Whole-genome Identification of the PP2C Gene Family in citrus and its response to Abiotic Stresses

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Although the PP2C gene family has been extensively characterized in plants such as Arabidopsis thaliana and Oryza sativa , its structural features and stress-responsive patterns remain rarely unexplored in citrus. This study aims to address this gap by conducting a genome-wide identification and functional characterization of PP2C genes in citrus, with a focus on their expression profiles under abiotic stress conditions. Results A comprehensive genome-wide analysis was performed in citrtus, and a total of 47 PP2C genes ( CsPP2C1 – CsPP2C47 ) were identified. All CsPP2C members possess conserved PP2C catalytic domains. Physicochemical characterization revealed that CsPP2C proteins exhibited a molecular weight range of 20.03–124.36 kDa, with the majority being acidic (pI 40), and hydrophilic (grand average of hydropathicity < 0). Phylogenetic analysis clustered them into 12 subgroups consistent with model species, indicating functional conservation of the gene family. These genes are unevenly distributed across 9 citrus chromosomes, and promoter cis-acting element analysis uncovered enrichment of stress-related motifs (MBS, LTR) and hormone-responsive elements (ABRE). Quantitative real-time PCR analysis revealed distinct expression patterns of CsPP2Cs in response to PEG, low temperature, salt, and hormone treatments at 0, 12, and 24h, suggesting the important roles in citrus growth and development as well as abiotic stresses. Conclusions This study presented a comprehensive characterization of the PP2C gene family in citrus, clarifying its evolutionary relationships, structural diversity, and stress-responsive expression profiles. The findings established a foundational framework for elucidating the molecular mechanisms underlying CsPP2C-mediated stress adaptation, and provided candidate genes for molecular breeding of stress-resilient citrus varieties. citrus Protein Phosphatase 2Cs bioinformatics stresses expression analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Citrus represents a vital agricultural resource around the world and holds significant economic value. With the continuous development of citrus industry, increasing attention is being paid to fruit yield and quality. However, due to climate issues, citrus plants are frequently threatened by multiple abiotic stresses, which usually results in a remarkbly decrease in yield and quality, and a significant economic losses[ 1 ]. To cope with adverse environmental conditions[ 2 ], plants have evolved sophisticated mechanisms to perceive and adapt to stresses through various physiological and biochemical provesses such as photosynthesis[ 3 ], glucose metabolism[ 4 ], and cell growth[ 5 ], many of which involve reversible protein phosphorylation[ 6 ]. Protein phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively, regulate nearly all aspects of cellular life, including the cell cycle, phytohormone signaling, and metabolic pathways[ 7 ]. PP2C is a class of Mg 2+ - or Mn 2+ -dependent monomeric enzymes, belonging to the serine/threonine protein phosphatases subfamily[ 8 ]. In guard cells, membrane-localized PP2Cs specifically mediate the dephosphorylation of plasma membrane (PM) H⁺-ATPase ; the functional activity of this enzyme is tightly controlled by the phosphorylation status of multiple residues in its C-terminal domain[ 9 ]. Among these phosphorylatable sites, the penultimate threonine (Thr) residue has remained a major focus of research. Phosphorylation of this specific Thr residue activates PM H⁺-ATPase by disrupting the autoinhibitory interaction of its