293.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e41.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e85.46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.346\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0023260\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS016125\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e406\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e46384.87\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e5.66\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e42.74\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e86.75\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.313\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0016074\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS011680\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e621\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e70605.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e6.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e37.90\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e77.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.648\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0018010\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS031618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e635\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e71683.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e6.36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e38.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e76.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.603\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0014865\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS011287\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e583\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e66525.65\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e6.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e45.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e81.63\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.494\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003eMAPK16-L (GenBank accession number: PP992974)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0023771\u003c/p\u003e\n \u003cp\u003e(95.75%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS009972\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e564\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e63919.95\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e8.73\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e36.68\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e76.13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.502\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003eMAPK10-L (GenBank accession number: PP992973)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0027166\u003c/p\u003e\n \u003cp\u003e(94.51%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS038791\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e618\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e70715.07\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e9.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e34.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e80.19\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.486\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0040595\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS024513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e608\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e68982.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e9.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e38.51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e80.53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.458\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003eEVM0020832\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 135px;\"\u003e\n \u003cp\u003eGWHPBAOS006687\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 106px;\"\u003e\n \u003cp\u003e598\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e67812.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 45px;\"\u003e\n \u003cp\u003e9.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 73px;\"\u003e\n \u003cp\u003e38.37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 82px;\"\u003e\n \u003cp\u003e79.93\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 95px;\"\u003e\n \u003cp\u003e-0.438\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 94px;\"\u003e\n \u003cp\u003eNucleus\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIn conclusion, the PpMAPK proteins from sand pear exhibit significant diversity in their physicochemical properties, which may be closely related to the differentiation of their functions within plant cells, providing a possible physiological basis for their roles in various biological processes.