Genome-wide identification and expression analysis of the PP2C gene family in Apocynum venetum and Apocynum hendersonii

Genome-wide identification and expression analysis of the PP2C gene family in Apocynum venetum and Apocynum hendersonii | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide identification and expression analysis of the PP2C gene family in Apocynum venetum and Apocynum hendersonii Jiayi Chen, Yue Wang, Yongmei Wu, Xiaoyu Huang, Xiaojun Qiu, Jikang Chen, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4268917/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Protein phosphatase class 2C (PP2C) is the largest protein phosphatase family in plants, which plays an important role in plant growth and development and response to adversity stress. Apocynum is a perennial persistent herb, divided into Apocynum venetum and Apocynum hendersonii . It mainly grows in saline soil, desert and other harsh environments, and is widely used in saline soil improvement, ecological restoration, textile and medicine. It was found that Apocynum hendersonii is more tolerant of adverse conditions. The main purposeof this study was to investigate the PP2C gene family and its expression pattern under salt stress and to identify important candidate genes related to salt tolerance. Results In this study, 68 AvPP2C genes and 68 AhPP2C genes were identified from the genomes of Apocynum venetum and Apocynum hendersonii , respectively. They were classified into 13 subgroups based on their phylogenetic relationships and further analyzed for their subcellular locations, gene structures, conserved structural domains and cis-acting elements. The results of qRT-PCR analyses of seven AvPP2C genes and seven AhPP2C genes proved that they differed significantly in gene expression under salt stress. It has been observed that the PP2C genes in Apocynum venetum and Apocynum hendersonii exhibit different expression patterns. Specifically, AvPP2C2, 6, 24, 27, 41 and AhPP2C2, 6, 24, 27, 42 have shown significant differences in expression under salt stress. This indicates that these genes may play a crucial role in the salt tolerance mechanism of Apocynum venetum and Apocynum hendersonii . Conclusions In this study, we conducted a genome-wide analysis of the AvPP2C and AhPP2C gene families in Apocynum , which provided a reference for further understanding the functional characteristics of these genes. Apocynum Protein phosphatase 2C Salt stress Figures Figure 1 Figure 2 Background Plants are exposed to different environmental adversities such as drought, salinity, and high temperature during growth and development, which can affect the balance of ions, regulation of hormones, and the reception and transmission of signals in the plant body. These factors ultimately impact the growth and development of the plant and can significantly reduce crop yield and quality[1-3]. Plants have evolved an array of response mechanisms to thrive in challenging environments. Among these is protein reversible phosphorylation, a complex process that entails two opposing chemical reactions: protein phosphorylation and dephosphorylation. This modification process is facilitated by protein kinases (PKs) and protein phosphatases (PPs), and can promptly adjust the biological properties of proteins based on external stimuli. It plays a crucial role in plant growth and development, signal transduction, and in mitigating the effects of stress due to unfavorable conditions[4]. Phosphorylation occurs at specific sites in proteins, including serine (Ser), threonine (Thr), and tyrosine (Tyr), and is regulated by protein kinases (PKs) and protein phosphatases (PPs). PPs are classified based on their substrate specificity, with Ser/Thr phosphatases (STPs), protein Tyr phosphatases (PTPs), and dual-specificity phosphatases (DSPTPs) being the main categories[5]. PTPs are further divided into two groups, phosphoprotein metal phosphatases (PPM) and phosphoprotein phosphatases (PPP), based on crystal structure, amino acid sequence, and response to inhibitors[6, 7]. The PPP family includes various types of protein phosphatases, such as PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7, while type 2C protein phosphatase (PP2C) is the largest protein phosphatase family in plants. Although closely related to the PPP family, PP2C has no sequence homology and belongs to the PPM family[6-9]. PP2C has a conserved catalytic structure at the C-terminus and a functional extension region at the N-terminal that determines its various functions[9]. In Arabidopsis , distinct subgroups of PP2C genes exhibit specific functions. It is worth noting that these genes belong to the PP2C family, which plays a vital role in regulating plant growth and development. Group A comprises genes that participate in abscisic acid (ABA) signaling, such as ABI1 and ABI2 . These genes interact with SnRK2 , binding and dephosphorylating it, which inhibits the ABA signaling pathway and stress response[10-13]. Group B is responsible for mitogen-activated protein kinase (MAPK) signaling, while Group C is mainly involved in flower development[14]. Members of Group D may have negative effects on cell elongation, but can also have positive effects on the ABA signaling pathway in response to saline stress[15-17]. Group E is responsible for regulating the opening and closing of plant stomata, while Group F induces stress responses in bacteria. The PP2C gene family has been extensively researched in various plants, such as rice[18, 19], maize[20, 21], and wheat[22], among others. Their roles in plant growth, development, and ability to combat negative stressors are increasingly being comprehended. Apocynum is a perennial, persistent herb that grows mainly in harsh environments such as saline soils and deserts, and is widely used in saline land improvement, ecological restoration, textile and medicine[23-29]. Apocynum is divided into Apocynum venetum and Apocynum hendersonii , and these two species differ in morphological characteristics and physiological properties. The former is characterized by red stems and flowers, while the latter is more salt tolerant and sports white flowers, making it an ideal choice for soil improvement and restoration[30, 31]. At present, no systematic analyses of the PP2C gene family in A. venetum and A. hendersonii . It is important to explore the PP2C genes of Apocynum for salt tolerance in plants. In this study, we performed genome-wide identification and analysis of PP2C gene family members in Apocynum . 68 AvPP2C genes and 68 AhPP2C genes were identified, and the physicochemical properties, gene structure, conserved structural domains, cis-acting elements, chromosomal location distribution, gene duplication, and phylogenetic relationships of PP2C gene family members were analyzed. And the expression patterns of PP2C genes in different tissues of Apocynum were analyzed based on the transcriptome data of our group. The response of the PP2C gene after NaCl stress was analyzed by qRT-PCR study. The results of this study laid the foundation for the study of PP2C gene function in Apocynum and provided a reference for the excavation and genetic improvement of Apocynum resistance genes. Results Identification, characteristics and phylogenetic analysis of the PP2C gene family in Apocynum . In this study, we used BLASTp to identify 68 members of the Av PP2C gene family in Apocynum venetum genome and 68 members of the Ah PP2C gene family in the Apocynum hendersonii genome, respectively, using the sequences of 80 AtPP2C proteins from Arabidopsis thaliana as a reference. We used TBtools to map the positional distribution of the PP2C genes on chromosomes of Apocynum venetum and Apocynum hendersonii respectively, which were named AvPP2C1-AvPP2C68 and AhPP2C1-AhPP2C68 according to their chromosomal positions(Fig. 1). Except for AhPP2C68 which was not assembled, the remaining 68 AvPP2C genes and 67 AhPP2C genes were located on 11 chromosomes, respectively. There were fewer genes on chromosomes 4, 8 and 10. We analyzed the physicochemical properties such as molecular weight (MW), theoretical isoelectric point (pI), and subcellular localization of the 68 AvPP2C proteins and 68 AhPP2C proteins (Table S1). The 68 AvPP2C genes encoded proteins were located at lengths ranging from 278-1086, theoretical isoelectric points ranging from 4.38-9.78, and molecular weights ranging from 30369.58-123024.02 Da. The 68 AhPP2C genes encoded proteins with lengths ranging from 138-1092, theoretical isoelectric points ranging from 4.62-9.8, and molecular weights of 15540.2-123582.74 Da. The results of subcellular localization show that PP2C genes have the ability to appear in various areas of the cell, including the nucleus, chloroplast, cell wall, cell membrane, cytoplasm, mitochondria, and peroxisome. Some specific PP2C genes, such as AvPP2C9, 10, 16, 19, 20, 21, 29, 31, 35, 38, 47, 53, 62 and AhPP2C9, 10, 21, 29, 31, 35, 37, 38, 39, 48, 53, 54, 58 are predicted to be present in multiple cellular structures. To investigate the phylogenetic relationship between Apocynum venetum , Apocynum hendersonii and Arabidopsis thaliana PP2C genes, a phylogenetic tree was constructed using the neighbor-joining (NJ) method based on 80 At PP2C genes of Arabidopsis thaliana , 68 Av PP2C genes of Apocynum venetum and 68 Ah PP2C genes of Apocynum hendersonii (Fig. 2). Based on phylogenetic analysis, the PP2C genes in Apocynum were categorized into 13 subgroups (A-L). These subgroups contained 9, 4, 4, 8, 7, 6, 4, 6, 3, 2, 2, 7, 2 AvPP2C genes and 9, 4, 4, 9, 7, 6, 3, 6, 3, 2, 2, 7, 2 AhPP2C genes, with similar groupings to those found in Arabidopsis . Notably, PP2C proteins from both Apocynum and Arabidopsis were present in each subfamily, suggesting a common ancestor. Interestingly, AvPP2C3 , AvPP2C7 , AvPP2C31 , AvPP2C61 , AhPP2C3 , AhPP2C7 , AhPP2C31 , and AhPP2C60 of Apocynum did not cluster with any other group. The evolutionary tree also showed that genes in Apocynum venetum and Apocynum hendersonii were tightly clustered, indicating high homology and similar gene functions. Gene structures and conserved motifs analyses of the AvPP2C genes and the AhPP2C genes Based on their phylogenetic relationships, the structures and conserved domains of the AvPP2C and AhPP2C genes were studied. The gene structures include positional information such as coding sequence (CDS), untranslated regions (UTR), introns and PP2C structural domains(Fig. 3A and C). Genes within the same subgroup share similar gene structures, but there are variations in the length and arrangement of their CDSs and UTRs. For instance, all PP2C genes in group C contained UTR and CDS regions, but their lengths varied. AvPP2C42 had the lengthiest UTR region, while 16 genes in Apocynum venetum had no UTR regions and 14 genes in Apocynum hendersonii had no UTR regions. AvPP2C39 and AhPP2C40 had the highest number of CDS regions, totaling 16. After examining the number of introns, it was found that three AvPP2C genes and four AhPP2C genes in Apocynum had no introns, while the remaining 129 PP2C genes contained introns. In order to study the structure and function of PP2C proteins and their evolutionary relationships, the conserved domains of AvPP2Cs proteins and AhPP2Cs proteins were analyzed using the online MEME website. The analysis revealed 15 different conserved domains, which were given the names motif 1 to motif 15(Fig. 3A and B, Table S2). The results demonstrated that PP2C proteins in the same subgroups possess a similar distribution that may be linked to their functions. Motif 1 (except AvPP2C62, AhPP2C67, AhPP2C68), motif 2 (except AvPP2C31, AvPP2C44, AhPP2C45, AhPP2C31), and motif 3 (except AhPP2C68, AhPP2C19, AvPP2C19, AvPP2C48) were present in all subgroups. Motifs 6, 7, 11, 13 and 15 were absent in subgroups C and D, while motif 5 was only present in subgroups C and D, except AhPP2C68. Motif 9 and 10 were present only in subgroup D, and motif 11 was present only in subgroup E, but not in other groups. Cis-element analysis of the PP2Cs promoter in Apocynum venetum and Apocynum hendersonii The cis-acting elements were predicted in the promoter regions of Apocynum venetum and Apocynum hendersonii using analysis through the PlantCARE online website pairs and visualized using TBtools software, both of which retrieved a total of 68 cis-acting elements. Among them, there were 32 light-responsive elements, 13 phytohormone-responsive elements, 12 stress-responsive elements, and 11 elements related to plant growth and development (Fig. 4, Table S3). It was found that all the promoters of AvPP2C s and AhPP2C s contained light-responsive elements, phytohormone-responsive elements, and stress-responsive elements. However, elements associated with plant growth and development were missing in AvPP2C18 , AvPP2C33 , AvPP2C59 , AvPP2C61 , AvPP2C65 , AhPP2C18 , AhPP2C19 , AhPP2C32 , AhPP2C58 , AhPP2C60 , and AhPP2C66 . Box 4 was identified as the most abundant light-responsive element, with 263 in Apocynum venetum and 290 in Apocynum hendersonii . Among the phytohormone response elements, a large number of abscisic acid response elements (ABRE) and AAGAA-motifs were found, with AvPP2C s having 148 ABREs and 132 AAGAA-motifs, and AhPP2C s having 141 ABREs and 129 AAGAA-motifs. The ethylene response element (ERE) was also found in large numbers, with AvPP2C s having 147 and AhPP2C s having 156. The stress-responsive elements MYB and MYC were found to be the most abundant with 247 and 235 in Apocynum venetum and 242 and 238 in Apocynum hendersonii , respectively. These elements are involved in drought and ABA-induced responses. Another stress-responsive element, ARE, was found to be essential for anaerobic induction, with 133 in Apocynum venetum and 130 in Apocynum hendersonii . The large number of these elements suggests that most PP2C genes may respond to various stresses and are closely related to plant stress resistance. Collinear analysis and evolutionary analysis of AvPP 2 Cs proteins and AhPP2Cs proteins The expansion of gene families in the genome is predominantly caused by tandem and segmental duplications. To gain insight into the PP2C gene family, 68 AvPP2C genes and 68 AhPP2C genes were analyzed for duplication (Fig. 5A and B, Table S4). It was discovered that in Apocynum venetum , one tandem duplication pair was located on chromosome LG06 (39/40), whereas 17 pairs of segmental duplication genes were unevenly distributed on chromosomes other than LG08 and LG10. In Apocynum hendersonii , there were 2 tandem replication gene pairs located on chromosome LG06 (38/39, 40/41), and 17 fragment replication gene pairs were unevenly distributed on 9 chromosomes except for LG08 and LG10. These findings suggest that the evolution of PP2Cs in Apocynum was primarily driven by segmental duplication events, as the number of segmental duplication gene pairs was significantly greater than that of tandem duplications. Furthermore, the study investigated the homology between Apocynum venetum , Apocynum hendersonii , and Arabidopsis thaliana to understand the evolutionary relationship of PP2C genes(Fig. 5C, Table S5). The results indicated that there were 84 homologous gene pairs between Apocynum venetum and Arabidopsis thaliana , and 82 homologous gene pairs between Apocynum hendersonii and Arabidopsis thaliana . Among these pairs, Apocynum venetum and Apocynum hendersonii had the highest number of homologous gene pairs on chromosome LG06, with 13 pairs each. These findings suggest that Apocynum venetum and Apocynum hendersonii may have a high degree of homology with