C-terminal domain, thereby participating in key physiological processes including light-induced stomatal opening and auxin-driven cell expansion[ 10 ]. Notably, the isoforms belonging to protein phosphatase 2C (PP2C) clade D (PP2C.Ds) play a role in facilitating the dephosphorylation of PM H⁺-ATPase. This dephosphorylation process is fundamental to auxin - induced hypocotyl elongation and cell expansion events[ 11 ]. PP2Cs are widely distributed across various taxa, including archaea[ 12 ], bacteria[ 13 ], fungi[ 14 ], plants[ 15 ] and animals[ 16 ]. To date, numerous PP2C genes have been identified in various plant species including Arabidopsis , rice[ 17 ], paulownia[ 18 ], strawberry[ 19 ], wheat[ 20 ] and hemp[ 21 ]. For instance, 80 PP2C members have been reported in Arabidopsis thaliana [ 22 ], 194 in Oat ( AsaPP2C )[ 23 ], 87 in banana[ 24 ], 92 in tomato[ 25 ], and 56 in cucumber[ 26 ]. Based on protein sequence aimilarity, PP2Cs can be classfied into multiple subfamilies. In Arabidopsis , the PP2C gene family members of can be divided into 12 subfamilies (A-L)[ 27 ], each with distinct functional characteristics. Briefly, subfamily A members are involved in abscisic acid (ABA) signaling and act as negative regulators in ABA responses[ 28 ]. Several subfamily B proteins participate in mitogen-activated protein kinase (MAPK) signaling pathways, also serving as negative regulators[ 29 ]; Proteins in subfamily C are associated with floral organ development[ 30 ]; while subfamily D is implicated in saline-alkali stress response[ 31 ]. Subfamily E regulates stomatal signaling to control transiration[ 32 ], and subfamily F is involved in the induction of bacterial stress response[ 33 ]. PP2Cs widely participate in plant growth and development, wound response, and adaptation to biotic and abiotic stresses. Some studies have revealed that the ABA-mediated reduction in PP2C activity enhances protein kinase phosphorylation, thereby influencing plant development and stresse adaptation[ 34 ]. For example, in Poncirus trifoliate , 10 PP2C genes exhibited notable alterations in their expression levels when subjected to low temperature, high temperature or drought stresses, and PtrPP2C29 was especially sensitive[ 35 ]. In Saccharum spontaneum , 37 out of 89 PP2Cs displayed distinct expression patterns under drought conditions, underscoring their importance in drought tolerance[ 36 ]. Among the 172 PP2C members in peanut ( Arachis hypogaea L.), AhPP2C45 and AhPP2C134 were markedly upregulated under salt stress[ 37 ]. Similarly, in Broussonetia papyrifera , 4 out of 18 BpPP2Cs genes were induced by low temperature (4°C), and exhibited high phosphorylation levels, indicating a role in cold stress reponse[ 38 ]. In this study, we obtained 47 PP2C gene members in Citrus. Bioinformatics analysis was conducted on these 47 PP2C members, including physical and chemical property, phylogenetic relationship, chromosome localization, gene structure, conserved domain, and cis-acting element prediction. In addition, the gene expression profiles under abiotic stress and hormone treatments were investigated. This study sorted out the molecular traits and stress-regulation ability of citrus PP2C members via whole-genome identification and stress tests, making the plant PP2C family research system more complete, and it also offers a base for understanding CsPP2C's roles in citrus stress responses. 