\u003c/p\u003e\n\u003cp\u003eTo further explore the distribution of \u003cem\u003ePpMAPK\u003c/em\u003e gene family members in the genome, we conducted chromosome mapping and visualization analysis using TBtools software (Fig. 3). The analysis revealed that the \u003cem\u003ePpMAPK\u003c/em\u003e genes are randomly distributed across 11 chromosomes of the sand pear genome, located on chromosomes 1, 2, 3, 6, 7, 8, 9, 11, 13, 14, and 15. However, the gene \u003cem\u003eEVM0026334\u003c/em\u003e could not be successfully mapped to any chromosome. The distribution of \u003cem\u003ePpMAPK\u003c/em\u003e genes on each chromosome showed significant variation, with the highest number of genes found on chromosomes 2, 11, and 15, each containing three genes. On other chromosomes, the number of \u003cem\u003ePpMAPK\u003c/em\u003e genes was relatively low, particularly on chromosomes 1, 7, 8, and 9, which each contained only one \u003cem\u003ePpMAPK\u003c/em\u003e gene: \u003cem\u003eEVM0000507\u003c/em\u003e, \u003cem\u003eEVM0033302\u003c/em\u003e, \u003cem\u003eEVM0008427\u003c/em\u003e, and \u003cem\u003ePpMAPK10-L\u003c/em\u003e, respectively. These results indicate an uneven distribution of \u003cem\u003ePpMAPK\u003c/em\u003e genes in the sand pear genome, with the majority of genes located on specific chromosomes. This provides important genomic insights for further studying the functions and evolution of \u003cem\u003ePpMAPK\u003c/em\u003e genes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCis-element analysis of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003ePpMAPK\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;gene promoters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the functional roles of the \u003cem\u003ePpMAPK\u003c/em\u003e gene family in greater depth, we extracted the 2 kb upstream sequences of 22 \u003cem\u003ePpMAPK\u003c/em\u003e genes in sand pear and conducted a systematic analysis of their hormone-responsive cis-regulatory elements (Fig. 4). Notably, no hormone response elements were identified in the upstream sequence of the \u003cem\u003eEVM0020832\u003c/em\u003e gene. In contrast, the upstream sequences of the remaining 21 \u003cem\u003ePpMAPK\u003c/em\u003e genes contain five distinct types of hormone-responsive cis-regulatory elements, namely abscisic acid, auxin, gibberellin, jasmonic acid, and SA response elements. Among these five hormone response elements, the abscisic acid response element was the most common. Except for \u003cem\u003eEVM0020253\u003c/em\u003e, \u003cem\u003eEVM0023260\u003c/em\u003e, \u003cem\u003eEVM0018010\u003c/em\u003e, and \u003cem\u003eEVM0014865\u003c/em\u003e, nearly all \u003cem\u003ePpMAPK\u003c/em\u003e genes contain this element. The auxin response element, on the other hand, was relatively rare and was found in only seven genes, including \u003cem\u003eEVM0015322\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003eEVM0020253\u003c/em\u003e, \u003cem\u003eEVM0016652\u003c/em\u003e, \u003cem\u003eEVM0009261\u003c/em\u003e, \u003cem\u003eEVM0040595\u003c/em\u003e, and \u003cem\u003eEVM0027754\u003c/em\u003e. The gibberellin response element, however, was more widely distributed, being present in the promoter sequences of 11 genes: \u003cem\u003eEVM0008427\u003c/em\u003e, \u003cem\u003eEVM0020253\u003c/em\u003e, \u003cem\u003eEVM0016652\u003c/em\u003e, \u003cem\u003eEVM0027707\u003c/em\u003e, \u003cem\u003eEVM0001504\u003c/em\u003e, \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003eEVM000926\u003c/em\u003e, \u003cem\u003eEVM0023260\u003c/em\u003e, \u003cem\u003eEVM0014865\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e, and \u003cem\u003eEVM0027754\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe Methyl Jasmonate response element also showed a relatively broad distribution, with only a few genes lacking this cis-regulatory element: \u003cem\u003eEVM0033302\u003c/em\u003e, \u003cem\u003eEVM0027707\u003c/em\u003e, \u003cem\u003eEVM0026334\u003c/em\u003e, \u003cem\u003eEVM0001504\u003c/em\u003e, and \u003cem\u003eEVM0018010\u003c/em\u003e. The SA response element, however, was more limited in distribution, being present in only seven genes: \u003cem\u003eEVM0008427\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003eEVM0000507\u003c/em\u003e, \u003cem\u003eEVM0027707\u003c/em\u003e, \u003cem\u003eEVM0038345\u003c/em\u003e, \u003cem\u003eEVM0023260\u003c/em\u003e, and \u003cem\u003eEVM0018010\u003c/em\u003e. In conclusion, the presence of various hormone-responsive cis-regulatory elements in the \u003cem\u003ePpMAPK\u003c/em\u003e gene family of sand pear suggests that these genes may play important roles in plant hormone signaling pathways. The different hormone response elements likely reflect the functional differences of these genes in various physiological processes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression patterns of \u003cem\u003ePpMAPKs\u003c/em\u003e in sand pear (\u003cem\u003ePyrus pyrifolia\u003c/em\u003e)\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpecific quantitative primers for \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e genes were designed based on transcriptome data, with their primers provided in Additional file 1 (Table S1). Using these primers, detailed expression pattern analyses of the four \u003cem\u003ePpMAPK\u003c/em\u003e genes were conducted across different tissue types (Fig. 5). The results revealed significant tissue-specific expression of the \u003cem\u003ePpMAPK\u003c/em\u003e genes. Specifically, \u003cem\u003ePpMAPK3-L\u003c/em\u003e exhibited the highest expression in petals and the lowest in leaves (Fig. 5A). \u003cem\u003ePpMAPK7-L\u003c/em\u003e showed the lowest expression in leaves but peaked in shoot tips (Fig. 5B). \u003cem\u003ePpMAPK10-L\u003c/em\u003e was predominantly expressed in anthers, with minimal expression in leaves (Fig. 5C). Similarly, \u003cem\u003ePpMAPK16-L\u003c/em\u003e exhibited the weakest expression in leaves but showed the strongest expression in shoots (Fig. 5D). Furthermore, all four \u003cem\u003ePpMAPK\u003c/em\u003e genes were expressed to varying degrees in fruit tissues, suggesting their potential involvement in fruit development.