Arabidopsis. Tissue-specific expression of AvPP2C genes and AhPP2C genes in different tissues To gain a better understanding of the function of PP2C genes in the growth and development of Apocynum venetum and Apocynum hendersonii , we analyzed the expression patterns of PP2C genes using RNA-Seq data of roots, stems, and leaves of both species(Fig. 6). Most of the PP2C genes were expressed in all tissues, except for four genes ( AvPP2C19 , AvPP2C38 , AhPP2C39 , and AhPP2C68 ), which were barely expressed. In Apocynum venetum , AvPP2C47 was highly expressed in roots, stems, and leaves. AvPP2C9 , AvPP2C24 , AvPP2C49 , AvPP2C60 , and AvPP2C68 were moderately expressed in roots; AvPP2C13 and AvPP2C60 were highly expressed in stems; AvPP2C13 was highly expressed in leaves, and AvPP2C7 and AvPP2C60 were moderately expressed in leaves. In Apocynum hendersonii , AhPP2C29 and AhPP2C67 were highly expressed in roots, while AhPP2C24 , AhPP2C13 , AhPP2C49 , AhPP2C48 , and AhPP2C59 were moderately expressed in roots. AhPP2C9 , AhPP2C13 , AhPP2C59 , and AhPP2C48 were highly expressed in stems, and AhPP2C13 , AhPP2C48 , and AhPP2C59 were highly expressed in leaves. Relative expression changes of AvPP2C genes and AhPP2C genes under NaCl treatment In order to study the expression of AvPP2C genes and AhPP2C genes under salt stress conditions and to identify important PP2C genes related to salt tolerance. Based on phylogenetic analysis and homology of known PP2C genes in Arabidopsis thaliana , five genes in subgroup D and two genes in subgroup G were selected in Apocynum hendersonii and their expression patterns in different tissues of Apocynum venetum under salt stress were investigated by qRT-PCR(Fig. 7). The results showed that the AvPP2C1 gene was down-regulated in roots after NaCl stress, with the lowest expression at 50 mM concentration. It was up-regulated in leaves and phloem, with the highest expression at 200 mM and 100 mM concentrations, respectively, reaching 2.14-fold and 1.40-fold of CK. AhPP2C1 gene was up-regulated in leaves and increased with increasing NaCl concentration; it showed down-regulation in bast and decreased with increasing NaCl concentration. AvPP2C2 gene showed up-regulation under NaCl stress, in which the expression of the gene in leaves varied the most with NaCl concentration, which was 10.44 times that of CK. The expression trend of AvPP2C2 in root, xylem and phloem was the same, and all of them reached the maximum expression at 100 mM concentration. While the AhPP2C2 gene was down-regulated in roots and up-regulated in leaves after NaCl stress, there was little change in the xylem and phloem, but the expression reached the maximum at 50 mM concentration in all. AvPP2C6 gene did not change significantly in roots, but showed up-regulation in leaves and xylem phloem, and it is noteworthy that its expression in leaves increased with the increase of NaCl concentration and reached the maximum at 200 mM, which was 13.55 times that of CK. And AhPP2C6 was different from AvPP2C6 in that it showed down-regulation in roots. The expression of AvPP2C14 was different in roots, leaves, xylem and phloem, with the highest in leaves and the lowest in roots; after NaCl stress, the AvPP2C14 gene showed a tendency of increasing and then de-creasing with the increase of NaCl concentration, and its expression reached the maximum in all tissues at the concentration of 50 mM. While the expression of AhPP2C14 did not change significantly under NaCl treatment, it only showed up-regulation in leaves, and the expression of the gene was the highest at 50 mM concentration, and its expression gradually declined with the increase of concentration. AvPP2C24 gene had the highest expression in leaves and showed up-regulation in all tissues after NaCl stress. AhPP2C24 gene was the same as AvPP2C24 gene in that the expression of AhPP2C24 gene was up-regulated in all tissues after NaCl stress, except in phloem at 50 mM concentration. The difference was that the expression of AhPP2C24 gene changed more significantly in leaves after NaCl treatment. Both AvPP2C27 and AhPP2C27 genes showed up-regulation after NaCl treatment, but the expression of AvPP2C27 gene in roots and leaves increased with increasing NaCl concentration, while the expression of AhPP2C27 gene in leaves reached a maximum at 50 mM concentration and then decreased with increasing concentration. The expression of AvPP2C41 and AhPP2C42 genes in roots increased with increasing NaCl concentration, AvPP2C41 gene showed down-regulation in leaves at 100 mM and up-regulation at 50 mM and 200 mM concentrations. While AhPP2C42 gene showed up-regulation after NaCl stress, the expression was maximum in leaves at 50 mM concentration, which was 8.20 times of CK, and then decreased with the increase of NaCl concentration. Discussion PP2C genes are closely related to abscisic acid ABA signaling pathway, plant adversity stress, and stomatal opening and closing, etc. The PP2C gene families of Arabidopsis thaliana , rice, maize, and wheat have been investigated by previous authors. In this study, we comprehensively analyzed the AvPP2C genes of Apocynum venetum and the AhPP2C genes of Apocynum hendersonii , including the identification of the gene families, phylogenetic relationships, and chromosomal locations, collinear relationship, gene structure, conserved motifs, and expression patterns. A total of 136 PP2C genes, including 68 AvPP2C genes and 68 AhPP2C genes, were identified in Apocynum , and the physicochemical properties and subcellular localization of the genes were predicted, and it was found that the vast majority of PP2C genes were located in the nucleus and chloroplasts, and a few genes might be present in the mitochondria, cell wall, cytoplasm, peroxisomes, and cell membrane. Therefore, it is hypothesized that they may be related to photosynthesis, cell growth and development, and respiration. According to phylogenetic and evolutionary relationship analysis, Apocynum hendersonii and Apocynum venetum are closely related to each other, and a total of 136 PP2C genes in Apocynum hendersonii and Apocynum venetum were divided into 13 subgroups (A-L), which is consistent with the grouping of Arabidopsis thaliana . Previous studies found that genes in the A subgroup of PP2C genes in Arabidopsis mainly inhibit ABA receptor activity and negatively regulate ABA signaling[32]; the B subgroup is involved in the mitogen-activated protein kinase signaling pathway and participates in the process of MAPK phosphorylation, and is capable of dephosphorylating and inactivating MAPK[33]; Subgroup C gene family members are involved in stem cell maintenance and differentiation[34]; Members of subgroup D negatively regulate small auxin up RNA (SAUR)-mediated cell elongation, and PP2C.D1, PP2C.D2, and PP2C.D5 in Arabidopsis act during leaf greening, and PP2C.D1 regulates hook formation by affecting ethylene accumulation[15, 35, 36]. Moreover, members of this group respond to saline and alkaline stress, which may be related to stomatal movement and function, by inhibiting plasma membrane H-ATPase activity. It has been shown that Arabidopsis PP2C.D negatively regulates aluminium resistance by modulating malate secretion[16, 37]; Members of the E subgroup gene family may be associated with inflorescence stem growth, regulation of stomatal signaling, and so on[38-40]. Based on the phylogenetic relationship between Apocynum and Arabidopsis , it is hypothesized that the functions of gene family members located in the same subgroup in Apocynum may be similar to those in Arabidopsis . The prediction of the subcellular localization of PP2C gene family members in Apocynum showed that most of the PP2C gene family in Apocynum venetum and Apocynum hendersonii were predicted to be located in the nucleus and chloroplasts, in addition to which some of the genes might be located in the cell wall, cytoplasm, cell membrane, mitochondria, and peroxisomes. The predictions of PP2C gene family in Apocynum venetum and Apocynum hendersonii were partially different. For example, AvPP2C9 was predicted to be located in mitochondria and the nucleus, whereas AhPP2C9 was predicted to be located in chloroplasts, mitochondria, and the nucleus. AvPP2C20 was located in the nucleus and chloroplasts, whereas AhPP2C20 was predicted to be located in chloroplasts only. This may imply differences in the location and function of some PP2C genes in Apocynum hendersonii and Apocynum venetum . The diversity of gene structures, conserved structural domains, and cis-acting elements is also closely related to the functions of gene families. Genes in the same subgroup are similar in structure, which may imply that they have the same function[41]. The gene structures of Apocynum hendersonii and Apocynum venetum genes from the same evolutionary tree branching were similar, except for the AhPP2C68 gene. However, the gene structures of individual PP2C genes were different again, for example, AvPP2C18 and AhPP2C18 in subgroup L had significantly more CDS regions compared with AvPP2C33 and AhPP2C33 , which might be related to the increase of introns during the evolutionary process. The PP2C gene family is evolutionarily conserved, and most PP2Cs have a highly conserved structural domain with a unique non-catalytic N-terminal extension region. The specificity of the function of each sub-group of PP2C genes may be related to an N-terminal substrate docking domain[42]. A total of 15 conserved motifs were identified in the amino acid sequences of AvPP2C and AhPP2C genes, and the distribution of conserved motifs was similar for genes in the same subgroup, which may be closely related to the function of PP2C proteins. Cis-acting elements are involved in the regulation of gene expression and are closely related to plant growth and development, hormone responses and responses to various stresses. Among AvPP2C and AhPP2C genes, Box4 elements were the most abundant (AvPP2Cs: 263; AhPP2Cs: 290), followed by MYB elements (AvPP2Cs: 247; AhPP2Cs: 242). Among all the response elements, light responsive and stress responsive were the most numerous elements, with a high number of 1,318 stress responsive in Apocynum hendersonii . This may be related to the characteristics of Apocynum hendersonii high-stress tolerance. In this study, chromosomal position analysis and covariance analysis of PP2C gene family of Apocynum venetum and Apocynum hendersonii were carried out, which is important to understand the amplification mechanism of the genes. It was found that both AvPP2Cs and AhPP2Cs were distributed on 11 chromosomes. Seventeen pairs of segmental duplications and one pair of tandem duplications were identified in Apocynum venetum ; 17 pairs of segmental duplications and two pairs of tandem duplications were identified in Apocynum hendersonii . Gene duplication is the main driver of gene family amplification, which mainly includes segmental duplication, tandem duplication, and genomic duplication, and segmental duplication is more conducive to maintaining gene function than tandem duplication[43]. Therefore, we hypothesize that segmental duplication is the main force driving the evolution and expansion of the PP2C gene family, which is similar to the results found for PP2C in Arabidopsis [44], woodland and pineapple strawberry[45], and cucumber[4]. AvPP2C genes and AhPP2C genes showed specific expression in different tissues, and most of the PP2C genes were lowly expressed in roots, stems and leaves. However, among all the PP2C genes, AvPP2C13 , AvPP2C47 , AvPP2C60 , AhPP2C48 , and AhPP2C59 showed higher expression in all three tissues: roots, stems, and leaves, whereas AhPP2C29 , and AhPP2C67 showed high expression in roots and lower expression in stems and leaves. In cucumber, most of the CsPP2C genes were highly expressed in fertilized ovaries, male, female, and leaf (except CsPP2C11, 41, 5, 33, 50 ), and low in other tissues [4]. In maize, most of the ZmPP2C genes were expressed in all tissues, with ZmPP2C42 and ZmPP2C47 being the most highly expressed in mature pollen, and ZmPP2C59 being expressed in primary roots and root cortex[46]. This suggests that PP2C genes in different plants may play different roles at different stages of plant growth, and some of these PP2C genes may have special functions in specific tissues. Studies on Arabidopsis have shown that members of subgroup D respond to saline stress, and AtPP2C.D6 and AtPP2C.D7 interact with SCaBP8 of the SOS pathway, whose phosphatase activity is inhibited under salt stress. AtPP2C . G1 in group G is dependent on ABA to positively regulate salt tolerance. Our study showed that the expression of some AvPP2C genes differed significantly from that of AhPP2C genes after NaCl stress, in which the expression of AvPP2C2 gene was significantly up-regulated after salt stress, and the expression was significantly higher in roots, xylem, phloem, and leaves. It indicated that AvPP2C2 gene might play an important role in these four tissues when Apocynum venetum resisted salt stress. The expression of AhPP2C2 gene was down-regulated in roots and up-regulated in leaves after salt stress, while it showed a similar expression pattern in xylem and phloem, both of which peaked at a concentration of 50 mM. AvPP2C2 and AhPP2C2 showed different expression patterns in Apocynum venetum and Apocynum hendersonii under salt stress, which was hypothesized to be related to their differences in salt tolerance. The expression of AvPP2C6 and AhPP2C6 was up-regulated in leaves, xylem and phloem, and the change of AvPP2C6 was not obvious in roots, while AhPP2C6 showed down-regulation in roots. AvPP2C24, 27 and 41 in roots showed similar trends after salt stress. In summary, PP2C genes located in the same subgroup responded differently in the face of salt stress, while there may be different expression patterns in Apocynum venetum and Apocynum hendersonii , where AvPP2C2, 6, 24, 27, 41 and AhPP2C2, 6, 24, 27, 42 showed significant differences in expression under salt stress, suggesting that these genes may play important roles in Apocynum venetum and Apocynum hendersonii when facing salt stress. Conclusions Genome-wide identification and analysis of PP2C family members in Apocynum venetum genomes and Apocynum hendersonii genomes. In this study, a total of 68 AvPP2C genes and 68 AhPP2C genes were identified and classified into 13 subgroups, which were further analyzed for subcellular localization, gene structure, conserved structural domains, and cis-acting elements. By analyzing the expression patterns of PP2C genes in Apocynum venetum and Apocynum hendersonii under salt stress, it was found that the PP2C genes in subgroup D and subgroup G showed significant differences under salt stress, which might be related to the differences in salt tolerance between Apocynum venetum and Apocynum hendersonii . This study provides a reference for the subsequent research on the function of PP2C gene. Materials and methods Identification of PP2C genes in Apocynum The protein sequences of 80 At PP2C genes of Arabidopsis were downloaded from Tair (https://www.arabidopsis.org/). Protein sequences of Apocynum venetum and Apocynum hendersonii were obtained from the whole genome data sequenced in our laboratory. The PP2C genes were identified by two methods. First, the putative PP2C protein present in the genome was identified using BLASTp. Then, the PP2C structural domain (PF00481) of the PP2C protein was downloaded from Pfam (http://pfam.xfam.org/) and searched against the local protein database using HMMER 3.0[47, 48]. Meanwhile, the SMART (https://smart.embl.de/) was used to