2. Results 2.1. Identification of PP2Cs genes in citrus A total of 47 PP2C members have been identified in the citrus and citrus-relative genomes, one unique to Poncirus trifoliata . As shown in Fig. 1, the 47 CsPP2C genes are unevenly distributed on 9 scaffolds with the detailed distribution as follows: 7 genes on scaffold_1, 3 genes on scaffold_2, 7 genes on scaffold_3, 3 genes on scaffold_4, 6 gene on scaffold_5, 4 genes on scaffold_6,5 genes on scaffold_7, 5 genes on scaffold_8, and 7 genes on scaffold_9. Scaffold_1, scaffold_3 and scaffold_9 contain the most CsPP2C genes, while scaffold_4 contains the least. Most CsPP2Cs are located at ends of the chromosomes. 2.2. Phylogenetic analysis The NJ tree showed the evolutionary relationship of PP2C gene families among citrus, Arabidopsis and rice (Fig. 2). The NJ tree divided the PP2C family members into 12 groups (Group I ~ XII), each group containing at least two PP2C members from different species; Some groups (such as Group VII and X) had a larger number of members, while others (such as Group I) were comprosed of a smaller number of members; The PP2C members of the same species were dispersed across multiple group. These results indicated that PP2C gene family is highly conserved in plants. 2.3. Synteny analysis The collinearity analysis between C.clementina and two representative species including A.thaliana and O.sativa showed that 4 CsPP2Cs were homologous to OsPP2Cs found in O.sativa , 7 CsPP2Cs are homologous to AtPP2Cs found in A.thaliana (Fig. 3). In these CsPP2Cs , gene demonstrated one-to-many homozygosity. For example, C.clementina Chr1 corresponds to both O.sativa Chr3 and Chr7, and C.clementina Chr2 corresponds to both A.thaliana Chr1 and Chr3. 2.4. Physical and chemical properties The physicochemical properties of 47 CsPP2Cs were analyzed (Table 1 ). The number of amino acid ranged from 184 to 1101 amino acid residues, corresponding to relative molecular weights of 20.03 to 124.36 kDa, with an average molecular weight of approximately 47.26 kDa. The theoretical isoelectric points (pI) ranged from 4.76 to 9.41 and most of CsPP2Cs were classified as acidic proteins, due to their pI values below 7. Based on the instability index, most CsPP2C protein were predicted to be unstable. The aliphatic index ranged from 65.14 to 95.53, averaging about 83.07. Among them, CsPP2C47 had the smallest aliphatic index, and CsPP2C35 had the largest. The grand average of hydropathicity values ranged from − 0.795 to 0.058. With the exception of CsPP2C21, all other CsPP2C proteins had a grand average of hydropathicity below 0, indicating that most of them were hydrophilic proteins. Table 1 Physicochemical property of PP2C proteins Gene Locus ID Number of amino acid Molecularweight (g/mol) pI Instability index Aliphatic index Grand average of hydropathicity CsPP2C1 Ciclev10001476 384 41661.17 5.22 51.95 81.80 -0.246 CsPP2C2 Ciclev10005100 401 43771.80 5.16 51.48 92.39 -0.092 CsPP2C3 Ciclev10018806 879 97646.61 5.88 42.10 68.63 -0.554 CsPP2C4 Ciclev10008623 383 42031.36 5.10 38.84 85.80 -0.258 CsPP2C5 Ciclev10020453 401 44418.38 4.76 56.52 87.21 -0.151 CsPP2C6 Ciclev10025600 446 46966.67 6.86 46.82 78.72 -0.124 CsPP2C7 Ciclev10005158 383 42358.12 6.26 42.22 86.53 -0.248 CsPP2C8 Ciclev10020547 387 41558.31 5.05 42.36 85.43 -0.217 CsPP2C9 Ciclev10004613 584 64391.48 6.19 45.59 91.01 -0.191 CsPP2C10 Ciclev10009047 298 32624.76 8.64 40.49 78.86 -0.432 CsPP2C11 Ciclev10018605 1101 124357.23 5.77 40.55 80.13 -0.381 CsPP2C12 Ciclev10001891 316 34236.27 5.20 34.01 80.00 -0.279 CsPP2C13 Ciclev10027924 704 78971.56 5.59 36.43 77.13 -0.514 CsPP2C14 Ciclev10031727 400 43948.90 8.86 44.38 76.05 -0.391 CsPP2C15 Ciclev10022462 184 20031.58 5.35 43.79 82.55 -0.194 CsPP2C16 Ciclev10011223 679 75471.30 5.41 36.55 75.10 -0.543 CsPP2C17 Ciclev10025810 390 42217.60 5.91 46.10 76.54 -0.400 CsPP2C18 Ciclev10028649 382 42561.81 8.83 49.40 91.10 -0.259 CsPP2C19 Ciclev10004981 440 48347.43 5.41 65.46 73.86 -0.469 CsPP2C20 Ciclev10006433 265 29650.73 9.41 43.05 76.94 -0.457 CsPP2C21 