\u003c/p\u003e\n\u003cp\u003eTo further explore the roles of \u003cem\u003ePpMAPK\u003c/em\u003e genes in fruit development, their expression patterns were dynamically tracked across different stages of fruit development (Fig. 6). The results indicated that these genes were expressed post-harvest, each displaying distinct expression peaks. Specifically, \u003cem\u003ePpMAPK3-L\u003c/em\u003e and \u003cem\u003ePpMAPK10-L\u003c/em\u003e reached their highest expression levels 25 days after harvest (DAH), while \u003cem\u003ePpMAPK7-L\u003c/em\u003e peaked at 30 DAH. In contrast, \u003cem\u003ePpMAPK16-L\u003c/em\u003e exhibited its highest expression as early as 5 DAH.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRegulatory analysis of \u003cem\u003ePpMAPK\u003c/em\u003e genes by SA and ETH\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the potential roles of \u003cem\u003ePpMAPK\u003c/em\u003e genes in the process of fruit senescence, pears were treated post-harvest with different concentrations of SA: 0.002, 0.02, 0.2, and 2 mM (Fig. 7A, B, C, D). The expression of the four \u003cem\u003ePpMAPK\u003c/em\u003e genes was then monitored. The experimental results showed that at a concentration of 0.002 mM SA, no significant differences in the expression levels of the four \u003cem\u003ePpMAPK\u003c/em\u003e genes were observed compared to the control group. Under the 0.02 mM SA treatment, the expression level of \u003cem\u003ePpMAPK10-L\u003c/em\u003e significantly increased, suggesting that it may play an important regulatory role under this condition. In contrast, the expression changes of other \u003cem\u003ePpMAPK\u003c/em\u003e genes were relatively small. When the SA concentration was increased to 0.2 mM, only \u003cem\u003ePpMAPK3-L\u003c/em\u003e exhibited significantly higher expression compared to the control group, suggesting that this concentration of SA may stimulate the expression of \u003cem\u003ePpMAPK3-L\u003c/em\u003e, while the expression levels of the other three genes (\u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e) showed no significant differences from the control, indicating that these genes might be less sensitive or less responsive to this concentration of SA. At 2 mM SA, the expression of \u003cem\u003ePpMAPK3-L\u003c/em\u003e was significantly lower than that of the control group, suggesting that this high concentration of SA may inhibit the expression of \u003cem\u003ePpMAPK3-L\u003c/em\u003e. In contrast, \u003cem\u003ePpMAPK7-L\u003c/em\u003e exhibited significantly higher expression, indicating that this concentration of SA may upregulate \u003cem\u003ePpMAPK7-L\u003c/em\u003e expression through some mechanism. However, the expression of \u003cem\u003ePpMAPK10-L\u003c/em\u003e and \u003cem\u003ePpMAPK16-L\u003c/em\u003e did not differ significantly from the control group, suggesting that these two genes might be less responsive or unaffected by the 2 mM SA treatment.\u003c/p\u003e\n\u003cp\u003eTo further elucidate the role of \u003cem\u003ePpMAPK\u003c/em\u003e genes in fruit senescence and based on previous experimental designs, the flesh of post-harvest \u0026lsquo;Whangkeumbae\u0026rsquo; pear was treated with 1 \u0026micro;/L ethephon (ETH), and the expression of \u003cem\u003ePpMAPK\u003c/em\u003e genes was analyzed at 3, 6, 12, 24, 36, and 48 h after treatment (Fig. 7E, F, G, H). The results showed that the expression of \u003cem\u003ePpMAPK3-L\u003c/em\u003e was significantly lower at 3, 6, 36, and 48 h post-treatment compared to the control group, suggesting that ETH may inhibit the expression of this gene through some mechanism. The expression of \u003cem\u003ePpMAPK7-L\u003c/em\u003e was significantly higher at 12 h post-treatment and also showed a significant increase at 36 h, indicating that ETH may activate the expression of this gene after prolonged treatment. The expression of \u003cem\u003ePpMAPK10-L\u003c/em\u003e was significantly higher at 3, 24, and 48 h post-treatment, suggesting that ETH may participate in the fruit senescence process by up-regulating the expression of this gene at these time points. Finally, the expression of \u003cem\u003ePpMAPK16-L\u003c/em\u003e was significantly increased at all time points (3, 12, 24, 36, and 48 h) after ETH treatment, indicating that ETH treatment may enhance the role of \u003cem\u003ePpMAPK16-L\u003c/em\u003e in fruit senescence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInteraction between PpMAPK3-L and PpbZIP2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the aforementioned experimental results, the four PpMAPK genes each play distinct roles in fruit senescence. To further investigate the potential interaction mechanisms of these MAPKs during fruit senescence, we employed the online tool STRING to predict their potential interacting proteins. The prediction results suggested that PpMAPK3-L may interact with PpbZIP2 (Gene ID: EVM0004317.1), while PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L may interact with PpbZIP3 (Additional file 2: Fig. S1).