predict its structural domain with E-value <e-5[49]. The results obtained by both methods were intersected and the protein sequences were extracted from the local protein database using TBtools and the gene IDs identified by screening. Sequence analysis and basic information of the Apocynum PP2C gene family Physicochemical property analysis of PP2C proteins including number of amino acid, molecular weight, theoretical pI, instability index, aliphatic index, grand average of hydropathicity were analyzed by Protein Paramter Calc in TBtools. Prediction of the subcellular location of PP2C protein by Plant-mPloc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) of Cell-Ploc 2.0. Analysis of chromosome location and collinearity analysis Chromosome position information was obtained from the annotation file of the Apocynum genome and the chromosome position distribution was mapped using TBtools. Homology and collinearity within the Apocynum venetum and Apocynum hendersonii genomes and between them and Arabidopsis thaliana were analyzed using the MCScanX software and visualized using the Advanced Circos and Multiple Synteny Plot functions in TBtools[50, 51]. Construction of phylogenetic tree The phylogenetic tree of the PP2C gene family of Arabidopsis and Apocynum was constructed using MEGA-X with the Neighbor-Joining method, Bootstrap value set to 1000 and other default parameters[52, 53]. Finally, the phylogenetic tree was embellished with iTOL(https://itol.embl.de/)[54]. Analysis of gene structures and protein conserved motifs The gene structure of Apocynum PP2C was analyzed using the Batch CD-search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) on the NCBI website. The MEME website (https://meme-suite.org/meme/tools/meme) was used to predict the conserved structural domains of the PP2C genes in Apocynum , setting the motif number to 15 and the rest to default values[55]. Gene Structure View in TBtools was used to generate the final overall map of the phylogenetic tree, gene structure and conserved structural domains of PP2C s . Analysis of cis-acting elements in PP2C gene promoters The sequences of 2.0 kb upstream of AvPP2C genes and AhPP2C genes were extracted using TBtools and then the results were submitted to PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for cis-acting element prediction[55]. Expression Analysis of PP2C Genes From the transcriptome data of roots, stems and leaves of Apocynum venetum and Apocynum hendersonii , the FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values of AvPP2C s and AhPP2C s were extracted and heat maps were generated using TBtools[51]. Plant material treatment methods Seedlings grown under hydroponic conditions for 6-8 weeks were divided into four groups and treated with different concentrations (0, 50, 100, and 200 mM) of NaCl stress, and after 14 days of treatment, respectively, the roots, leaves, xylem and phloem were sampled and stored in a refrigerator at -80°C. RNA Isolation and q RT- PCR Total RNA was extracted using the SteadyPure Plant RNA Extraction Kit (Accurate Biotechnology (Changsha, China) Co., Ltd.), and the RNA was reverse transcribed into cDNA using the Evo M-MLV One Step RT-PCR Kit (Accurate Biotechnology (Changsha, China) Co., Ltd.)[56]. Specific primers were designed for qRT-PCR using Primer5(Table S6). ACT gene was used as an internal reference gene. qRT-PCR was performed on a CFX96 Touch Deep Well Real-Time Quantitative PCR System (Bio-Rad) using SYBR® Green Premix Pro Taq HS qPCR Kit II (Accurate Biotechnology (Changsha, China) Co., Ltd.)[56]. The relative expression of PP2C genes was calculated using the 2 -ΔΔCT method and histograms were plotted using GraphPad Prism 8. List of Abbreviations PP2C: Protein phosphatase class 2C qRT-PCR: Quantitative Real-time PCR MAPK: Mitogen-activated protein kinase MW: Molecular weight pI: Isoelectric point CDS: Coding sequence UTR: Untranslated regions CK: Control check SAUR: Small auxin up RNA Declarations Ethics approval and consent to participate The plant material used in this study was Apocynum venetum and Apocynum hendersonii , grown in the laboratory of the Institute of bast fiber crops, Chinese Academy of Agricultural Sciences (CAAS) and no permits were required to collect plant samples. In the present study, all methods were carried out following relevant guidelines and regulations. Ethical approval or consent was not required for this study because no endangered or protected species were involved. Consent for publication Not applicable. Availability of data and materials The sequence information of Arabidopsis PP2C family genes were collected from The Arabidopsis Information Resoure (https://www.arabidopsis.org/). The PP2C family expression data were generated by qRT-PCR. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by Hunan Provincial Department of Education general project (No.22C0674)，ESI Discipline Special Project of Changsha Medical University (No.2022CYY023 and No.2022CYY006)，Science Research Project of Hunan Provincial Department of Education (No.23A0662)，National Undergraduate Innovation and Entrepreneurship Training Program Project under Grant (No.202210823006). Contributions Jiayi Chen: Methodology, writing-review. Yue Wang: Conceived, designed the experiments and writing, Methodology. Xiaoyu Huang, Xiaojun Qiu: Methodology, writing-review & editing. Jikang Chen, Qian Lin: Methodology. Haohan Zhao, Yongmei Wu: Edit improves the manuscript. Gang Gao, Fengming Chen: Concept of study, Supervision and revised the manuscript. Acknowledgements The authors thank laboratory colleagues for their technical assistance. The authors thank the editors and reviewers for their rigorous evaluation of the manuscript. References Yang S, Gong Z, Guo Y, Gong J, Zheng S, Lin R, Yang H, Mao L, Qin F, Luo L et al : Studies on plant responses to environmental change in China:the past and the future . Scientia Sinica Vitae 2019, 49 (11):1457-1478. Zhu JK: Abiotic stress signaling and responses in plants . Cell 2016, 167 (2):313-324. Waadt R, Seller CA, Hsu PK, Takahashi Y, Munemasa S, Schroeder J: Plant hormone regulation of abiotic stress responses . Nature Reviews Molecular Cell Biology 2022, 23 (10):680-694. Zhang G, Zhang Z, Luo S, Li X, Lyu J, Liu Z, Wan Z, Yu J: Genome-wide identification and expression analysis of the cucumber PP2C gene family . Bmc Genomics 2022, 23 (1). Cao J, Jiang M, Li P, Chu Z: Genome-wide identification and evolutionary analyses of the PP2C gene family with their expression profiling in response to multiple stresses in Brachypodium distachyon . Bmc Genomics 2016, 17 . Luan S: Protein phosphatases in plants . Annual Review of Plant Biology 2003, 54 :63-92. Shazadee H, Khan N, Wang J, Wang C, Zeng J, Huang Z, Wang X: Identification and expression profiling of protein phosphatases ( PP2C ) gene family in Gossypium hirsutum L . International Journal of Molecular Sciences 2019, 20 (6). Schweighofer A, Hirt H, Meskiene L: Plant PP2C phosphatases: emerging functions in stress signaling . Trends in Plant Science 2004, 9 (5):236-243. Zhang J, Tao N: Research progress of plant PP2C-type protein phosphatase in ABA signal transduction and adversity stress regulation mechanism . Guangxi Zhiwu / Guihaia 2015, 35 (6):935-941. Peirats-Llobet M, Han SK, Gonzalez-Guzman M, Jeong CW, Rodriguez L, Belda-Palazon B, Wagner D, Rodriguez PL: A direct link between abscisic acid sensing and the chromatin-remodeling ATPase BRAHMA via Core ABA signaling pathway components . Molecular Plant 2016, 9 (1):136-147. Hsu PK, Dubeaux G, Takahashi Y, Schroeder JI: Signaling mechanisms in abscisic acid-mediated stomatal closure . Plant Journal 2021, 105 (2):307-321. Li ZX, Waadt R, Schroeder JI: Release of GTP exchange factor mediated down-regulation of abscisic acid signal transduction through ABA-induced rapid degradation of RopGEFs . Plos Biology 2016, 14 (5). Shi Y, Liu XN, Zhao SS, Guo Y: The PYR-PP2C-CKL2 module regulates ABA-mediated actin reorganization during stomatal closure . New Phytologist 2022, 233 (5):2168-2184. Wu P, Wang W, Li Y, Hou X: Divergent evolutionary patterns of the MAPK cascade genes in Brassica rapa and plant phylogenetics . Horticulture Research 2017, 4 . Rovira A, Sentandreu M, Nagatani A, Leivar P, Monte E: The sequential action of MIDA9/PP2C.D1, PP2C.D2, and PP2C.D5 is necessary to form and maintain the hook after germination in the dark . Frontiers in Plant Science 2021, 12 . Chen C, Yu Y, Ding X, Liu B, Duanmu H, Zhu D, Sun X, Cao L, Zaib un N, Li Q et al : Genome-wide analysis and expression profiling of PP2C clade D under saline and alkali stresses in wild soybean and Arabidopsis . Protoplasma 2018, 255 (2):643-654. Xue TT, Wang D, Zhang SZ, Ehlting J, Ni F, Jakab S, Zheng CC, Zhong Y: Genome-wide and expression analysis of protein phosphatase 2C in rice and Arabidopsis . Bmc Genomics 2008, 9 . Singh A, Jha SK, Bagri J, Pandey GK: ABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis . Plos One 2015, 10 (4). Xie W, Liu S, Gao H, Wu J, Liu D, Kinoshita T, Huang C-F: PP2C.D phosphatase SAL1 positively regulates aluminum resistance via restriction of aluminum uptake in rice . Plant Physiology 2023, 192 (2):1498-1516. He Z, Wu J, Sun X, Dai M: The maize clade a PP2C phosphatases play critical roles in multiple abiotic stress responses . International Journal of Molecular Sciences 2019, 20 (14). Guo YZ, Shi YB, Wang YL, Liu F, Li Z, Qi JS, Wang Y, Zhang JB, Yang SH, Wang Y et al : The clade F PP2C phosphatase ZmPP84 negatively regulates drought tolerance by repressing stomatal closure in maize . New Phytologist 2023, 237 (5):1728-1744. Wang JY, Li CA, Li L, Gao LF, Hu G, Zhang YF, Reynolds MP, Zhang XY, Jia JZ, Mao XG et al : DIW1 encoding a clade I PP2C phosphatase negatively regulates drought tolerance by de-phosphorylating TaSnRK1.1 in wheat . Journal of Integrative Plant Biology 2023. Guo X, Chai W, Bai J, Ma Z: Cloning and bioinformatics analysis of AvFLS gene from Apocynum venetum . Molecular Plant Breeding 2019, 17 (15):4978-4985. Huang X, Qiu X, Wang Y, Abubakar AS, Chen P, Chen J, Chen K, Yu C, Wang X, Gao G et al : Genome-wide investigation of the NAC transcription factor family in Apocynum venetum revealed their synergistic roles in abiotic stress response and trehalose metabolism . International Journal of Molecular Sciences 2023, 24 (5). Zhang M, Lu X, Ren T, Marowa P, Meng C, Wang J, Yang H, Li C, Zhang L, Xu Z: Heterologous overexpression of Apocynum venetum flavonoids synthetase genes improves Arabidopsis thaliana salt tolerance by activating the IAA and JA biosynthesis pathways . Frontiers in Plant Science 2023, 14 . Cao C, Lin DF, Zhou YJ, Li N, Wang YW, Gong WB, Zhu ZH, Liu CW, Yan L, Hu ZX et al : Solid-state fermentation of Apocynum venetum L. by Aspergillus niger: Effect on phenolic compounds, antioxidant activities and metabolic syndrome-associated enzymes . Frontiers in Nutrition 2023, 10 . Li X, Li JJ, Su HY, Sun P, Zhang Z, Li MF, Xing H: Physiological and transcriptional responses of Apocynum venetum to salt stress at the seed germination stage . International Journal of Molecular Sciences 2023, 24 (4). Zhang Y, Liu S, Ma JL, Chen C, Huang P, Ji JH, Wu D, Ren LQ: Apocynum venetum leaf extract alleviated doxorubicin-induced cardiotoxicity through the AKT/Bcl-2 signaling pathway . Phytomedicine 2022, 94 . Jiang L, Wang L, Tian CY: High lithium tolerance of Apocynum venetum seeds during germination . Environmental Science and Pollution Research 2018, 25 (5):5040-5046. Gao G, Abubakar AS, Chen J, Wang Y, Chen P, Chen K, Yu C, Wang X, Qiu X, Huang X et al : Comparative genome and metabolome analyses uncover the evolution and flavonoid biosynthesis between Apocynum venetum and Apocynum hendersonii . Iscience 2023, 26 (5). Yuan N, Li MM, Jia CL: De novo transcriptome assembly and population genetic analyses of an important coastal shrub, Apocynum venetum L . Bmc Plant Biology 2020, 20 (1). Ma Y: Regulators of PP2C phosphatase activity function as abscisic acid sensors (vol 324, pg 1064, 2009) . Science 2009, 324 (5932):1266-1266. Umbrasaite J, Schweighofer A, Meskiene I: Substrate analysis of Arabidopsis PP2C-type protein phosphatases . In: Plant Kinases: Methods and Protocols. Edited by Dissmeyer N, Schnittger A, vol. 779; 2011: 149-161. Yu LP, Miller AK, Clark SE: POLTERGEIST encodes a protein phosphatase 2C that regulates CLAVATA pathways controlling stem cell identity at Arabidopsis shoot and flower meristems . Current Biology 2003, 13 (3):179-188. Sentandreu M, Martín G, González-Schain N, Leivar P, Soy J, Tepperman JM, Quail PH, Monte E: Functional profiling identifies genes involved in organ-specific branches of the PIF3 regulatory network in Arabidopsis . Plant Cell 2011, 23 (11):3974-3991. Spartz AK, Ren H, Park MY, Grandt KN, Lee SH, Murphy AS, Sussman MR, Overvoorde PJ, Gray WM: SAUR inhibition of PP2C-D phosphatases activates plasma membrane H + -ATPases to promote cell expansion in Arabidopsis . Plant Cell 2014, 26 (5):2129-2142. Fu H, Yu X, Jiang Y, Wang Y, Yang Y, Chen S, Chen Q, Guo Y: SALT OVERLY SENSITIVE 1 is inhibited by clade D Protein phosphatase 2C D6 and D7 in Arabidopsis thaliana . Plant Cell 2023, 35 (1):279-297. Mishra G, Zhang WH, Deng F, Zhao J, Wang XM: A bifurcating pathway directs abscisic acid effects on stomatal closure and opening in Arabidopsis . Science 2006, 312 (5771):264-266. Sugimoto H, Kondo S, Tanaka T, Imamura C, Muramoto N, Hattori E, Ogawa Ki, Mitsukawa N, Ohto C: Overexpression of a novel Arabidopsis PP2C isoform, AtPP2CF1 , enhances plant biomass production by increasing inflorescence stem growth . Journal of Experimental Botany 2014, 65 (18):5385-5400. Wong JH, Klejchová M, Snipes SA, Nagpal P, Bak G, Wang B, Dunlap S, Park MY, Kunkel EN, Trinidad B et al : SAUR proteins and PP2C.D phosphatases regulate H + -ATPases and K + channels to control stomatal movements . Plant Physiology 2021, 185 (1):256-273. Yang J, Chen R, Hu W, Wu Q, Tong X, Li X: Identification and expression analysis of PP2C gene family in Poncirus trifoliata . Journal of Fruit Science 2022, 39 (4):532-547. Meskiene I, Baudouin E, Schweighofer A, Liwosz A, Jonak C, Rodriguez PL, Jelinek H, Hirt H: Stress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase . Journal of Biological Chemistry 2003, 278 (21):18945-18952. Lynch M, Conery JS: The evolutionary fate and consequences of duplicate genes . Science 2000, 290 (5494):1151-1155. Cannon SB, Mitra A, Baumgarten A, Young ND, May G: The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana . BMC plant biology 2004, 4 :10-10. Guo L, Lu S, Liu T, Nai G, Ren J, Gou H, Chen B, Mao J: Genome-wide identification and abiotic stress response analysis of PP2C gene family in Woodland and Pineapple Strawberries . International Journal of Molecular Sciences 2023, 24 (4). Wu H, Zhu L, Cai G, Lv C, Yang H, Ren X, Hu B, Zhou X, Jiang T, Xiang Y et al : Genome-wide identification and characterization of the PP2C family from Zea mays and its role in long-distance signaling . Plants-Basel 2023, 12 (17). Chen CJ, Chen H, Zhang Y, Thomas HR, Frank MH, He YH, Xia R: TBtools: An integrative toolkit developed for interactive analyses of big biological data . Molecular Plant 2020, 13 (8):1194-1202. Mistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer ELL, Tosatto SCE, Paladin L, Raj S, Richardson LJ et al : Pfam: The protein families database in 2021 . Nucleic Acids Res 2021, 49 (D1):D412-D419. Ren M, Wang Q, Zhang F-h, Wang Y-l, Wang Y-y, Li W, Qi K-j, Xie Z-h, Zhang S-l, Tao S-t: Genome-wide identification of the GAox gene family and functional characterization of PbGA3ox4 during stone cell formation in Chinese white pear . Scientia Horticulturae 2024, 330 . Wang YP, Tang HB, DeBarry JD, Tan X, Li JP, Wang XY, Lee TH, Jin HZ, Marler B, Guo H et al : MCScanX : a toolkit for detection and evolutionary analysis of gene synteny and collinearity . Nucleic Acids Res 2012, 40 (7). Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R: TBtools: An integrative toolkit developed for interactive analyses of big biological data . Molecular Plant 2020, 13 (8):1194-1202. Kumar S, Stecher G, Li M, Knyaz C, Tamura K: MEGA X: Molecular Evolutionary genetics analysis across computing platforms . Molecular Biology and Evolution 2018, 35 (6):1547-1549. Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees . Molecular biology and evolution 1987, 4 (4):406-425. Letunic I, Bork P: Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation . Nucleic Acids Res 2021, 49 (W1):W293-W296. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren JY, Li WW, Noble WS: MEME SUITE: tools for motif discovery and searching . Nucleic Acids Res 2009, 37 :W202-W208. Qiu X, Zhao H, Abubakar AS, Shao D, Chen J, Chen P, Yu C, Wang X, Chen K, Zhu A: Genome-wide analysis of AP2/ERF gene superfamily in Ramie ( Boehmeria nivea L.) revealed their synergistic roles in regulating abiotic stress resistance and ramet development . International Journal of Molecular Sciences 2022, 23 (23). Additional Declarations No competing interests reported. Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx TableS6.xlsx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 07 May, 2024 Reviews received at journal 07 May, 2024 Reviews received at journal 06 May, 2024 Reviewers agreed at journal 25 Apr, 2024 Reviewers agreed at journal 24 Apr, 2024 Reviewers invited by journal 24 Apr, 2024 Editor invited by journal 19 Apr, 2024 Editor assigned by journal 19 Apr, 2024 Submission checks completed at journal 19 Apr, 2024 First submitted to journal 15 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-4268917","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":293799002,"identity":"a000e822-97e9-4c51-8be2-c961d70e7a4a","order_by":0,"name":"Jiayi Chen","email":"","orcid":"","institution":"Hunan Provincial Key Laboratory of the Traditional Chinese Medicine Agricultural Biogenomics, Changsha Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Chen","suffix":""},{"id":293799004,"identity":"78175e4b-d858-4162-9ff7-67de39022834","order_by":1,"name":"Yue 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10:07:58","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4268917/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4268917/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55240308,"identity":"9db70ab0-d2e7-4cf1-92a2-50aeaed36193","added_by":"auto","created_at":"2024-04-24 14:53:56","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":242682,"visible":true,"origin":"","legend":"\u003cp\u003eChromosomal distribution and localization of the \u003cem\u003ePP2C\u003c/em\u003e gene family in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e. The blue rectangular bars represent chromosomes of \u003cem\u003eApocynum\u003c/em\u003e, and chromosome names are shown on the left side of each chromosome. The 0-27 Mb scale bar on the left represents chromosome length. (A) Chromosome distribution and localization of \u003cem\u003eAvPP2Cs\u003c/em\u003e. (B) Chromosome distribution and localization of \u003cem\u003eAhPP2Cs\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/dde46a2b76cdc5b4ed06f8e0.jpeg"},{"id":55241116,"identity":"8feefc3d-9763-4b2d-8772-8e175662fb4b","added_by":"auto","created_at":"2024-04-24 15:01:57","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5382539,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of \u003cem\u003ePP2C\u003c/em\u003e family between \u003cem\u003eApocynum venetum\u003c/em\u003e, \u003cem\u003eApocynum hendersonii\u003c/em\u003e with \u003cem\u003eArabidopsis\u003c/em\u003e. The phylogenetic tree was constructed by MEGA 7 using the neighbor-joining method. The subgroups (A-L) are represented by different colors.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/d533ded0c35f8b63fcebac81.png"},{"id":55241645,"identity":"3eb78d16-fc58-4922-b876-086d9be95146","added_by":"auto","created_at":"2024-04-24 15:10:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2514332,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/1c1a956f-83fc-4a15-a876-32f6e211ec74.pdf"},{"id":55240310,"identity":"6222b03f-cfab-41cb-849d-c6d914225cd6","added_by":"auto","created_at":"2024-04-24 14:53:57","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":22300,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/3c35713a77a8543322b61ad0.xlsx"},{"id":55241117,"identity":"fe346d9e-bf5f-4c58-8472-8c4f465dde08","added_by":"auto","created_at":"2024-04-24 15:01:57","extension":"xlsx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":10812,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/38b709dededf46d3ad771920.xlsx"},{"id":55240319,"identity":"1b617d7c-0f1d-482b-ab66-afaa675be6b2","added_by":"auto","created_at":"2024-04-24 14:53:57","extension":"xlsx","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":27916,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/edb4ad7484637e33583511c1.xlsx"},{"id":55240316,"identity":"b34e4216-d631-496e-bfe6-37f1c35dbf86","added_by":"auto","created_at":"2024-04-24 14:53:57","extension":"xlsx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":14213,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/b9c2eb24490b7b4409f041ed.xlsx"},{"id":55241118,"identity":"a2e158f0-8508-49e4-88a0-72d37822a3d0","added_by":"auto","created_at":"2024-04-24 15:01:57","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":20470,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/34056f4a97e2022d07b54c03.xlsx"},{"id":55240320,"identity":"fb7ca649-8e8e-4a24-ac25-1df1ea319006","added_by":"auto","created_at":"2024-04-24 14:53:57","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":11437,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4268917/v1/9734a21ea2622c4df41dd7ed.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification and expression analysis of the PP2C gene family in Apocynum venetum and Apocynum hendersonii","fulltext":[{"header":"Background","content":"\u003cp\u003ePlants are exposed to different environmental adversities such as drought, salinity, and high temperature during growth and development, which can affect the balance of ions, regulation of hormones, and the reception and transmission of signals in the plant body. These factors ultimately impact the growth and development of the plant and can significantly reduce crop yield and quality[1-3]. Plants have evolved an array of response mechanisms to thrive in challenging environments. Among these is protein reversible phosphorylation, a complex process that entails two opposing chemical reactions: protein phosphorylation and dephosphorylation. This modification process is facilitated by protein kinases (PKs) and protein phosphatases (PPs), and can promptly adjust the biological properties of proteins based on external stimuli. It plays a crucial role in plant growth and development, signal transduction, and in mitigating the effects of stress due to unfavorable conditions[4]. Phosphorylation occurs at specific sites in proteins, including serine (Ser), threonine (Thr), and tyrosine (Tyr), and is regulated by protein kinases (PKs) and protein phosphatases (PPs). PPs are classified based on their substrate specificity, with Ser/Thr phosphatases (STPs), protein Tyr phosphatases (PTPs), and dual-specificity phosphatases (DSPTPs) being the main categories[5]. PTPs are further divided into two groups, phosphoprotein metal phosphatases (PPM) and phosphoprotein phosphatases (PPP), based on crystal structure, amino acid sequence, and response to inhibitors[6, 7]. The PPP family includes various types of protein phosphatases, such as PP1, PP2A, PP2B, PP4, PP5, PP6, and PP7, while type 2C protein phosphatase (PP2C) is the largest protein phosphatase family in plants. Although closely related to the PPP family, PP2C has no sequence homology and belongs to the PPM family[6-9]. PP2C has a conserved catalytic structure at the C-terminus and a functional extension region at the N-terminal that determines its various functions[9]. In \u003cem\u003eArabidopsis\u003c/em\u003e, distinct subgroups of \u003cem\u003ePP2C\u003c/em\u003e genes exhibit specific functions. It is worth noting that these genes belong to the \u003cem\u003ePP2C\u003c/em\u003e family, which plays a vital role in regulating plant growth and development. Group A comprises genes that participate in abscisic acid (ABA) signaling, such as \u003cem\u003eABI1\u003c/em\u003e and \u003cem\u003eABI2\u003c/em\u003e. These genes interact with \u003cem\u003eSnRK2\u003c/em\u003e, binding and dephosphorylating it, which inhibits the ABA signaling pathway and stress response[10-13]. Group B is responsible for mitogen-activated protein kinase (MAPK) signaling, while Group C is mainly involved in flower development[14]. Members of Group D may have negative effects on cell elongation, but can also have positive effects on the ABA signaling pathway in response to saline stress[15-17]. Group E is responsible for regulating the opening and closing of plant stomata, while Group F induces stress responses in bacteria. The \u003cem\u003ePP2C\u003c/em\u003e gene family has been extensively researched in various plants, such as rice[18, 19], maize[20, 21], and wheat[22], among others. Their roles in plant growth, development, and ability to combat negative stressors are increasingly being comprehended.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eApocynum\u003c/em\u003e is a perennial, persistent herb that grows mainly in harsh environments such as saline soils and deserts, and is widely used in saline land improvement, ecological restoration, textile and medicine[23-29]. \u003cem\u003eApocynum\u003c/em\u003e is divided into \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e, and these two species differ in morphological characteristics and physiological properties. The former is characterized by red stems and flowers, while the latter is more salt tolerant and sports white flowers, making it an ideal choice for soil improvement and restoration[30, 31]. At present, no systematic analyses of the \u003cem\u003ePP2C\u003c/em\u003e gene family in \u003cem\u003eA. venetum\u003c/em\u003e and \u003cem\u003eA. hendersonii\u003c/em\u003e. It is important to explore the \u003cem\u003ePP2C\u003c/em\u003e genes of \u003cem\u003eApocynum\u003c/em\u003e for salt tolerance in plants.\u003c/p\u003e\n\u003cp\u003eIn this study, we performed genome-wide identification and analysis of \u003cem\u003ePP2C\u003c/em\u003e gene family members in \u003cem\u003eApocynum\u003c/em\u003e. 68 \u003cem\u003eAvPP2C\u003c/em\u003e genes and 68 \u003cem\u003eAhPP2C\u003c/em\u003e genes were identified, and the physicochemical properties, gene structure, conserved structural domains, cis-acting elements, chromosomal location distribution, gene duplication, and phylogenetic relationships of \u003cem\u003ePP2C\u003c/em\u003e gene family members were analyzed. And the expression patterns of \u003cem\u003ePP2C\u003c/em\u003e genes in different tissues of \u003cem\u003eApocynum\u003c/em\u003e were analyzed based on the transcriptome data of our group. The response of the \u003cem\u003ePP2C\u003c/em\u003e gene after NaCl stress was analyzed by qRT-PCR study. The results of this study laid the foundation for the study of \u003cem\u003ePP2C\u003c/em\u003e gene function in \u003cem\u003eApocynum\u003c/em\u003e and provided a reference for the excavation and genetic improvement of \u003cem\u003eApocynum\u003c/em\u003e resistance genes.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIdentification, characteristics and phylogenetic analysis of the \u003cem\u003ePP2C\u003c/em\u003e gene family \u003c/strong\u003e\u003cstrong\u003ein\u003c/strong\u003e\u003cstrong\u003e \u003cem\u003eApocynum\u003c/em\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we used BLASTp to identify 68 members of the \u003cem\u003eAv\u003c/em\u003e\u003cem\u003ePP2C\u003c/em\u003e gene family in \u003cem\u003eApocynum venetum\u003c/em\u003e genome and 68 members of the \u003cem\u003eAh\u003c/em\u003e\u003cem\u003ePP2C\u003c/em\u003e gene family in the \u003cem\u003eApocynum hendersonii\u003c/em\u003e genome, respectively, using the sequences of 80 AtPP2C proteins from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e as a reference. We used TBtools to map the positional distribution of the \u003cem\u003ePP2C\u003c/em\u003e genes on chromosomes of \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e respectively, which were named \u003cem\u003eAvPP2C1-AvPP2C68\u003c/em\u003e and \u003cem\u003eAhPP2C1-AhPP2C68\u003c/em\u003e according to their chromosomal positions(Fig. 1). Except for \u003cem\u003eAhPP2C68\u003c/em\u003e which was not assembled, the remaining 68 \u003cem\u003eAvPP2C\u003c/em\u003e genes and 67 \u003cem\u003eAhPP2C\u003c/em\u003e genes were located on 11 chromosomes, respectively. There were fewer genes on chromosomes 4, 8 and 10.\u003c/p\u003e\n\u003cp\u003eWe analyzed the physicochemical properties such as molecular weight (MW), theoretical isoelectric point (pI), and subcellular localization of the 68 AvPP2C proteins and 68 AhPP2C proteins (Table S1). The 68 \u003cem\u003eAvPP2C\u003c/em\u003e genes encoded proteins were located at lengths ranging from 278-1086, theoretical isoelectric points ranging from 4.38-9.78, and molecular weights ranging from 30369.58-123024.02 Da. The 68 \u003cem\u003eAhPP2C\u003c/em\u003e genes encoded proteins with lengths ranging from 138-1092, theoretical isoelectric points ranging from 4.62-9.8, and molecular weights of 15540.2-123582.74 Da. The results of subcellular localization show that \u003cem\u003ePP2C\u003c/em\u003e genes have the ability to appear in various areas of the cell, including the nucleus, chloroplast, cell wall, cell membrane, cytoplasm, mitochondria, and peroxisome. Some specific \u003cem\u003ePP2C\u003c/em\u003e genes, such as \u003cem\u003eAvPP2C9, 10, 16, 19, 20, 21, 29, 31, 35, 38, 47, 53, 62\u003c/em\u003e and \u003cem\u003eAhPP2C9, 10, 21, 29, 31, 35, 37, 38, 39, 48, 53, 54, 58\u003c/em\u003e are predicted to be present in multiple cellular structures.