Ciclev10028878 312 33856.41 5.15 38.12 88.69 0.058 CsPP2C22 Ciclev10011931 390 43954.19 6.89 53.39 90.72 -0.207 CsPP2C23 Ciclev10031682 411 44810.69 6.23 56.39 80.39 -0.367 CsPP2C24 Ciclev10005200 372 40744.16 6.97 46.61 81.40 -0.403 CsPP2C25 Ciclev10027412 261 29000.94 6.16 54.95 91.57 -0.312 CsPP2C26 Ciclev10025781 397 44283.42 8.49 49.66 87.88 -0.283 CsPP2C27 Ciclev10001565 368 40842.46 6.42 41.38 77.09 -0.216 CsPP2C28 Ciclev10007997 523 57186.26 5.24 40.90 78.53 -0.364 CsPP2C29 Ciclev10011576 491 53862.52 5.69 44.02 75.48 -0.438 CsPP2C30 Ciclev10007908 550 59725.47 4.86 46.71 90.35 -0.111 CsPP2C31 Ciclev10009076 292 31361.74 4.90 39.57 79.52 -0.279 CsPP2C32 Ciclev10013751 517 57718.55 6.90 42.25 83.77 -0.372 CsPP2C33 Ciclev10031798 386 42841.71 5.93 39.13 90.65 -0.237 CsPP2C34 Ciclev10015132 470 51912.11 5.38 44.63 68.43 -0.497 CsPP2C35 Ciclev10020542 389 42761.65 5.56 40.93 95.53 -0.060 CsPP2C36 Ciclev10028600 397 44096.10 8.17 49.38 88.34 -0.286 CsPP2C37 Ciclev10010157 329 36032.15 6.09 34.80 92.10 -0.181 CsPP2C38 Ciclev10023343 301 32406.95 6.28 48.42 89.07 -0.092 CsPP2C39 Ciclev10005185 375 41865.84 7.30 38.44 91.23 -0.226 CsPP2C40 Ciclev10001232 429 46747.09 5.18 40.97 80.02 -0.369 CsPP2C41 Ciclev10025314 544 58862.87 5.21 43.07 94.45 -0.131 CsPP2C42 Ciclev10001248 427 45785.51 7.51 30.80 88.36 -0.122 CsPP2C43 Ciclev10028495 429 46985.89 5.52 41.29 75.66 -0.425 CsPP2C44 Ciclev10016165 284 31368.91 7.24 34.95 88.91 -0.312 CsPP2C45 Ciclev10000910 511 54983.89 7.58 42.68 83.42 -0.099 CsPP2C46 Ciclev10008648 380 41376.74 5.19 58.93 81.61 -0.234 CsPP2C47 Ptrif.0002s2428 259 28567.47 4.97 49.88 65.14 -0.795 2.5. Gene structure and conserved domains To determine the quantity and positions of introns and exons, gene structure analysis was performed by comparing the full-length cDNA sequences with their corresponding genomic DNA sequences. As showed in Fig. 4, the number of exon varied considerably ranged from 1 ( CsPP2C8 ) to 20 ( CsPP2C11 ), revealing diverse intron-exon architectures. Among the 47 CsPP2Cs , approximately 78.7% (37 genes) contained 3 to 6 exons, indicating that most family members possess a moderate number of exons. CsPP2C9 , CsPP2C11 , CsPP2C21 , CsPP2C35 had a considerably higher number of exons than most of the other genes in the family. It was worthy to note that CsPP2C8 had only a single exon (intronless genes), whereas CsPP2C11 contained the highest number of exons. The conserved motifs in 47 CsPP2Cs were analysized, which revealed that all 47 CsPP2C proteins contained conserved motifs with high sequence similarity (Fig. 5). The number of motifs varied considerably among different CsPP2C proteins, with the majority harboring either 6 or 7 motifs. Motif 3 and motif 1 were present in nearly all CsPP2C proteins, and motif 1, motif 2, motif 4 and motif 5 were also widely distributed. Notably, the positions of motif 1 and motif 3 were highly conserved. 45 members (95.7%) contained motif 1 and 44 members (93.6%) contained motif 3. In contrast, some motifs (such us motif 8) exhibited restricted distribution patterns, occurring only in specific subsets of CsPP2C proteins. The substantial variation in motif composition and copy number suggested significant functional diversification within the CsPP2C family. 