\u003c/p\u003e\n\u003cp\u003eTo further confirm these predictions, we established a Y2H system to analyze the interactions between PpMAPK and PpbZIP. PpMAPK3-L, PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L were individually cloned into the pGBKT7 (BD) vector to generate the recombinant vectors PpMAPK-BD. Concurrently, PpbZIP2 and PpbZIP3 were cloned into the pGADT7 (AD) vector to yield the recombinant vectors PpbZIP-AD. Initially, self-activation assays were conducted to verify that the constructed vectors did not possess the ability to spontaneously activate transcription. The experimental results demonstrated that after co-transforming the four PpMAPK-BD and PpbZIP-AD vectors, cells were able to grow normally on synthetic dropout medium/-tryptophan/-leucine (SD/-Leu/-Trp) medium, confirming the successful introduction of these genes into AH109 competent cells. Further tests revealed no growth on SD/-Trp/-Leu/-histones/-adenine/X-alpha gal (SD/-Trp/-Leu/-His/-Ade/X-\u0026alpha;-gal) medium, confirming the absence of self-activation in the PpMAPK genes (Additional file 3: Fig. S2). Next, after co-transforming the four PpMAPK-BD vectors with the two PpbZIP-AD vectors into AH109 cells, the results revealed normal growth on SD/-Leu/-Trp medium, confirming the successful introduction of PpMAPK and PpbZIP into the competent cells. Among these experiments, only PpMAPK3-L-BD and PpbZIP2-AD exhibited significant growth on SD/-Leu/-Trp/-Ade/-His medium, further confirming the existence of an interaction between PpMAPK3-L and PpbZIP2. This study offers compelling evidence for understanding the molecular mechanisms of PpMAPK and PpbZIP proteins in fruit senescence and provides new insights for future functional research and regulation of fruit senescence (Fig. 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression Pattern of \u003cem\u003ePpbZIP2\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the interaction mechanism between \u003cem\u003ePpMAPK3-L\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePpbZIP2\u003c/em\u003e, this study comprehensively analyzed the expression pattern of \u003cem\u003ePpbZIP2\u003c/em\u003e. The results revealed that \u003cem\u003ePpbZIP2\u0026nbsp;\u003c/em\u003eis expressed in various tissues of sand pear, with the highest expression observed in the leaves and the lowest in the petals. This suggests that \u003cem\u003ePpbZIP2\u003c/em\u003e may play distinct physiological roles in different tissues (Fig. 9A). To explore the role of \u003cem\u003ePpbZIP2\u003c/em\u003e during fruit development, the expression dynamics of \u003cem\u003ePpbZIP2\u003c/em\u003e throughout fruit development were monitored. The analysis showed that the expression of \u003cem\u003ePpbZIP2\u003c/em\u003e fluctuated during fruit development, peaking at 30 DAH and reaching its lowest level of 60 days after full bloom (DAFB) (Fig. 9B). Notably, the expression of \u003cem\u003ePpbZIP2\u003c/em\u003e is induced by SA (Fig. 9C), while ETH significantly inhibits its expression (Fig. 9D). This finding provides crucial insights into the role of \u003cem\u003ePpbZIP2\u003c/em\u003e in fruit senescence and reveals potential molecular mechanisms by which SA and ETH regulate \u003cem\u003ePpbZIP2\u003c/em\u003e expression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, four \u003cem\u003ePpMAPK\u003c/em\u003e genes were initially identified from the transcriptome of sand pear (\u003cem\u003ePyrus pyrifolia\u003c/em\u003e) and named \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e. Subsequently, using 20 \u003cem\u003eArabidopsis thaliana\u003c/em\u003e MAPK protein sequences (AtMAPK1\u0026ndash;AtMAPK20) as query sequences [8], an additional 22 MAPK family members were identified in the sand pear genome database through BLAST comparison and domain validation. The results revealed that PpMAPK7-L shows a 100% similarity with the EVM0004426 sequence in the genome. Based on the combined transcriptomic and genomic data analysis, a total of 25 \u003cem\u003ePpMAPK\u003c/em\u003e genes were identified in sand pear, providing a solid foundation for a comprehensive study of the PpMAPK family. Comparative analysis revealed a remarkable diversity of \u003cem\u003eMAPK\u003c/em\u003e genes across plant species. MAPK family members were identified in various plants, including rice [32], maize [9], chickpea [33], kiwifruit [34], barley [35], \u003cem\u003eGossypium raimondii\u003c/em\u003e [36], bread wheat [37], \u003cem\u003eFragaria vesca\u003c/em\u003e [38], \u003cem\u003eFagopyrum tataricum\u003c/em\u003e [39], \u003cem\u003eBrachypodium distachyon\u003c/em\u003e [40], tomato [13], tobacco [41], poplar [42], apple [12], soybean [43] with varying numbers of family members (Additional file 4: Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). This indicates that, although the MAPK signaling pathway is highly conserved in eukaryotes, the evolution and function of its family members exhibit considerable species-specific variation.\u003c/p\u003e \u003cp\u003eThis study conducted an in-depth analysis of the tissue-specific expression patterns of the \u003cem\u003ePpMAPK\u003c/em\u003e genes. \u003cem\u003ePpMAPK3-L\u003c/em\u003e is highly expressed in petals, \u003cem\u003ePpMAPK7-L\u003c/em\u003e shows dominant expression in the shoots, \u003cem\u003ePpMAPK10-L\u003c/em\u003e is mainly expressed in the anthers, while \u003cem\u003ePpMAPK16-L\u003c/em\u003e exhibits the highest expression in the shoots. Furthermore, all four genes are expressed the least in the leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As observed in other plants, the expression patterns of the \u003cem\u003eMAPK\u003c/em\u003e genes in sand pear exhibit significant tissue specificity. For example, in buckwheat, \u003cem\u003eFtMAPK1\u003c/em\u003e and \u003cem\u003eFtMAPK3\u003c/em\u003e are highly expressed in the roots, stems, and leaves. In watermelon, distinct expression patterns of \u003cem\u003eMAPK\u003c/em\u003e genes are observed in the roots, stems, and leaves [44]. In addition, the \u003cem\u003ePpMAPK\u003c/em\u003e genes also exhibit distinct dynamic expression profiles during the senescence process of sand pear fruit. \u003cem\u003ePpMAPK3-L\u003c/em\u003e and \u003cem\u003ePpMAPK10-L\u003c/em\u003e peak at 25 DAH, \u003cem\u003ePpMAPK7-L\u003c/em\u003e peaks at 30 DAH, while \u003cem\u003ePpMAPK16-L\u003c/em\u003e shows the highest expression at 5 DAH (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). These results suggest that these genes may be involved in the fruit senescence process through different temporal regulatory mechanisms.