\u003c/p\u003e\n\u003cp\u003eTo investigate the phylogenetic relationship between \u003cem\u003eApocynum venetum\u003c/em\u003e, \u003cem\u003eApocynum hendersonii\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e \u003cem\u003ePP2C\u003c/em\u003e genes, a phylogenetic tree was constructed using the neighbor-joining (NJ) method based on 80 \u003cem\u003eAt\u003c/em\u003e\u003cem\u003ePP2C\u003c/em\u003e genes of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, 68 \u003cem\u003eAv\u003c/em\u003e\u003cem\u003ePP2C\u003c/em\u003e genes of Apocynum venetum and 68 \u003cem\u003eAh\u003c/em\u003e\u003cem\u003ePP2C\u003c/em\u003e genes of \u003cem\u003eApocynum hendersonii\u003c/em\u003e (Fig. 2). Based on phylogenetic analysis, the \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eApocynum\u003c/em\u003e were categorized into 13 subgroups (A-L). These subgroups contained 9, 4, 4, 8, 7, 6, 4, 6, 3, 2, 2, 7, 2 \u003cem\u003eAvPP2C\u003c/em\u003e genes and 9, 4, 4, 9, 7, 6, 3, 6, 3, 2, 2, 7, 2 \u003cem\u003eAhPP2C\u003c/em\u003e genes, with similar groupings to those found in \u003cem\u003eArabidopsis\u003c/em\u003e. Notably, PP2C proteins from both \u003cem\u003eApocynum\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e were present in each subfamily, suggesting a common ancestor. Interestingly, \u003cem\u003eAvPP2C3\u003c/em\u003e, \u003cem\u003eAvPP2C7\u003c/em\u003e, \u003cem\u003eAvPP2C31\u003c/em\u003e, \u003cem\u003eAvPP2C61\u003c/em\u003e, \u003cem\u003eAhPP2C3\u003c/em\u003e, \u003cem\u003eAhPP2C7\u003c/em\u003e, \u003cem\u003eAhPP2C31\u003c/em\u003e, and \u003cem\u003eAhPP2C60\u003c/em\u003e of \u003cem\u003eApocynum\u003c/em\u003e did not cluster with any other group. The evolutionary tree also showed that genes in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e were tightly clustered, indicating high homology and similar gene functions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene structures and conserved motifs analyses of the \u003cem\u003eAvPP2C\u003c/em\u003e genes and the \u003cem\u003eAhPP2C\u003c/em\u003e genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on their phylogenetic relationships, the structures and conserved domains of the \u003cem\u003eAvPP2C\u003c/em\u003e and \u003cem\u003eAhPP2C\u003c/em\u003e genes were studied. The gene structures include positional information such as coding sequence (CDS), untranslated regions (UTR), introns and \u003cem\u003ePP2C\u003c/em\u003e structural domains(Fig. 3A and C). Genes within the same subgroup share similar gene structures, but there are variations in the length and arrangement of their CDSs and UTRs. For instance, all \u003cem\u003ePP2C\u003c/em\u003e genes in group C contained UTR and CDS regions, but their lengths varied. \u003cem\u003eAvPP2C42\u003c/em\u003e had the lengthiest UTR region, while 16 genes in \u003cem\u003eApocynum venetum\u003c/em\u003e had no UTR regions and 14 genes in \u003cem\u003eApocynum hendersonii\u003c/em\u003e had no UTR regions. \u003cem\u003eAvPP2C39\u003c/em\u003e and \u003cem\u003eAhPP2C40\u003c/em\u003e had the highest number of CDS regions, totaling 16. After examining the number of introns, it was found that three \u003cem\u003eAvPP2C\u003c/em\u003e genes and four \u003cem\u003eAhPP2C\u003c/em\u003e genes in \u003cem\u003eApocynum \u003c/em\u003ehad no introns, while the remaining 129 \u003cem\u003ePP2C\u003c/em\u003e genes contained introns.\u003c/p\u003e\n\u003cp\u003eIn order to study the structure and function of PP2C proteins and their evolutionary relationships, the conserved domains of AvPP2Cs proteins and AhPP2Cs proteins were analyzed using the online MEME website. The analysis revealed 15 different conserved domains, which were given the names motif 1 to motif 15(Fig. 3A and B, Table S2). The results demonstrated that PP2C proteins in the same subgroups possess a similar distribution that may be linked to their functions. Motif 1 (except AvPP2C62, AhPP2C67, AhPP2C68), motif 2 (except AvPP2C31, AvPP2C44, AhPP2C45, AhPP2C31), and motif 3 (except AhPP2C68, AhPP2C19, AvPP2C19, AvPP2C48) were present in all subgroups. Motifs 6, 7, 11, 13 and 15 were absent in subgroups C and D, while motif 5 was only present in subgroups C and D, except AhPP2C68. Motif 9 and 10 were present only in subgroup D, and motif 11 was present only in subgroup E, but not in other groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCis-element analysis of the \u003cem\u003ePP2Cs\u003c/em\u003e promoter in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe cis-acting elements were predicted in the promoter regions of \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e using analysis through the PlantCARE online website pairs and visualized using TBtools software, both of which retrieved a total of 68 cis-acting elements. Among them, there were 32 light-responsive elements, 13 phytohormone-responsive elements, 12 stress-responsive elements, and 11 elements related to plant growth and development (Fig. 4, Table S3). It was found that all the promoters of \u003cem\u003eAvPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e and \u003cem\u003eAhPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e contained light-responsive elements, phytohormone-responsive elements, and stress-responsive elements. However, elements associated with plant growth and development were missing in \u003cem\u003eAvPP2C18\u003c/em\u003e, \u003cem\u003eAvPP2C33\u003c/em\u003e, \u003cem\u003eAvPP2C59\u003c/em\u003e, \u003cem\u003eAvPP2C61\u003c/em\u003e, \u003cem\u003eAvPP2C65\u003c/em\u003e, \u003cem\u003eAhPP2C18\u003c/em\u003e, \u003cem\u003eAhPP2C19\u003c/em\u003e, \u003cem\u003eAhPP2C32\u003c/em\u003e, \u003cem\u003eAhPP2C58\u003c/em\u003e, \u003cem\u003eAhPP2C60\u003c/em\u003e, and \u003cem\u003eAhPP2C66\u003c/em\u003e. Box 4 was identified as the most abundant light-responsive element, with 263 in \u003cem\u003eApocynum venetum\u003c/em\u003e and 290 in \u003cem\u003eApocynum hendersonii\u003c/em\u003e. Among the phytohormone response elements, a large number of abscisic acid response elements (ABRE) and AAGAA-motifs were found, with \u003cem\u003eAvPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e having 148 ABREs and 132 AAGAA-motifs, and \u003cem\u003eAhPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e having 141 ABREs and 129 AAGAA-motifs. The ethylene response element (ERE) was also found in large numbers, with \u003cem\u003eAvPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e having 147 and \u003cem\u003eAhPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e having 156. The stress-responsive elements MYB and MYC were found to be the most abundant with 247 and 235 in \u003cem\u003eApocynum venetum\u003c/em\u003e and 242 and 238 in \u003cem\u003eApocynum hendersonii\u003c/em\u003e, respectively. These elements are involved in drought and ABA-induced responses. Another stress-responsive element, ARE, was found to be essential for anaerobic induction, with 133 in \u003cem\u003eApocynum venetum\u003c/em\u003e and 130 in \u003cem\u003eApocynum hendersonii\u003c/em\u003e. The large number of these elements suggests that most \u003cem\u003ePP2C\u003c/em\u003e genes may respond to various stresses and are closely related to plant stress resistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCollinear analysis and evolutionary analysis of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eAvPP\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e2\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCs\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e proteins \u003c/strong\u003e\u003cstrong\u003eand \u003cem\u003eAhPP2Cs\u003c/em\u003e proteins\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expansion of gene families in the genome is predominantly caused by tandem and segmental duplications. To gain insight into the \u003cem\u003ePP2C\u003c/em\u003e gene family, 68 \u003cem\u003eAvPP2C\u003c/em\u003e genes and 68 \u003cem\u003eAhPP2C\u003c/em\u003e genes were analyzed for duplication (Fig. 5A and B, Table S4). It was discovered that in \u003cem\u003eApocynum venetum\u003c/em\u003e, one tandem duplication pair was located on chromosome LG06 (39/40), whereas 17 pairs of segmental duplication genes were unevenly distributed on chromosomes other than LG08 and LG10. In \u003cem\u003eApocynum hendersonii\u003c/em\u003e, there were 2 tandem replication gene pairs located on chromosome LG06 (38/39, 40/41), and 17 fragment replication gene pairs were unevenly distributed on 9 chromosomes except for LG08 and LG10. These findings suggest that the evolution of \u003cem\u003ePP2Cs\u003c/em\u003e in \u003cem\u003eApocynum\u003c/em\u003e was primarily driven by segmental duplication events, as the number of segmental duplication gene pairs was significantly greater than that of tandem duplications.\u003c/p\u003e\n\u003cp\u003eFurthermore, the study investigated the homology between \u003cem\u003eApocynum venetum\u003c/em\u003e, \u003cem\u003eApocynum hendersonii\u003c/em\u003e, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e to understand the evolutionary relationship of \u003cem\u003ePP2C\u003c/em\u003e genes(Fig. 5C, Table S5). The results indicated that there were 84 homologous gene pairs between \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, and 82 homologous gene pairs between \u003cem\u003eApocynum hendersonii\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Among these pairs, \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e had the highest number of homologous gene pairs on chromosome LG06, with 13 pairs each. These findings suggest that \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e may have a high degree of homology with \u003cem\u003eArabidopsis.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue-specific expression of \u003cem\u003eAvPP2C\u003c/em\u003e genes and \u003cem\u003eAhPP2C\u003c/em\u003e genes in different tissues\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo gain a better understanding of the function of \u003cem\u003ePP2C\u003c/em\u003e genes in the growth and development of \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e, we analyzed the expression patterns of \u003cem\u003ePP2C\u003c/em\u003e genes using RNA-Seq data of roots, stems, and leaves of both species(Fig. 6). Most of the \u003cem\u003ePP2C\u003c/em\u003e genes were expressed in all tissues, except for four genes (\u003cem\u003eAvPP2C19\u003c/em\u003e, \u003cem\u003eAvPP2C38\u003c/em\u003e, \u003cem\u003eAhPP2C39\u003c/em\u003e, and \u003cem\u003eAhPP2C68\u003c/em\u003e), which were barely expressed. In \u003cem\u003eApocynum venetum\u003c/em\u003e, \u003cem\u003eAvPP2C47\u003c/em\u003e was highly expressed in roots, stems, and leaves. \u003cem\u003eAvPP2C9\u003c/em\u003e, \u003cem\u003eAvPP2C24\u003c/em\u003e, \u003cem\u003eAvPP2C49\u003c/em\u003e, \u003cem\u003eAvPP2C60\u003c/em\u003e, and \u003cem\u003eAvPP2C68\u003c/em\u003e were moderately expressed in roots; \u003cem\u003eAvPP2C13\u003c/em\u003e and \u003cem\u003eAvPP2C60\u003c/em\u003e were highly expressed in stems; \u003cem\u003eAvPP2C13\u003c/em\u003e was highly expressed in leaves, and \u003cem\u003eAvPP2C7\u003c/em\u003e and \u003cem\u003eAvPP2C60\u003c/em\u003e were moderately expressed in leaves. In \u003cem\u003eApocynum hendersonii\u003c/em\u003e, \u003cem\u003eAhPP2C29\u003c/em\u003e and \u003cem\u003eAhPP2C67\u003c/em\u003e were highly expressed in roots, while \u003cem\u003eAhPP2C24\u003c/em\u003e, \u003cem\u003eAhPP2C13\u003c/em\u003e, \u003cem\u003eAhPP2C49\u003c/em\u003e, \u003cem\u003eAhPP2C48\u003c/em\u003e, and \u003cem\u003eAhPP2C59\u003c/em\u003e were moderately expressed in roots. \u003cem\u003eAhPP2C9\u003c/em\u003e, \u003cem\u003eAhPP2C13\u003c/em\u003e, \u003cem\u003eAhPP2C59\u003c/em\u003e, and \u003cem\u003eAhPP2C48\u003c/em\u003e were highly expressed in stems, and \u003cem\u003eAhPP2C13\u003c/em\u003e, \u003cem\u003eAhPP2C48\u003c/em\u003e, and \u003cem\u003eAhPP2C59\u003c/em\u003e were highly expressed in leaves.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRelative expression changes of \u003cem\u003eAvPP2C\u003c/em\u003e \u003c/strong\u003e\u003cstrong\u003egenes\u003c/strong\u003e\u003cstrong\u003e and \u003cem\u003eAhPP2C\u003c/em\u003e genes under NaCl treatment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to study the expression of \u003cem\u003eAvPP2C\u003c/em\u003e genes and \u003cem\u003eAhPP2C\u003c/em\u003e genes under salt stress conditions and to identify important \u003cem\u003ePP2C\u003c/em\u003e genes related to salt tolerance. Based on phylogenetic analysis and homology of known \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, five genes in subgroup D and two genes in subgroup G were selected in \u003cem\u003eApocynum hendersonii\u003c/em\u003e and their expression patterns in different tissues of \u003cem\u003eApocynum venetum\u003c/em\u003e under salt stress were investigated by qRT-PCR(Fig. 7). \u003c/p\u003e\n\u003cp\u003eThe results showed that the \u003cem\u003eAvPP2C1\u003c/em\u003e gene was down-regulated in roots after NaCl stress, with the lowest expression at 50 mM concentration. It was up-regulated in leaves and phloem, with the highest expression at 200 mM and 100 mM concentrations, respectively, reaching 2.14-fold and 1.40-fold of CK. \u003cem\u003eAhPP2C1\u003c/em\u003e gene was up-regulated in leaves and increased with increasing NaCl concentration; it showed down-regulation in bast and decreased with increasing NaCl concentration. \u003cem\u003eAvPP2C2\u003c/em\u003e gene showed up-regulation under NaCl stress, in which the expression of the gene in leaves varied the most with NaCl concentration, which was 10.44 times that of CK. The expression trend of \u003cem\u003eAvPP2C2\u003c/em\u003e in root, xylem and phloem was the same, and all of them reached the maximum expression at 100 mM concentration. While the \u003cem\u003eAhPP2C2\u003c/em\u003e gene was down-regulated in roots and up-regulated in leaves after NaCl stress, there was little change in the xylem and phloem, but the expression reached the maximum at 50 mM concentration in all. \u003cem\u003eAvPP2C6 \u003c/em\u003egene did not change significantly in roots, but showed up-regulation in leaves and xylem phloem, and it is noteworthy that its expression in leaves increased with the increase of NaCl concentration and reached the maximum at 200 mM, which was 13.55 times that of CK. And \u003cem\u003eAhPP2C6\u003c/em\u003e was different from \u003cem\u003eAvPP2C6\u003c/em\u003e in that it showed down-regulation in roots. The expression of \u003cem\u003eAvPP2C14\u003c/em\u003e was different in roots, leaves, xylem and phloem, with the highest in leaves and the lowest in roots; after NaCl stress, the \u003cem\u003eAvPP2C14\u003c/em\u003e gene showed a tendency of increasing and then de-creasing with the increase of NaCl concentration, and its expression reached the maximum in all tissues at the concentration of 50 mM. While the expression of \u003cem\u003eAhPP2C14\u003c/em\u003e did not change significantly under NaCl treatment, it only showed up-regulation in leaves, and the expression of the gene was the highest at 50 mM concentration, and its expression gradually declined with the increase of concentration. \u003cem\u003eAvPP2C24\u003c/em\u003e gene had the highest expression in leaves and showed up-regulation in all tissues after NaCl stress. \u003cem\u003eAhPP2C24\u003c/em\u003e gene was the same as \u003cem\u003eAvPP2C24\u003c/em\u003e gene in that the expression of \u003cem\u003eAhPP2C24\u003c/em\u003e gene was up-regulated in all tissues after NaCl stress, except in phloem at 50 mM concentration. The difference was that the expression of \u003cem\u003eAhPP2C24\u003c/em\u003e gene changed more significantly in leaves after NaCl treatment. Both \u003cem\u003eAvPP2C27\u003c/em\u003e and \u003cu\u003eAhPP2C27\u003c/u\u003e genes showed up-regulation after NaCl treatment, but the expression of \u003cem\u003eAvPP2C27\u003c/em\u003e gene in roots and leaves increased with increasing NaCl concentration, while the expression of \u003cem\u003eAhPP2C27\u003c/em\u003e gene in leaves reached a maximum at 50 mM concentration and then decreased with increasing concentration. The expression of \u003cem\u003eAvPP2C41\u003c/em\u003e and \u003cem\u003eAhPP2C42\u003c/em\u003e genes in roots increased with increasing NaCl concentration, \u003cem\u003eAvPP2C41\u003c/em\u003e gene showed down-regulation in leaves at 100 mM and up-regulation at 50 mM and 200 mM concentrations. While \u003cem\u003eAhPP2C42\u003c/em\u003e gene showed up-regulation after NaCl stress, the expression was maximum in leaves at 50 mM concentration, which was 8.20 times of CK, and then decreased with the increase of NaCl concentration.