2.6. Cis -acting elements in the promoters of CsPP2Cs Putative cis -acting regulatory elements in the promoter of CsPP2C genes were predicted using PlantCARE (Fig. 6). These cis -acting elements were classified into three primary categories: hormone response, stress response, and plant growth/photoregulatory elements. Hormone-responsive elements included the CGTCA-motif and TGACG-motif (involved in MeJA-responsiveness), ABRE (abscisic acid responsiveness), auxin-responsive element, the TCA-element (salicylic acid responsiveness), and gibberellin-responsive element (P-box and GARE-motif). Stress-responsive elements encompassed LTR (low-temperature responsiveness), MBS (drought-inducibility), TC-rich repeats (defense and stress responsiveness), and the GC-motif (involved in anaerobic induction). Photoregulatory and circadian-related elements included the G-box, ACE, GT1-motif, AE-box, TCT-motif (all involved in light responsiveness),and the circadian control element. Notably, a significant abundance of ABA-responsive elements (ABRE) was detected within the promoter regions of the majority of CsPP2C genes, indicating the extensive participation of this gene family in the ABA signaling pathway.. MeJA-responsive elements (CGTCA-motif and TGACG-motif) were also widely distributed among members, such as CsPP2C1 , CsPP2C15 , CsPP2C20 , CsPP2C29 , and CsPP2C38 , indicating their potential roles in JA-mediated stress responses. Furthermore, a significant number of elements related to light responsiveness (e.g., G-box, ACE) were detected in the promoters of CsPP2C5 , CsPP2C13 , CsPP2C27 , and CsPP2C44 , implying possible regulation by light signals. Elements involved in low-temperature (LTR) and drought (MBS) responsiveness were identified in several members, including CsPP2C3 , CsPP2C9 , CsPP2C24 , and CsPP2C41 . Collectively, these results suggested that most CsPP2C s were involved in diverse biological processes, responding to various phytohormone (e.g., ABA, MeJA,SA, Auxin, GA) and environmental stimuli (e.g., low temperature, drought, light, and anaerobic stress). 2.7. Expression analysis Expression profiling of CsPP2C genes under various treatments (including NaCl, SA, ABA, low temperature, ETH, and PEG) revealed distinct response patterns among members (Fig. 7). Genes including CsPP2C1, CsPP2C5, CsPP2C9, CsPP2C13, CsPP2C15, CsPP2C17, CsPP2C21, CsPP2C25, CsPP2C29, CsPP2C33, CsPP2C37, CsPP2C41, and CsPP2C45 showed low or negligible expression changes across most conditions, suggesting limited involvement in the tested stress or hormone responses. In contrast, a subset of genes exhibited notably treatment-specific expression dynamics. Under NaCl treatment, CsPP2C3 expression increased sharply at the beginning of treatment (1h) but returned to baseline levels thereafter; CsPP2C7 was strongly induced by ABA, peaking at 6h, and showed continuously increasing expression under low temperature; CsPP2C11 expression was suppressed by ETH treatment for 24h. CsPP2C19 was up-regulated under both NaCl and low temperature but down-regulated by ETH; CsPP2C27 was steadily up-regulated during low temperature exposure but significantly down regulated by ETH; CsPP2C31 was significantly induced under ABA and low temperature treatments, thouth it exhibited no clear trend under SA; CsPP2C35 expression increased under multiple treatments (such as SA, low temperature, NaCl), particularly under low temperature. CsPP2C39 was induced by low temperature and showed transient up-regulation followed by a decline under NaCl and SA; CsPP2C43 expression rose then fell under ABA, SA and low temperature conditions; CsPP2C47 was down-regulated in response to SA and PEG. These results indicated that CsPP2C3, CsPP2C7, CsPP2C11, CsPP2C19, CsPP2C23, CsPP2C27, CsPP2C31, CsPP2C35, CsPP2C39, CsPP2C43 , and CsPP2C47 were likely involved in biological processes related to hormones (e.g., ABA, ETH) and abiotic stresses (e.g., low temperature, PEG, and NaCl stress), thereby highlighting their potential roles in citrus growth, development, and stress adaptation. 