\u003c/p\u003e \u003cp\u003eThis study further verified the importance of the MAPK signaling pathway in hormone response, and revealed the response mechanism of four \u003cem\u003ePpMAPK\u003c/em\u003e genes under different hormone conditions. The results showed that these genes were significantly regulated by SA and ETH, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), suggesting that the PpMAPK signaling pathway may play a key role in the physiological regulation of fruit through hormone-mediated mechanisms. The expression levels of four \u003cem\u003ePpMAPK\u003c/em\u003e genes also showed significant differences between diseased fruit and control fruit. Specifically, the expression levels of PpMAPK3-L and PpMAPK10-L in fruit were significantly up-regulated, which may be related to disease-induced defense response. In contrast, the expression of PpMAPK7-L and PpMAPK16-L in diseased fruit was significantly lower than that in control, suggesting that they may play an important role in the maintenance of healthy fruit (Additional file 5: Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). These differences indicate that different \u003cem\u003ePpMAPK\u003c/em\u003e genes may play a role in the mechanism of pear fruit disease resistance.\u003c/p\u003e \u003cp\u003eTo further investigate the potential interaction mechanisms of MAPK genes in fruit senescence, this study first used the online tool STRING to predict possible interacting proteins of these MAPK genes (Additional file 2: Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The predicted interactions were then validated using Y2H technology. The experimental results demonstrated an interaction between PpMAPK3-L and PpbZIP2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This finding is consistent with previous studies, which have shown that many MAPK members interact with transcription factors to play critical roles in various physiological processes in plants. For instance, the interaction between MPK4 and MYB1 is crucial for regulating anthocyanin accumulation [45], while the interaction between MdERF17 and MdMPK4 is important at different stages of plant growth [46]. In sand pear fruit, the expression of \u003cem\u003ePpbZIP2\u003c/em\u003e peaked at 30 DAH (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Further experiments revealed that SA significantly induced \u003cem\u003ePpbZIP2\u003c/em\u003e expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC), while ETH treatment significantly suppressed it (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). These findings suggest that the expression of \u003cem\u003ePpbZIP2\u003c/em\u003e is regulated by various signaling molecules. Additional mechanistic studies revealed that the interaction between PpbZIP2 and PpMAPK3-L may delay fruit senescence through synergistic effects, regulating the expression of senescence-related genes, thus providing new molecular mechanisms and research directions for preserving sand pear fruit.\u003c/p\u003e \u003cp\u003eIn summary, this study revealed the potential function of the \u003cem\u003eMAPK\u003c/em\u003e genes in fruit senescence and disease resistance regulation, and provided valuable clues for resolving the biological role of the \u003cem\u003eMAPK\u003c/em\u003e genes in complex physiological processes of pear fruit. More importantly, these findings laid an important theoretical foundation for improving stress resistance, extending the shelf life, and improving the quality of fruit by molecular breeding in the future, and pointed out the direction for further research on the MAPK signaling pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study provides an in-depth analysis of the PpMAPKs in \u0026lsquo;Whangkeumbae\u0026rsquo; (\u003cem\u003ePyrus pyrifolia\u003c/em\u003e) through the integration of transcriptomic and genomic data. A total of 25 \u003cem\u003ePpMAPK\u003c/em\u003e genes were identified, including four from transcriptomic data (\u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e) and 22 from genomic data. Further homologous sequence alignment revealed that the transcriptomic PpMAPKs (PpMAPK3-L, PpMAPK7-L, PpMAPK10-L, and PpMAPK16-L) shared high sequence identity with their corresponding genomic counterparts (EVM0007944.1, EVM0004426.1, EVM0027166.1, EVM0023771, EVM0028755.1, and EVM0015862.1), with identity values of 99.19%, 100%, 94.51%, and 95.75%, respectively. This indicates a high degree of consistency in gene identification between the transcriptomic and genomic data. These findings provide a solid data foundation for understanding the genetic characteristics of \u003cem\u003eMAPK\u003c/em\u003e genes in \u003cem\u003ePyrus pyrifolia\u003c/em\u003e. Phylogenetic analysis revealed that the 25 PpMAPKs were classified into four subfamilies (A, B, C, and D). Subfamilies A and B each contained six members, subfamily C contained four members, and subfamily D comprised nine members. The potential functional differences among the gene members of each subfamily provide valuable insights for the study of MAPK signaling pathways. Further interaction analysis identified a significant interaction between PpMAPK3-L and the transcription factor PpbZIP2. Based on this finding, it is hypothesized that PpMAPK3-L and PpbZIP2 may jointly regulate fruit senescence in \u003cem\u003ePyrus pyrifolia\u003c/em\u003e, offering new perspectives for regulating the MAPK signaling pathway in plant senescence processes.