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cem\u003ePP2C\u003c/em\u003e genes are closely related to abscisic acid ABA signaling pathway, plant adversity stress, and stomatal opening and closing, etc. The \u003cem\u003ePP2C\u003c/em\u003e gene families of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, rice, maize, and wheat have been investigated by previous authors. In this study, we comprehensively analyzed the \u003cem\u003eAvPP2C\u003c/em\u003e genes of \u003cem\u003eApocynum venetum\u003c/em\u003e and the \u003cem\u003eAhPP2C\u003c/em\u003e genes of \u003cem\u003eApocynum hendersonii\u003c/em\u003e, including the identification of the gene families, phylogenetic relationships, and chromosomal locations, collinear relationship, gene structure, conserved motifs, and expression patterns. A total of 136 \u003cem\u003ePP2C\u003c/em\u003e genes, including 68 \u003cem\u003eAvPP2C\u003c/em\u003e genes and 68 \u003cem\u003eAhPP2C\u003c/em\u003e genes, were identified in \u003cem\u003eApocynum\u003c/em\u003e, and the physicochemical properties and subcellular localization of the genes were predicted, and it was found that the vast majority of \u003cem\u003ePP2C\u003c/em\u003e genes were located in the nucleus and chloroplasts, and a few genes might be present in the mitochondria, cell wall, cytoplasm, peroxisomes, and cell membrane. Therefore, it is hypothesized that they may be related to photosynthesis, cell growth and development, and respiration. According to phylogenetic and evolutionary relationship analysis, \u003cem\u003eApocynum hendersonii\u003c/em\u003e and \u003cem\u003eApocynum venetum\u003c/em\u003e are closely related to each other, and a total of 136 \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eApocynum hendersonii\u003c/em\u003e and \u003cem\u003eApocynum venetum\u003c/em\u003e were divided into 13 subgroups (A-L), which is consistent with the grouping of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Previous studies found that genes in the A subgroup of \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eArabidopsis\u003c/em\u003e mainly inhibit ABA receptor activity and negatively regulate ABA signaling[32]; the B subgroup is involved in the mitogen-activated protein kinase signaling pathway and participates in the process of MAPK phosphorylation, and is capable of dephosphorylating and inactivating MAPK[33]; Subgroup C gene family members are involved in stem cell maintenance and differentiation[34]; Members of subgroup D negatively regulate small auxin up RNA (SAUR)-mediated cell elongation, and PP2C.D1, PP2C.D2, and PP2C.D5 in Arabidopsis act during leaf greening, and PP2C.D1 regulates hook formation by affecting ethylene accumulation[15, 35, 36]. Moreover, members of this group respond to saline and alkaline stress, which may be related to stomatal movement and function, by inhibiting plasma membrane H-ATPase activity. It has been shown that \u003cem\u003eArabidopsis\u003c/em\u003e PP2C.D negatively regulates aluminium resistance by modulating malate secretion[16, 37]; Members of the E subgroup gene family may be associated with inflorescence stem growth, regulation of stomatal signaling, and so on[38-40]. Based on the phylogenetic relationship between \u003cem\u003eApocynum\u003c/em\u003e and \u003cem\u003eArabidopsis\u003c/em\u003e, it is hypothesized that the functions of gene family members located in the same subgroup in \u003cem\u003eApocynum\u003c/em\u003e may be similar to those in \u003cem\u003eArabidopsis\u003c/em\u003e. The prediction of the subcellular localization of \u003cem\u003ePP2C\u003c/em\u003e gene family members in \u003cem\u003eApocynum\u003c/em\u003e showed that most of the \u003cem\u003ePP2C\u003c/em\u003e gene family in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e were predicted to be located in the nucleus and chloroplasts, in addition to which some of the genes might be located in the cell wall, cytoplasm, cell membrane, mitochondria, and peroxisomes. The predictions of \u003cem\u003ePP2C\u003c/em\u003e gene family in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e were partially different. For example, \u003cem\u003eAvPP2C9\u003c/em\u003e was predicted to be located in mitochondria and the nucleus, whereas \u003cem\u003eAhPP2C9\u003c/em\u003e was predicted to be located in chloroplasts, mitochondria, and the nucleus. \u003cem\u003eAvPP2C20\u003c/em\u003e was located in the nucleus and chloroplasts, whereas \u003cem\u003eAhPP2C20\u003c/em\u003e was predicted to be located in chloroplasts only. This may imply differences in the location and function of some \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eApocynum hendersonii\u003c/em\u003e and \u003cem\u003eApocynum venetum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eThe diversity of gene structures, conserved structural domains, and cis-acting elements is also closely related to the functions of gene families. Genes in the same subgroup are similar in structure, which may imply that they have the same function[41]. The gene structures of \u003cem\u003eApocynum hendersonii\u003c/em\u003e and \u003cem\u003eApocynum venetum\u003c/em\u003e genes from the same evolutionary tree branching were similar, except for the \u003cem\u003eAhPP2C68\u003c/em\u003e gene. However, the gene structures of individual \u003cem\u003ePP2C\u003c/em\u003e genes were different again, for example, \u003cem\u003eAvPP2C18\u003c/em\u003e and \u003cem\u003eAhPP2C18\u003c/em\u003e in subgroup L had significantly more CDS regions compared with \u003cem\u003eAvPP2C33\u003c/em\u003e and \u003cem\u003eAhPP2C33\u003c/em\u003e, which might be related to the increase of introns during the evolutionary process. The \u003cem\u003ePP2C\u003c/em\u003e gene family is evolutionarily conserved, and most \u003cem\u003ePP2Cs\u003c/em\u003e have a highly conserved structural domain with a unique non-catalytic N-terminal extension region. The specificity of the function of each sub-group of \u003cem\u003ePP2C\u003c/em\u003e genes may be related to an N-terminal substrate docking domain[42]. A total of 15 conserved motifs were identified in the amino acid sequences of \u003cem\u003eAvPP2C\u003c/em\u003e and \u003cem\u003eAhPP2C\u003c/em\u003e genes, and the distribution of conserved motifs was similar for genes in the same subgroup, which may be closely related to the function of PP2C proteins. Cis-acting elements are involved in the regulation of gene expression and are closely related to plant growth and development, hormone responses and responses to various stresses. Among \u003cem\u003eAvPP2C\u003c/em\u003e and \u003cem\u003eAhPP2C\u003c/em\u003e genes, Box4 elements were the most abundant (AvPP2Cs: 263; AhPP2Cs: 290), followed by MYB elements (AvPP2Cs: 247; AhPP2Cs: 242). Among all the response elements, light responsive and stress responsive were the most numerous elements, with a high number of 1,318 stress responsive in \u003cem\u003eApocynum hendersonii\u003c/em\u003e. This may be related to the characteristics of \u003cem\u003eApocynum hendersonii\u003c/em\u003e high-stress tolerance.\u003c/p\u003e\n\u003cp\u003eIn this study, chromosomal position analysis and covariance analysis of \u003cem\u003ePP2C\u003c/em\u003e gene family of\u0026nbsp;\u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e were carried out, which is important to understand the amplification mechanism of the genes. It was found that both \u003cem\u003eAvPP2Cs\u003c/em\u003e and \u003cem\u003eAhPP2Cs\u003c/em\u003e were distributed on 11 chromosomes. Seventeen pairs of segmental duplications and one pair of tandem duplications were identified in \u003cem\u003eApocynum venetum\u003c/em\u003e; 17 pairs of segmental duplications and two pairs of tandem duplications were identified in \u003cem\u003eApocynum hendersonii\u003c/em\u003e. Gene duplication is the main driver of gene family amplification, which mainly includes segmental duplication, tandem duplication, and genomic duplication, and segmental duplication is more conducive to maintaining gene function than tandem duplication[43]. Therefore, we hypothesize that segmental duplication is the main force driving the evolution and expansion of the \u003cem\u003ePP2C\u003c/em\u003e gene family, which is similar to the results found for \u003cem\u003ePP2C\u003c/em\u003e in \u003cem\u003eArabidopsis\u003c/em\u003e[44], woodland and pineapple strawberry[45], and cucumber[4].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvPP2C\u003c/em\u003e genes and \u003cem\u003eAhPP2C\u003c/em\u003e genes showed specific expression in different tissues, and most of the \u003cem\u003ePP2C\u003c/em\u003e genes were lowly expressed in roots, stems and leaves. However, among all the \u003cem\u003ePP2C\u003c/em\u003e genes, \u003cem\u003eAvPP2C13\u003c/em\u003e, \u003cem\u003eAvPP2C47\u003c/em\u003e, \u003cem\u003eAvPP2C60\u003c/em\u003e, \u003cem\u003eAhPP2C48\u003c/em\u003e, and \u003cem\u003eAhPP2C59\u003c/em\u003e showed higher expression in all three tissues: roots, stems, and leaves, whereas \u003cem\u003eAhPP2C29\u003c/em\u003e, and \u003cem\u003eAhPP2C67\u003c/em\u003e showed high expression in roots and lower expression in stems and leaves. In cucumber, most of the \u003cem\u003eCsPP2C\u003c/em\u003e genes were highly expressed in fertilized ovaries, male, female, and leaf (except \u003cem\u003eCsPP2C11, 41, 5, 33, 50\u003c/em\u003e), and low in other tissues [4]. In maize, most of the \u003cem\u003eZmPP2C\u003c/em\u003e genes were expressed in all tissues, with \u003cem\u003eZmPP2C42\u003c/em\u003e and \u003cem\u003eZmPP2C47\u003c/em\u003e being the most highly expressed in mature pollen, and \u003cem\u003eZmPP2C59\u003c/em\u003e being expressed in primary roots and root cortex[46]. This suggests that \u003cem\u003ePP2C\u003c/em\u003e genes in different plants may play different roles at different stages of plant growth, and some of these \u003cem\u003ePP2C\u003c/em\u003e genes may have special functions in specific tissues.\u003c/p\u003e\n\u003cp\u003eStudies on \u003cem\u003eArabidopsis\u003c/em\u003e have shown that members of subgroup D respond to saline stress, and \u003cem\u003eAtPP2C.D6\u003c/em\u003e and \u003cem\u003eAtPP2C.D7\u003c/em\u003e interact with \u003cem\u003eSCaBP8\u003c/em\u003e of the SOS pathway, whose phosphatase activity is inhibited under salt stress. \u003cem\u003eAtPP2C\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003eG1\u003c/em\u003e in group G is dependent on ABA to positively regulate salt tolerance. Our study showed that the expression of some \u003cem\u003eAvPP2C\u003c/em\u003e genes differed significantly from that of \u003cem\u003eAhPP2C\u003c/em\u003e genes after NaCl stress, in which the expression of \u003cem\u003eAvPP2C2\u003c/em\u003e gene was significantly up-regulated after salt stress, and the expression was significantly higher in roots, xylem, phloem, and leaves. It indicated that \u003cem\u003eAvPP2C2\u003c/em\u003e gene might play an important role in these four tissues when \u003cem\u003eApocynum venetum\u003c/em\u003e resisted salt stress. The expression of \u003cem\u003eAhPP2C2\u003c/em\u003e gene was down-regulated in roots and up-regulated in leaves after salt stress, while it showed a similar expression pattern in xylem and phloem, both of which peaked at a concentration of 50 mM. \u003cem\u003eAvPP2C2\u003c/em\u003e and \u003cem\u003eAhPP2C2\u003c/em\u003e showed different expression patterns in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e under salt stress, which was hypothesized to be related to their differences in salt tolerance. The expression of \u003cem\u003eAvPP2C6\u003c/em\u003e and \u003cem\u003eAhPP2C6\u003c/em\u003e was up-regulated in leaves, xylem and phloem, and the change of \u003cem\u003eAvPP2C6\u003c/em\u003e was not obvious in roots, while \u003cem\u003eAhPP2C6\u003c/em\u003e showed down-regulation in roots. \u003cem\u003eAvPP2C24, 27\u003c/em\u003e and \u003cem\u003e41\u003c/em\u003e in roots showed similar trends after salt stress. In summary, \u003cem\u003ePP2C\u003c/em\u003e genes located in the same subgroup responded differently in the face of salt stress, while there may be different expression patterns in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e, where \u003cem\u003eAvPP2C2, 6, 24, 27, 41\u003c/em\u003e and \u003cem\u003eAhPP2C2, 6, 24, 27, 42\u003c/em\u003e showed significant differences in expression under salt stress, suggesting that these genes may play important roles in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e when facing salt stress.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eGenome-wide identification and analysis of \u003cem\u003ePP2C\u003c/em\u003e family members in \u003cem\u003eApocynum venetum\u003c/em\u003e genomes and \u003cem\u003eApocynum hendersonii\u003c/em\u003e genomes. In this study, a total of 68 \u003cem\u003eAvPP2C\u003c/em\u003e genes and 68 \u003cem\u003eAhPP2C\u003c/em\u003e genes were identified and classified into 13 subgroups, which were further analyzed for subcellular localization, gene structure, conserved structural domains, and cis-acting elements. By analyzing the expression patterns of \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e under salt stress, it was found that the \u003cem\u003ePP2C\u003c/em\u003e genes in subgroup D and subgroup G showed significant differences under salt stress, which might be related to the differences in salt tolerance between \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e. This study provides a reference for the subsequent research on the function of \u003cem\u003ePP2C\u003c/em\u003e gene.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eIdentification of \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eApocynum\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protein sequences of 80\u0026nbsp;\u003cem\u003eAt\u003c/em\u003e\u003cem\u003ePP2C\u003c/em\u003e genes of \u003cem\u003eArabidopsis\u003c/em\u003e were downloaded from Tair (https://www.arabidopsis.org/). Protein sequences of \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e were obtained from the whole genome data sequenced in our laboratory. The \u003cem\u003ePP2C\u003c/em\u003e genes were identified by two methods. First, the putative PP2C protein present in the genome was identified using BLASTp. Then, the PP2C structural domain (PF00481) of the PP2C protein was downloaded from Pfam (http://pfam.xfam.org/) and searched against the local protein database using HMMER 3.0[47, 48]. Meanwhile, the SMART (https://smart.embl.de/) was used to predict its structural domain with E-value \u0026lt;e-5[49]. The results obtained by both methods were intersected and the protein sequences were extracted from the local protein database using TBtools and the gene IDs identified by screening.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence analysis and basic information of the \u003cem\u003eApocynum\u003c/em\u003e \u003cem\u003ePP2C\u003c/em\u003e gene family\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePhysicochemical property analysis of PP2C proteins including number of amino acid, molecular weight, theoretical pI, instability index, aliphatic index, grand average of hydropathicity were analyzed by Protein Paramter Calc in TBtools. Prediction of the subcellular location of PP2C protein by Plant-mPloc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/) of Cell-Ploc 2.0.