3. Discussion In this study, a comprehensive genome-wide analysis obtained a total of 47 PP2C genes across three citrus species: Citrus clementina (46), Citrus sinensis (46), and Poncirus trifoliate (46). After removing redundancies, 47 unique members ( CsPP2C1 – CsPP2C47 ) were retained for further characterization. The number of PP2C genes in citrus is relatively small compared to A.thaliana (80)[ 39 ], rice (87)[ 40 ], and oat (194)[ 41 ]. This variation may result from differences in genome size, lineage-specific evolutionary events, or gene family expansion/contraction. Members clustered within the same subgroup exhibit shorter branch lengths, indicative of closer genetic affinities. By contrast, longer branch lengths between distinct subgroups reflect substantial genetic divergence among them. The PP2C members from Oryza sativa ( OsPP2C ), Citrus ( CsPP2C ), and Arabidopsis thaliana ( AtPP2C ) were consistently categorized into 12 subgroups. This result suggests that the PP2C family had already established a conserved classification framework prior to the speciation and differentiation of these three plant species. Each subgroup encompasses members derived from three plant species, providing evidence of the presence of cross-species homologous genes within the PP2C family. Specifically, PP2C homologs that possess conserved sequence characteristics and likely similar biological functions are preserved across these distinct taxonomic groups. In addition, PP2C members from the same species are scattered across multiple subgroups; this distribution pattern implies that the PP2C family has undergone extensive gene duplication events accompanied by functional divergence during the evolutionary process of individual species, and that family expansion is primarily mediated via a mechanism of dispersed clustering. To verify the evolutionary relationships inferred from the phylogenetic tree, collinearity analysis was performed. The results revealed a notable collinearity between the PP2C genes in citrus and those in O.sativa and A.thaliana , thereby corroborating the evolutionary homology among the PP2C families of these three species. Collinearity was predominantly manifested in a one-to-many pattern, implying that lineage-specific duplication events occurred in the PP2C gene family after speciation. This evolutionary process would have resulted in a single ancestral PP2C gene corresponding to multiple homologous genes in derived species. To further contextualize the exon-intron architectural characteristics of CsPP2Cs (Fig. 1), we compared their structural profiles with those of Arabidopsis thaliana (AtPP2Cs) and Oryza sativa (OsPP2Cs). Our findings unveiled a considerable degree of diversity in the gene structures of CsPP2Cs, with exon numbers spanning from 1 to 20. This range surpassed that observed in Arabidopsis thaliana (AtPP2Cs), where exons ranged from 0 to 13[ 42 ], and in Oryza sativa (OsPP2Cs), where exons ranged from 0 to 18[ 43 ]. Such an expanded structural variation suggested that the citrus PP2C family exhibited greater evolutionary plasticity compared to these two model species. Notably, one CsPP2C member ( CsPP2C8 ) was identified as intronless (Fig. 1)—a feature also reported in a subset of AtPP2C-A subfamily members (e.g., ABI1 , ABI2 ), but rare in OsPP2Cs. Intronless genes are widely recognized to enable rapid transcriptional activation in stress responses, suggesting that CsPP2C8 may function as fast-acting regulators in abiotic stress signaling[ 43 – 44 ]. Approximately 78.7% of CsPP2Cs contained 3–6 exons (Fig. 1), a proportion consistent with that of AtPP2Cs but higher than that of OsPP2Cs. This enrichment in moderate exon range was hypothesized to hypothesized to represent an evolutionary trade-off between functional versatility and transcriptional efficiency[ 45 ], potentially explaining