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003ePear (\u003cem\u003ePyrus pyrifolia\u003c/em\u003e) fruit were selected as materials from Hebei Agricultural University. Flowers were marked at the flowering stage to assess the fruit ripening stage. Fruit were harvested at these stages: 30, 60, 90, 120, 130, 140, and 145 DAFB. The harvested flesh were quickly frozen with liquid nitrogen and stored in the refrigerator at -80℃ for later use. Similarly, shoots, stems, young leaves, petals and anthers taken from pear trees were taken back to the laboratory and placed in a -80℃ refrigerator.\u003c/p\u003e \u003cp\u003eThe ripe pear fruit of 10 DAH were immediately brought back to the laboratory. The pulp were treated with 0.002, 0.02, 0.2 and 2 mM SA for 12 h, respectively. Double-distilled water treatment as the control. The fresh meat slices of the fruit were immediately frozen in liquid nitrogen and stored at -80℃ for further analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGenome-wide identification of MAPK in sand pear\u003c/h2\u003e \u003cp\u003eFirst, all of the DNA sequences (FASTA format), cDNA sequences (FASTA format), CDS sequences (FASTA Format), the entire protein sequence (FASTA format), and the GFF3 gene annotation file were downloaded from the national genome data center (NGDC) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bigd.big.ac.cn/gwh\u003c/span\u003e\u003cspan address=\"https://bigd.big.ac.cn/gwh\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). From the classification of protein database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/interpro/download/Pfam/\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/interpro/download/Pfam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) download the Hidden Markov Model (HMM). TBtools' simple HMM search tool was used to obtain all protein sequences with protein kinase domains, set the confidence to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e [47]. Only one transcript is kept for each gene and the others are deleted. At NCBI-CDD (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/-cdd/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/-cdd/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and SMART test conservative (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) integrity of the domain.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eConstruction and beautification of phylogenetic tree\u003c/h2\u003e \u003cp\u003eA total of 22 MAPK family members were identified in sand pear, and 4 MAPK members were identified in transcriptome. The sequence similarity between PpMAPK7-L and EVM0004426.1 was 100%. NCBI blast was used to find the protein sequences of apple, tomato, tobacco and \u003cem\u003eArabidopsis\u003c/em\u003e that were closely related to the four MAPKs. The protein sequences of all MAPK members were sorted into FASTA format, and the FASTA file was put into MEGA7.0 software [48]. Multiple sequences were compared by ClustalW, the phylogenetic tree was constructed by neighbor-joining (NJ) [49]. The Artificial Intelligence (AI) software was used to add branching colors and grouping information to the constructed evolutionary tree.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of physical and chemical properties of MAPK family members\u003c/h2\u003e \u003cp\u003eTBtools was used to extract CDS sequences, protein sequences and promoter sequences of MAPK family members for subsequent analysis. On Expasy website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) protein hydrophobicity, molecular weight and pI were predicted. The online site Plant-mPLoc (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict the location of PpMAPKs. STRING ( \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was used to predict interacting proteins of PpMAPKs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of motifs, conserved domains and gene structure of PpMAPKs\u003c/h2\u003e \u003cp\u003eThe evolutionary relationship of PpMAPK was analyzed and identified by using MEGA 7.0 software. The PpMAPK protein sequences were submitted to NCBI-CDD and the online MEME tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/index.html\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for domain and motif identification. The genomic annotation file of \u003cem\u003ePyrus pyrifolia\u003c/em\u003e was obtained from NGDC and the gene structure was analyzed. The CDS sequences were compared with the corresponding genomic DNA sequences using the Gene Structure Display Server (GSDS) tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to analyze the gene structure. The phylogenetic tree, conserved motifs, domains and gene structure diagrams of PpMAPK were constructed by using TBtools software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eChromosome localization and promoter cis-acting elements of PpMAPK family\u003c/h2\u003e \u003cp\u003eTBtools software was used to analyze the pear genome annotation file, determine the chromosome location information of each \u003cem\u003ePpMAPK\u003c/em\u003e sequence, and draw the corresponding chromosome location map. Place the sequence obtained in TBtools at the first 2000 bp of the start codon ATG in the Plant CARE website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"https://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Hormone-related