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of chromosome location and collinearity analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChromosome position information was obtained from the annotation file of the \u003cem\u003eApocynum\u003c/em\u003e genome and the chromosome position distribution was mapped using TBtools. Homology and collinearity within the \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e genomes and between them and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e were analyzed using the MCScanX software and visualized using the Advanced Circos and Multiple Synteny Plot functions in TBtools[50, 51].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of phylogenetic tree\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phylogenetic tree of the \u003cem\u003ePP2C\u003c/em\u003e gene family of \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eApocynum\u003c/em\u003e was constructed using MEGA-X with the Neighbor-Joining method, Bootstrap value set to 1000 and other default parameters[52, 53]. Finally, the phylogenetic tree was embellished with iTOL(https://itol.embl.de/)[54].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of gene structures and protein conserved motifs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe gene structure of \u003cem\u003eApocynum\u003c/em\u003e \u003cem\u003ePP2C\u003c/em\u003e was analyzed using the Batch CD-search (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) on the NCBI website. The MEME website (https://meme-suite.org/meme/tools/meme) was used to predict the conserved structural domains of the \u003cem\u003ePP2C\u003c/em\u003e genes\u0026nbsp;in \u003cem\u003eApocynum\u003c/em\u003e, setting the motif number to 15 and the rest to default values[55]. Gene Structure View in TBtools was used to generate the final overall map of the phylogenetic tree, gene structure and conserved structural domains of \u003cem\u003ePP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of cis-acting elements in \u003cem\u003ePP2C\u003c/em\u003e gene promoters\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequences of 2.0 kb upstream of \u003cem\u003eAvPP2C\u003c/em\u003e genes\u0026nbsp;and \u003cem\u003eAhPP2C\u003c/em\u003e genes\u0026nbsp;were extracted using TBtools and then the results were submitted to PlantCARE website (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for cis-acting element prediction[55].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression Analysis of \u003cem\u003ePP2C\u003c/em\u003e Genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFrom the transcriptome data of roots, stems and leaves of \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e, the FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values of \u003cem\u003eAvPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e and \u003cem\u003eAhPP2C\u003c/em\u003e\u003cem\u003es\u003c/em\u003e were extracted and heat maps were generated using TBtools[51].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlant material treatment methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSeedlings grown under hydroponic conditions for 6-8 weeks were divided into four groups and treated with different concentrations (0, 50, 100, and 200 mM) of NaCl stress, and after 14 days of treatment, respectively, the roots, leaves, xylem and phloem were sampled and stored in a refrigerator at -80\u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Isolation and q\u003c/strong\u003e\u003cstrong\u003eRT-\u003c/strong\u003e\u003cstrong\u003ePCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using the \u003cem\u003eSteadyPure\u003c/em\u003e Plant RNA Extraction Kit (Accurate Biotechnology (Changsha, China) Co., Ltd.), and the RNA was reverse transcribed into cDNA using the \u003cem\u003eEvo M-MLV\u003c/em\u003e One Step RT-PCR Kit (Accurate Biotechnology (Changsha, China) Co., Ltd.)[56]. Specific primers were designed for qRT-PCR using Primer5(Table S6). ACT gene was used as an internal reference gene. qRT-PCR was performed on a CFX96 Touch Deep Well Real-Time Quantitative PCR System (Bio-Rad) using SYBR\u0026reg; Green Premix \u003cem\u003ePro Taq\u003c/em\u003e HS qPCR Kit II (Accurate Biotechnology (Changsha, China) Co., Ltd.)[56]. The relative expression of \u003cem\u003ePP2C\u003c/em\u003e genes was calculated using the 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e method and histograms were plotted using GraphPad Prism 8.\u003c/p\u003e"},{"header":"List of Abbreviations","content":"\u003cp\u003e\u003cstrong\u003ePP2C:\u003c/strong\u003e Protein phosphatase class 2C\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT-PCR:\u003c/strong\u003e Quantitative Real-time PCR\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMAPK:\u003c/strong\u003e Mitogen-activated protein kinase\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMW:\u003c/strong\u003e Molecular weight\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003epI:\u003c/strong\u003e Isoelectric point\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCDS:\u003c/strong\u003e Coding sequence\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUTR:\u003c/strong\u003e Untranslated regions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCK:\u003c/strong\u003e Control check\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSAUR:\u003c/strong\u003e Small auxin up RNA\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plant material used in this study was \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e, grown in the laboratory of the Institute of bast fiber crops, Chinese Academy of Agricultural Sciences (CAAS) and no permits were required to collect plant samples. In the present study, all methods were carried out following relevant guidelines and regulations. Ethical approval or consent was not required for this study because no endangered or protected species were involved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sequence information of \u003cem\u003eArabidopsis\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ePP2C\u003c/em\u003e family genes were collected from The\u003cem\u003e\u0026nbsp;Arabidopsis\u0026nbsp;\u003c/em\u003eInformation Resoure (https://www.arabidopsis.org/). The \u003cem\u003ePP2C\u003c/em\u003e family expression data were generated by qRT-PCR. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Hunan Provincial Department of Education general project (No.22C0674)，ESI Discipline Special Project of Changsha Medical University (No.2022CYY023 and No.2022CYY006)，Science Research Project of Hunan Provincial Department of Education (No.23A0662)，National Undergraduate Innovation and Entrepreneurship Training Program Project under Grant (No.202210823006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJiayi Chen: Methodology, writing-review.\u003c/p\u003e\n\u003cp\u003eYue Wang: Conceived, designed the experiments and writing, Methodology.\u003c/p\u003e\n\u003cp\u003eXiaoyu Huang, Xiaojun Qiu: Methodology, writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eJikang Chen, Qian Lin: Methodology.\u003c/p\u003e\n\u003cp\u003eHaohan Zhao, Yongmei Wu: Edit improves the manuscript.\u003c/p\u003e\n\u003cp\u003eGang Gao, Fengming Chen: Concept of study, Supervision and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank laboratory colleagues for their technical assistance. The authors thank the editors and reviewers for their rigorous evaluation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYang S, Gong Z, Guo Y, Gong J, Zheng S, Lin R, Yang H, Mao L, Qin F, Luo L\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eStudies on plant responses to environmental change in China:the past and the future\u003c/strong\u003e. \u003cem\u003eScientia Sinica Vitae \u003c/em\u003e2019, \u003cstrong\u003e49\u003c/strong\u003e(11):1457-1478.\u003c/li\u003e\n\u003cli\u003eZhu JK: \u003cstrong\u003eAbiotic stress signaling and responses in plants\u003c/strong\u003e. \u003cem\u003eCell \u003c/em\u003e2016, \u003cstrong\u003e167\u003c/strong\u003e(2):313-324.\u003c/li\u003e\n\u003cli\u003eWaadt R, Seller CA, Hsu PK, Takahashi Y, Munemasa S, Schroeder J: \u003cstrong\u003ePlant hormone regulation of abiotic stress responses\u003c/strong\u003e. \u003cem\u003eNature Reviews Molecular Cell Biology \u003c/em\u003e2022, \u003cstrong\u003e23\u003c/strong\u003e(10):680-694.\u003c/li\u003e\n\u003cli\u003eZhang G, Zhang Z, Luo S, Li X, Lyu J, Liu Z, Wan Z, Yu J: \u003cstrong\u003eGenome-wide identification and expression analysis of the cucumber \u003cem\u003ePP2C\u003c/em\u003e gene family\u003c/strong\u003e. \u003cem\u003eBmc Genomics \u003c/em\u003e2022, \u003cstrong\u003e23\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eCao J, Jiang M, Li P, Chu Z: \u003cstrong\u003eGenome-wide identification and evolutionary analyses of the \u003cem\u003ePP2C\u003c/em\u003e gene family with their expression profiling in response to multiple stresses in Brachypodium distachyon\u003c/strong\u003e. \u003cem\u003eBmc Genomics \u003c/em\u003e2016, \u003cstrong\u003e17\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eLuan S: \u003cstrong\u003eProtein phosphatases in plants\u003c/strong\u003e. \u003cem\u003eAnnual Review of Plant Biology \u003c/em\u003e2003, \u003cstrong\u003e54\u003c/strong\u003e:63-92.\u003c/li\u003e\n\u003cli\u003eShazadee H, Khan N, Wang J, Wang C, Zeng J, Huang Z, Wang X: \u003cstrong\u003eIdentification and expression profiling of protein phosphatases (\u003cem\u003ePP2C\u003c/em\u003e) gene family in \u003cem\u003eGossypium hirsutum\u003c/em\u003e L\u003c/strong\u003e. \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2019, \u003cstrong\u003e20\u003c/strong\u003e(6).\u003c/li\u003e\n\u003cli\u003eSchweighofer A, Hirt H, Meskiene L: \u003cstrong\u003ePlant PP2C phosphatases: emerging functions in stress signaling\u003c/strong\u003e. \u003cem\u003eTrends in Plant Science \u003c/em\u003e2004, \u003cstrong\u003e9\u003c/strong\u003e(5):236-243.\u003c/li\u003e\n\u003cli\u003eZhang J, Tao N: \u003cstrong\u003eResearch progress of plant PP2C-type protein phosphatase in ABA signal transduction and adversity stress regulation mechanism\u003c/strong\u003e. \u003cem\u003eGuangxi Zhiwu / Guihaia \u003c/em\u003e2015, \u003cstrong\u003e35\u003c/strong\u003e(6):935-941.\u003c/li\u003e\n\u003cli\u003ePeirats-Llobet M, Han SK, Gonzalez-Guzman M, Jeong CW, Rodriguez L, Belda-Palazon B, Wagner D, Rodriguez PL: \u003cstrong\u003eA direct link between abscisic acid sensing and the chromatin-remodeling ATPase BRAHMA via Core ABA signaling pathway components\u003c/strong\u003e. \u003cem\u003eMolecular Plant \u003c/em\u003e2016, \u003cstrong\u003e9\u003c/strong\u003e(1):136-147.\u003c/li\u003e\n\u003cli\u003eHsu PK, Dubeaux G, Takahashi Y, Schroeder JI: \u003cstrong\u003eSignaling mechanisms in abscisic acid-mediated stomatal closure\u003c/strong\u003e. \u003cem\u003ePlant Journal \u003c/em\u003e2021, \u003cstrong\u003e105\u003c/strong\u003e(2):307-321.\u003c/li\u003e\n\u003cli\u003eLi ZX, Waadt R, Schroeder JI: \u003cstrong\u003eRelease of GTP exchange factor mediated down-regulation of abscisic acid signal transduction through ABA-induced rapid degradation of \u003cem\u003eRopGEFs\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003ePlos Biology \u003c/em\u003e2016, \u003cstrong\u003e14\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eShi Y, Liu XN, Zhao SS, Guo Y: \u003cstrong\u003eThe PYR-PP2C-CKL2 module regulates ABA-mediated actin reorganization during stomatal closure\u003c/strong\u003e. \u003cem\u003eNew Phytologist \u003c/em\u003e2022, \u003cstrong\u003e233\u003c/strong\u003e(5):2168-2184.\u003c/li\u003e\n\u003cli\u003eWu P, Wang W, Li Y, Hou X: \u003cstrong\u003eDivergent evolutionary patterns of the MAPK cascade genes in Brassica rapa and plant phylogenetics\u003c/strong\u003e. \u003cem\u003eHorticulture Research \u003c/em\u003e2017, \u003cstrong\u003e4\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eRovira A, Sentandreu M, Nagatani A, Leivar P, Monte E: \u003cstrong\u003eThe sequential action of MIDA9/PP2C.D1, PP2C.D2, and PP2C.D5 is necessary to form and maintain the hook after germination in the dark\u003c/strong\u003e. \u003cem\u003eFrontiers in Plant Science \u003c/em\u003e2021, \u003cstrong\u003e12\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eChen C, Yu Y, Ding X, Liu B, Duanmu H, Zhu D, Sun X, Cao L, Zaib un N, Li Q\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGenome-wide analysis and expression profiling of \u003cem\u003ePP2C\u003c/em\u003e clade D under saline and alkali stresses in wild soybean and \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eProtoplasma \u003c/em\u003e2018, \u003cstrong\u003e255\u003c/strong\u003e(2):643-654.\u003c/li\u003e\n\u003cli\u003eXue TT, Wang D, Zhang SZ, Ehlting J, Ni F, Jakab S, Zheng CC, Zhong Y: \u003cstrong\u003eGenome-wide and expression analysis of protein phosphatase 2C in rice and \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eBmc Genomics \u003c/em\u003e2008, \u003cstrong\u003e9\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eSingh A, Jha SK, Bagri J, Pandey GK: \u003cstrong\u003eABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003ePlos One \u003c/em\u003e2015, \u003cstrong\u003e10\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eXie W, Liu S, Gao H, Wu J, Liu D, Kinoshita T, Huang C-F: \u003cstrong\u003ePP2C.D phosphatase SAL1 positively regulates aluminum resistance via restriction of aluminum uptake in rice\u003c/strong\u003e. \u003cem\u003ePlant Physiology \u003c/em\u003e2023, \u003cstrong\u003e192\u003c/strong\u003e(2):1498-1516.\u003c/li\u003e\n\u003cli\u003eHe Z, Wu J, Sun X, Dai M: \u003cstrong\u003eThe maize clade a PP2C phosphatases play critical roles in multiple abiotic stress responses\u003c/strong\u003e. \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2019, \u003cstrong\u003e20\u003c/strong\u003e(14).\u003c/li\u003e\n\u003cli\u003eGuo YZ, Shi YB, Wang YL, Liu F, Li Z, Qi JS, Wang Y, Zhang JB, Yang SH, Wang Y\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eThe clade F PP2C phosphatase \u003cem\u003eZmPP84\u003c/em\u003e negatively regulates drought tolerance by repressing stomatal closure in maize\u003c/strong\u003e. \u003cem\u003eNew Phytologist \u003c/em\u003e2023, \u003cstrong\u003e237\u003c/strong\u003e(5):1728-1744.\u003c/li\u003e\n\u003cli\u003eWang JY, Li CA, Li L, Gao LF, Hu G, Zhang YF, Reynolds MP, Zhang XY, Jia JZ, Mao XG\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003e\u003cem\u003eDIW1\u003c/em\u003e encoding a clade I PP2C phosphatase negatively regulates drought tolerance by de-phosphorylating \u003cem\u003eTaSnRK1.1\u003c/em\u003e in wheat\u003c/strong\u003e. \u003cem\u003eJournal of Integrative Plant Biology \u003c/em\u003e2023.\u003c/li\u003e\n\u003cli\u003eGuo X, Chai W, Bai J, Ma Z: \u003cstrong\u003eCloning and bioinformatics analysis of \u003cem\u003eAvFLS\u003c/em\u003e gene from \u003cem\u003eApocynum venetum\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eMolecular Plant Breeding \u003c/em\u003e2019, \u003cstrong\u003e17\u003c/strong\u003e(15):4978-4985.