elements were screened among all cis-acting elements. The number of each cis-acting element was counted, the Heatmap tool of TBtools was used to classify and visually analyze promoters of PpMAPK family members. Map the Location of MAPK family members on chromosomes via using the Gene Location Visualize from GTF/GFF tool in TBtools. Artificial intelligence was used to beautify pictures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eY2H assay\u003c/h2\u003e \u003cp\u003eComplete coding DNA sequences (CDS) of \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e were obtained based on BLAST analysis of the sand pear transcriptome database [30]. The recombinant plasmid \u003cem\u003ePpMAPK3-L\u003c/em\u003e-pGBKT7, \u003cem\u003ePpMAPK7-L\u003c/em\u003e-pGBKT7, \u003cem\u003ePpMAPK10-L\u003c/em\u003e-pGBKT7, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e-pGBKT7 were successfully constructed by connecting PpMAPK3-L, PpMAPK7-L, PpMAPK10-L and PpMAPK16-L with the decoy vector pGBKT7, respectively. The CDS sequences of \u003cem\u003ePpbZIP2\u003c/em\u003e and \u003cem\u003ePpbZIP3\u003c/em\u003e were connected to the prey vector to construct \u003cem\u003ePpbZIP2\u003c/em\u003e-pGADT7 and \u003cem\u003ePpbZIP3\u003c/em\u003e-pGADT7. The constructed \u003cem\u003ePpMAPK3-L\u003c/em\u003e-pGBKT7, \u003cem\u003ePpMAPK7-L\u003c/em\u003e-pGBKT7, \u003cem\u003ePpMAPK10-L\u003c/em\u003e-pGBKT7, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e-pGBKT7 plasmids were fused with \u003cem\u003ePpbZIP2\u003c/em\u003e-pGADT7, PpbZIP3-pGADT7, respectively. The fusion plasmid was co-transferred to yeast AH109, and the transformed bacterial solution was coated on the yeast medium Trp and Leu. After incubation at 30℃ for 3 days, positive colonies were selected and strewed on SD/-Trp/-Leu and SD/-Trp/-Leu/-Ade/X-α-gal plates, respectively. Protein-protein interactions were observed after incubation at 30℃ for 3 days. Prey vectors and bait vectors without target sequences were used as negative control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003e According to the manufacturer instructions, use RNAprep Pure Plant Plus Kit (Tian Gen, Beijing, China) Kit to extract total RNA. Using FastQuant RT Kit (with gDNase) (Tian Gen, Beijing, China) Kit will RNA transcription for cDNA. Magic SYBR mixture (CoWin Biosciences, China) was used for the qRT-PCR reaction. The fluorescence quantitative PCR instrument employed in this study was the Mastercycler ep realplex 4 (Eppendorf AG, Hamburg, Germany). The internal references utilised in this study were the \u003cem\u003ePpUBI\u003c/em\u003e (GenBank number: AF195224) gene. Primers for RT-qPCR analysis are listed in Additional file 1 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe data represent the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of at least three replicates. Univariate analysis of variance (ANOVA)/Duncan's multiple range test were used for the overall significance of the differences. All data were analyzed in SPSS v27.0 (IBM Corp., Armonk, NY, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMAPK: Mitogen-activated protein kinase; SA: Salicylic acid; Y2H: yeast two-hybridization; TFs: Transcription factors; bZIP: basic leucine zipper; Leu: leucine; Ile: isoleucine; Val: valine; Phe: phenylalanine; Met: methionine; NCBI: National Center for Biotechnology Information; Da: Dalton; pI: isoelectric points; DAH: days after harvest; ETH: ethephon; BD: pGBKT7; AD: pGADT7; SD/-Trp/-Leu: synthetic dropout medium/-tryptophan/-leucine; SD/-Trp/-Leu/-His/-Ade/X-\u0026alpha;-gal: SD/-Trp/-Leu/-histones/-adenine/X-alpha gal; DAFB: days after full bloom; NGDC: national genome data center; HMM: Hidden Markov Model; NJ: neighbor-joining; GSDS: Gene Structure Display Server; AI: Artificial Intelligence; CDS: Complete coding DNA sequences; qRT-PCR: quantitative real-time polymerase chain reaction; ANOVA: Analysis of variance\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material availabie at\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors extend their gratitude to Professor Chen Liang (Chinese Academy of Sciences) for his valuable assistance with providing the yeast two-hybrid vectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003cstrong\u003e\u0026rsquo;\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003econtribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe work presented here was a collaborative effort among all the authors. SHY formulated the research topic. SHY, XY, and WHY analyzed data and interpreted the results. SHY and XY wrote the manuscript. SHY, XY and WHY revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (32272654), the Natural Science Foundation of Hebei Province, China (C2023204016), the Hebei Province Introduced Overseas-Scholar Fund, China (C20220361), and the Hebei Province Outstanding Youth Fund, China (2016, 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe genome sequence information contained in this study were obtained from following websites. \u003cem\u003ePyrus pyrifolia\u003c/em\u003e cultivar \u0026lsquo;Cuiguan\u0026rsquo; genome file from the National Genomics Date Center (NGDC): https://ngdc.cncb.ac.cn/. Arabidopsis protein sequences from the Arabidopsis information resource (TAIR) database: https://www.arabidopsis.org/.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u0026lsquo;Whangkeumbae\u0026rsquo; was used in the current study was obtained from the experimental farm of Hebei Agricultural University, China. All the required permissions have been obtained, and all the plant materials during the current study were provided free of charge and maintained following the international guidelines.