\u003c/li\u003e\n\u003cli\u003eHuang X, Qiu X, Wang Y, Abubakar AS, Chen P, Chen J, Chen K, Yu C, Wang X, Gao G\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGenome-wide investigation of the NAC transcription factor family in \u003cem\u003eApocynum venetum\u003c/em\u003e revealed their synergistic roles in abiotic stress response and trehalose metabolism\u003c/strong\u003e. \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2023, \u003cstrong\u003e24\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eZhang M, Lu X, Ren T, Marowa P, Meng C, Wang J, Yang H, Li C, Zhang L, Xu Z: \u003cstrong\u003eHeterologous overexpression of \u003cem\u003eApocynum venetum\u003c/em\u003e flavonoids synthetase genes improves \u003cem\u003eArabidopsis thaliana\u003c/em\u003e salt tolerance by activating the IAA and JA biosynthesis pathways\u003c/strong\u003e. \u003cem\u003eFrontiers in Plant Science \u003c/em\u003e2023, \u003cstrong\u003e14\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eCao C, Lin DF, Zhou YJ, Li N, Wang YW, Gong WB, Zhu ZH, Liu CW, Yan L, Hu ZX\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eSolid-state fermentation of \u003cem\u003eApocynum venetum\u003c/em\u003e L. by Aspergillus niger: Effect on phenolic compounds, antioxidant activities and metabolic syndrome-associated enzymes\u003c/strong\u003e. \u003cem\u003eFrontiers in Nutrition \u003c/em\u003e2023, \u003cstrong\u003e10\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eLi X, Li JJ, Su HY, Sun P, Zhang Z, Li MF, Xing H: \u003cstrong\u003ePhysiological and transcriptional responses of \u003cem\u003eApocynum venetum\u003c/em\u003e to salt stress at the seed germination stage\u003c/strong\u003e. \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2023, \u003cstrong\u003e24\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eZhang Y, Liu S, Ma JL, Chen C, Huang P, Ji JH, Wu D, Ren LQ: \u003cstrong\u003e\u003cem\u003eApocynum venetum\u003c/em\u003e leaf extract alleviated doxorubicin-induced cardiotoxicity through the AKT/Bcl-2 signaling pathway\u003c/strong\u003e. \u003cem\u003ePhytomedicine \u003c/em\u003e2022, \u003cstrong\u003e94\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eJiang L, Wang L, Tian CY: \u003cstrong\u003eHigh lithium tolerance of \u003cem\u003eApocynum venetum\u003c/em\u003e seeds during germination\u003c/strong\u003e. \u003cem\u003eEnvironmental Science and Pollution Research \u003c/em\u003e2018, \u003cstrong\u003e25\u003c/strong\u003e(5):5040-5046.\u003c/li\u003e\n\u003cli\u003eGao G, Abubakar AS, Chen J, Wang Y, Chen P, Chen K, Yu C, Wang X, Qiu X, Huang X\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eComparative genome and metabolome analyses uncover the evolution and flavonoid biosynthesis between \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eIscience \u003c/em\u003e2023, \u003cstrong\u003e26\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eYuan N, Li MM, Jia CL: \u003cstrong\u003eDe novo transcriptome assembly and population genetic analyses of an important coastal shrub, \u003cem\u003eApocynum venetum\u003c/em\u003e L\u003c/strong\u003e. \u003cem\u003eBmc Plant Biology \u003c/em\u003e2020, \u003cstrong\u003e20\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eMa Y: \u003cstrong\u003eRegulators of PP2C phosphatase activity function as abscisic acid sensors (vol 324, pg 1064, 2009)\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2009, \u003cstrong\u003e324\u003c/strong\u003e(5932):1266-1266.\u003c/li\u003e\n\u003cli\u003eUmbrasaite J, Schweighofer A, Meskiene I: \u003cstrong\u003eSubstrate analysis of \u003cem\u003eArabidopsis\u003c/em\u003e PP2C-type protein phosphatases\u003c/strong\u003e. In: \u003cem\u003ePlant Kinases: Methods and Protocols.\u003c/em\u003e Edited by Dissmeyer N, Schnittger A, vol. 779; 2011: 149-161.\u003c/li\u003e\n\u003cli\u003eYu LP, Miller AK, Clark SE: \u003cstrong\u003ePOLTERGEIST encodes a protein phosphatase 2C that regulates CLAVATA pathways controlling stem cell identity at \u003cem\u003eArabidopsis\u003c/em\u003e shoot and flower meristems\u003c/strong\u003e. \u003cem\u003eCurrent Biology \u003c/em\u003e2003, \u003cstrong\u003e13\u003c/strong\u003e(3):179-188.\u003c/li\u003e\n\u003cli\u003eSentandreu M, Mart\u0026iacute;n G, Gonz\u0026aacute;lez-Schain N, Leivar P, Soy J, Tepperman JM, Quail PH, Monte E: \u003cstrong\u003eFunctional profiling identifies genes involved in organ-specific branches of the PIF3 regulatory network in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003ePlant Cell \u003c/em\u003e2011, \u003cstrong\u003e23\u003c/strong\u003e(11):3974-3991.\u003c/li\u003e\n\u003cli\u003eSpartz AK, Ren H, Park MY, Grandt KN, Lee SH, Murphy AS, Sussman MR, Overvoorde PJ, Gray WM: \u003cstrong\u003eSAUR inhibition of PP2C-D phosphatases activates plasma membrane H\u003csup\u003e+\u003c/sup\u003e-ATPases to promote cell expansion in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003ePlant Cell \u003c/em\u003e2014, \u003cstrong\u003e26\u003c/strong\u003e(5):2129-2142.\u003c/li\u003e\n\u003cli\u003eFu H, Yu X, Jiang Y, Wang Y, Yang Y, Chen S, Chen Q, Guo Y: \u003cstrong\u003eSALT OVERLY SENSITIVE 1 is inhibited by clade D Protein phosphatase 2C D6 and D7 in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003ePlant Cell \u003c/em\u003e2023, \u003cstrong\u003e35\u003c/strong\u003e(1):279-297.\u003c/li\u003e\n\u003cli\u003eMishra G, Zhang WH, Deng F, Zhao J, Wang XM: \u003cstrong\u003eA bifurcating pathway directs abscisic acid effects on stomatal closure and opening in \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2006, \u003cstrong\u003e312\u003c/strong\u003e(5771):264-266.\u003c/li\u003e\n\u003cli\u003eSugimoto H, Kondo S, Tanaka T, Imamura C, Muramoto N, Hattori E, Ogawa Ki, Mitsukawa N, Ohto C: \u003cstrong\u003eOverexpression of a novel Arabidopsis PP2C isoform, \u003cem\u003eAtPP2CF1\u003c/em\u003e, enhances plant biomass production by increasing inflorescence stem growth\u003c/strong\u003e. \u003cem\u003eJournal of Experimental Botany \u003c/em\u003e2014, \u003cstrong\u003e65\u003c/strong\u003e(18):5385-5400.\u003c/li\u003e\n\u003cli\u003eWong JH, Klejchov\u0026aacute; M, Snipes SA, Nagpal P, Bak G, Wang B, Dunlap S, Park MY, Kunkel EN, Trinidad B\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eSAUR proteins and PP2C.D phosphatases regulate H\u003csup\u003e+\u003c/sup\u003e-ATPases and K\u003csup\u003e+\u003c/sup\u003e channels to control stomatal movements\u003c/strong\u003e. \u003cem\u003ePlant Physiology \u003c/em\u003e2021, \u003cstrong\u003e185\u003c/strong\u003e(1):256-273.\u003c/li\u003e\n\u003cli\u003eYang J, Chen R, Hu W, Wu Q, Tong X, Li X: \u003cstrong\u003eIdentification and expression analysis of \u003cem\u003ePP2C\u003c/em\u003e gene family in \u003cem\u003ePoncirus trifoliata\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eJournal of Fruit Science \u003c/em\u003e2022, \u003cstrong\u003e39\u003c/strong\u003e(4):532-547.\u003c/li\u003e\n\u003cli\u003eMeskiene I, Baudouin E, Schweighofer A, Liwosz A, Jonak C, Rodriguez PL, Jelinek H, Hirt H: \u003cstrong\u003eStress-induced protein phosphatase 2C is a negative regulator of a mitogen-activated protein kinase\u003c/strong\u003e. \u003cem\u003eJournal of Biological Chemistry \u003c/em\u003e2003, \u003cstrong\u003e278\u003c/strong\u003e(21):18945-18952.\u003c/li\u003e\n\u003cli\u003eLynch M, Conery JS: \u003cstrong\u003eThe evolutionary fate and consequences of duplicate genes\u003c/strong\u003e. \u003cem\u003eScience \u003c/em\u003e2000, \u003cstrong\u003e290\u003c/strong\u003e(5494):1151-1155.\u003c/li\u003e\n\u003cli\u003eCannon SB, Mitra A, Baumgarten A, Young ND, May G: \u003cstrong\u003eThe roles of segmental and tandem gene duplication in the evolution of large gene families in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eBMC plant biology \u003c/em\u003e2004, \u003cstrong\u003e4\u003c/strong\u003e:10-10.\u003c/li\u003e\n\u003cli\u003eGuo L, Lu S, Liu T, Nai G, Ren J, Gou H, Chen B, Mao J: \u003cstrong\u003eGenome-wide identification and abiotic stress response analysis of \u003cem\u003ePP2C\u003c/em\u003e gene family in \u003cem\u003eWoodland\u003c/em\u003e and \u003cem\u003ePineapple\u003c/em\u003e \u003cem\u003eStrawberries\u003c/em\u003e\u003c/strong\u003e. \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2023, \u003cstrong\u003e24\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eWu H, Zhu L, Cai G, Lv C, Yang H, Ren X, Hu B, Zhou X, Jiang T, Xiang Y\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eGenome-wide identification and characterization of the \u003cem\u003ePP2C\u003c/em\u003e family from Zea mays and its role in long-distance signaling\u003c/strong\u003e. \u003cem\u003ePlants-Basel \u003c/em\u003e2023, \u003cstrong\u003e12\u003c/strong\u003e(17).\u003c/li\u003e\n\u003cli\u003eChen CJ, Chen H, Zhang Y, Thomas HR, Frank MH, He YH, Xia R: \u003cstrong\u003eTBtools: An integrative toolkit developed for interactive analyses of big biological data\u003c/strong\u003e. \u003cem\u003eMolecular Plant \u003c/em\u003e2020, \u003cstrong\u003e13\u003c/strong\u003e(8):1194-1202.\u003c/li\u003e\n\u003cli\u003eMistry J, Chuguransky S, Williams L, Qureshi M, Salazar GA, Sonnhammer ELL, Tosatto SCE, Paladin L, Raj S, Richardson LJ\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003ePfam: The protein families database in 2021\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2021, \u003cstrong\u003e49\u003c/strong\u003e(D1):D412-D419.\u003c/li\u003e\n\u003cli\u003eRen M, Wang Q, Zhang F-h, Wang Y-l, Wang Y-y, Li W, Qi K-j, Xie Z-h, Zhang S-l, Tao S-t: \u003cstrong\u003eGenome-wide identification of the \u003cem\u003eGAox\u003c/em\u003e gene family and functional characterization of \u003cem\u003ePbGA3ox4\u003c/em\u003e during stone cell formation in Chinese white pear\u003c/strong\u003e. \u003cem\u003eScientia Horticulturae \u003c/em\u003e2024, \u003cstrong\u003e330\u003c/strong\u003e.\u003c/li\u003e\n\u003cli\u003eWang YP, Tang HB, DeBarry JD, Tan X, Li JP, Wang XY, Lee TH, Jin HZ, Marler B, Guo H\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003e\u003cem\u003eMCScanX\u003c/em\u003e: a toolkit for detection and evolutionary analysis of gene synteny and collinearity\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2012, \u003cstrong\u003e40\u003c/strong\u003e(7).\u003c/li\u003e\n\u003cli\u003eChen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, Xia R: \u003cstrong\u003eTBtools: An integrative toolkit developed for interactive analyses of big biological data\u003c/strong\u003e. \u003cem\u003eMolecular Plant \u003c/em\u003e2020, \u003cstrong\u003e13\u003c/strong\u003e(8):1194-1202.\u003c/li\u003e\n\u003cli\u003eKumar S, Stecher G, Li M, Knyaz C, Tamura K: \u003cstrong\u003eMEGA X: Molecular Evolutionary genetics analysis across computing platforms\u003c/strong\u003e. \u003cem\u003eMolecular Biology and Evolution \u003c/em\u003e2018, \u003cstrong\u003e35\u003c/strong\u003e(6):1547-1549.\u003c/li\u003e\n\u003cli\u003eSaitou N, Nei M: \u003cstrong\u003eThe neighbor-joining method: a new method for reconstructing phylogenetic trees\u003c/strong\u003e. \u003cem\u003eMolecular biology and evolution \u003c/em\u003e1987, \u003cstrong\u003e4\u003c/strong\u003e(4):406-425.\u003c/li\u003e\n\u003cli\u003eLetunic I, Bork P: \u003cstrong\u003eInteractive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2021, \u003cstrong\u003e49\u003c/strong\u003e(W1):W293-W296.\u003c/li\u003e\n\u003cli\u003eBailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren JY, Li WW, Noble WS: \u003cstrong\u003eMEME SUITE: tools for motif discovery and searching\u003c/strong\u003e. \u003cem\u003eNucleic Acids Res \u003c/em\u003e2009, \u003cstrong\u003e37\u003c/strong\u003e:W202-W208.\u003c/li\u003e\n\u003cli\u003eQiu X, Zhao H, Abubakar AS, Shao D, Chen J, Chen P, Yu C, Wang X, Chen K, Zhu A: \u003cstrong\u003eGenome-wide analysis of \u003cem\u003eAP2/ERF\u003c/em\u003e gene superfamily in Ramie (\u003cem\u003eBoehmeria nivea\u003c/em\u003e L.) revealed their synergistic roles in regulating abiotic stress resistance and ramet development\u003c/strong\u003e. \u003cem\u003eInternational Journal of Molecular Sciences \u003c/em\u003e2022, \u003cstrong\u003e23\u003c/strong\u003e(23).\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-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Apocynum, Protein phosphatase 2C, Salt stress","lastPublishedDoi":"10.21203/rs.3.rs-4268917/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4268917/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein phosphatase class 2C (PP2C) is the largest protein phosphatase family in plants, which plays an important role in plant growth and development and response to adversity stress. \u003cem\u003eApocynum \u003c/em\u003eis a perennial persistent herb, divided into \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e. It mainly grows in saline soil, desert and other harsh environments, and is widely used in saline soil improvement, ecological restoration, textile and medicine. It was found that \u003cem\u003eApocynum hendersonii\u003c/em\u003e is more tolerant of adverse conditions. The main purposeof this study was to investigate the \u003cem\u003ePP2C\u003c/em\u003e gene family and its expression pattern under salt stress and to identify important candidate genes related to salt tolerance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, 68 \u003cem\u003eAvPP2C\u003c/em\u003e genes and 68 \u003cem\u003eAhPP2C\u003c/em\u003e genes were identified from the genomes of \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e, respectively. They were classified into 13 subgroups based on their phylogenetic relationships and further analyzed for their subcellular locations, gene structures, conserved structural domains and cis-acting elements. The results of qRT-PCR analyses of seven \u003cem\u003eAvPP2C\u003c/em\u003e genes and seven \u003cem\u003eAhPP2C \u003c/em\u003egenes proved that they differed significantly in gene expression under salt stress. It has been observed that the \u003cem\u003ePP2C\u003c/em\u003e genes in \u003cem\u003eApocynum venetum\u003c/em\u003e and \u003cem\u003eApocynum hendersonii\u003c/em\u003e exhibit different expression patterns. Specifically, \u003cem\u003eAvPP2C2, 6, 24, 27, 41\u003c/em\u003e and \u003cem\u003eAhPP2C2, 6, 24, 27, 42\u003c/em\u003e have shown significant differences in expression under salt stress. This indicates that these genes may play a crucial role in the salt tolerance mechanism of \u003cem\u003eApocynum venetum\u003c/em\u003eand \u003cem\u003eApocynum hendersonii\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we conducted a genome-wide analysis of the \u003cem\u003eAvPP2C\u003c/em\u003e and \u003cem\u003eAhPP2C \u003c/em\u003egene families in \u003cem\u003eApocynum\u003c/em\u003e, which provided a reference for further understanding the functional characteristics of these genes.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification and expression analysis of the PP2C gene family in Apocynum venetum and Apocynum hendersonii","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-24 14:53:51","doi":"10.21203/rs.3.rs-4268917/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-07T17:55:13+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-07T05:36:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-06T10:24:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306011915495925564165691307862810122715","date":"2024-04-25T06:10:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"259641053924574223624253388418843812310","date":"2024-04-25T02:26:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-25T01:14:54+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-19T18:54:37+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-19T18:50:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-19T18:50:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-04-15T10:06:37+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6ab4ffb8-4fb8-4ec2-bcef-3f99eba44e15","owner":[],"postedDate":"April 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-06-24T13:00:01+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-24 14:53:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4268917","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4268917","identity":"rs-4268917","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}