\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\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLiang CZ, Zheng GY, Li WZ, Wang YQ, Hu B, Wang HR, et al. MT delays leaf senescence and enhances salt stress tolerance in rice. J Pineal Res. 2015;59:91\u0026ndash;101.\u003c/li\u003e\n \u003cli\u003eGapper NE, Mcquinn RP, Giovannoni JJ. Molecular and genetic regulation of fruit ripening. Plant Mol Biol. 2013;82:575\u0026ndash;591.\u003c/li\u003e\n \u003cli\u003eAdams-Phillips L, Barry C, Giovannoni J. 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Mol Biol Evol. 1987;4:406\u0026ndash;425.\u003c/li\u003e\n\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-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pyrus pyrifolia, MAPK, Molecular characterization, BZIP, Sand pear senescence","lastPublishedDoi":"10.21203/rs.3.rs-6141919/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6141919/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e ‘Whangkeumbae’, a highly regarded variety of sand pear, is celebrated in the market for its distinctive and superior flavor. However, the rapid production of ethylene after harvest significantly shortens its shelf life, becoming a major limiting factor for enhancing its commercial value. Mitogen-activated protein kinase (MAPK), a highly conserved family of transferases in eukaryote. Although the importance of this family has been extensively studied in other plants, the precise composition and functional mechanisms of MAPK members in sand pear remain elusive.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003eThis study conducted an in-depth analysis of four \u003cem\u003ePpMAPK\u003c/em\u003e genes identified in the transcriptome of the ‘Whangkeumbae’(\u003cem\u003ePyrus pyrifolia\u003c/em\u003e) and 22 \u003cem\u003ePpMAPKs\u003c/em\u003ein the \u003cem\u003ePyrus pyrifolia\u003c/em\u003e genome, demonstrating a high degree of concordance between the transcriptomic and genomic data. Specifically, the transcriptomic PpMAPK3-L (GenBank accession number: PP992971), PpMAPK7-L(GenBank accession number: PP992972), PpMAPK10-L (GenBank accession number: PP992973), and PpMAPK16-L (GenBank accession number: PP992974) exhibited sequence homology values of 99.19%, 100%, 94.51%, and 95.75%, respectively, with their corresponding genomic counterparts (EVM0007944.1, EVM0004426.1, EVM0027166.1, EVM0023771, EVM0028755.1, EVM0015862.1). These findings indicate that the integrated analysis of transcriptomic and genomic data provides critical genetic insights into the \u003cem\u003eMAPK \u003c/em\u003egenes in sand pear, culminating in the identification of a total of 25 \u003cem\u003ePpMAPK\u003c/em\u003e genes in this species. Further phylogenetic analysis classified these genes into four subfamilies (A, B, C, and D), with subfamilies A and B each comprising six members, subfamily C with four members, and subfamily D with nine members. The potential functional differences among the gene members of each subfamily provide valuable clues for future research into MAPK signaling pathways. Additionally, interaction analysis revealed a significant interaction between PpMAPK3-L and PpbZIP2, which coordinatively regulate the senescence traits of fruits in sand pear through their joint influence during the senescence process.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003eThe results of this study suggest that \u003cem\u003ePpMAPK3-L\u003c/em\u003e, \u003cem\u003ePpMAPK7-L\u003c/em\u003e, \u003cem\u003ePpMAPK10-L\u003c/em\u003e, and \u003cem\u003ePpMAPK16-L\u003c/em\u003e are likely to play pivotal roles in the maturation and senescence of sand pear fruit. Specifically, the interaction between PpMAPK3-L and PpbZIP2 could play a key role in the regulation of fruit senescence, indicating that the MAPK signaling pathway may modulate the fruit's physiological state through interactions with transcription factors. This finding offers significant insights for further investigation into the functions of \u003cem\u003eMAPK\u003c/em\u003e genes in the maturation and senescence of sand pear fruit and provides a new direction for investigating biotechnological approaches for delaying senescence and prolonging shelf life.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification and molecular characterization of the MAPK family members in sand pear (Pyrus pyrifolia)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-07 12:59:58","doi":"10.21203/rs.3.rs-6141919/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-03-07T01:21:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-05T06:19:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-05T06:16:22+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-03-03T01:56:25+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a57fbfb0-4769-4f83-8f61-e5a783eebf85","owner":[],"postedDate":"March 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-19T16:01:13+00:00","versionOfRecord":{"articleIdentity":"rs-6141919","link":"https://doi.org/10.1186/s12864-025-11672-0","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-05-15 15:57:40","publishedOnDateReadable":"May 15th, 2025"},"versionCreatedAt":"2025-03-07 12:59:58","video":"","vorDoi":"10.1186/s12864-025-11672-0","vorDoiUrl":"https://doi.org/10.1186/s12864-025-11672-0","workflowStages":[]},"version":"v1","identity":"rs-6141919","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6141919","identity":"rs-6141919","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}