its conservation of this exon range in eudicots (e.g., citrus and Arabidopsis ) and its relative depletion in monocots such as rice. The broader exon count range of CsPP2Cs likely reflected lineage-specific gene duplication and intron gain/loss events during citrus evolution, processes typically associated with functional divergence. Overall, the structural diversity of CsPP2Cs reflected conservative functional roles and lineage specific specialization compared to AtPP2Cs and OsPP2Cs[ 46 – 47 ]. Physicochemical characterization revealed that approximately 78.7% CsPP2C proteins were acidic, with isoelectric points (pI) ranging from 4.76 to 6.97. All CsPP2C members exhibited inherent instability (instability index > 40) and hydrophilicity (grand average of hydropathicity, GRAVY < 0), These characteristics were also evolutionarily conserved in Arabodopsis AtPP2Cs and rice OsPP2Cs[ 48 ]. In terms of molecular weight, CsPP2Cs displayed a broader range (20.03-124.36 kDa) compared to OsPP2Cs (35–62 kDa) and was comparable to the size diversity of AtPP2Cs. Additionally, CsPP2Cs showed significant changes in the fatty index (65.14–95.53) which is related to protein thermal stability[ 49 ]. Specifically, CsPP2C35 had the highest aliphatic index (95.53), indicating a greater degree of thermal stability, while CsPP2C47 presented the lowest lipid index (65.14), corresponding to the lowest level of thermal stability among the tested proteins. To investigate the functional divergence of CsPP2C genes in response to stresses, their expression profiles were analyzed under multiple abiotic stresses (NaCl, PEG, low temperature) and hormone treatments (ABA, ETH, SA). The results showed that a suite of CsPP2C genes, specifically CsPP2C3 , CsPP2C7 , CsPP2C19 , CsPP2C23 , CsPP2C27 , CsPP2C31 , CsPP2C35 , CsPP2C39 , CsPP2C43 , and CsPP2C47 , exhibited responsiveness to a range of stress conditions and hormone treatments, including NaCl, PEG-simulated drought, low temperature, as well as abscisic acid (ABA), ethylene (ETH), and salicylic acid (SA). Notably, these genes displayed distinct temporal expression patterns, suggesting potential functional specialization in their roles during stress responses.. For instance, CsPP2C7 was strongly induced by both ABA and low temperature—consistent with the expression behavior of its Arabidopsis homologs (e.g., AHG3/AtPP2CA of Arabidopsis plays a major role among PP2Cs in the ABA response in seeds[ 50 ]. CsPP2C27 showed a sustained up-regulation under cold stress but was suppressed by ETH, suggesting a specific regulatory role in low-temperaure adaptation. Such stress-responsive expression patterns of CsPP2Cs aligned with observations in other plant species: In Brachypodium distachyon , nearly all BdPP2C genes were up-regulated under abiotic stresses such as cold, heat, PEG and NaCl treatments[ 51 ]; in maize, most ZmPP2C-A clade genes (e.g., ZmPP2C-A2 , ZmPP2C-A6 ) were dramatically induced by drought, salt, and ABA, and further functional analysis confirmed their negative regulation of drought responses[ 52 ]. Similarly, BnPP2C1 , BnPP2C26 , and BnPP2C27 in ramie showed strong responses to drought, high salt, and ABA[ 53 ], while ZoPP2Cs in ginger exhibited diverse expression dynamics during maturation and under drought/salt stresses[ 54 ]. These cross-species similarities indicated the conserved functions of PP2Cs in orchestrating responses to abiotic stress and modulating hormone signaling pathways. Promoter analysis of CsPP2C genes uncovered a wealth of cis -acting elements intricately linked to stress responses and hormone signaling, such as ABRE, MBS, LTR, and MeJA-responsive motifs, which suggested the involvement of CsPP2C genes in a diverse array of signaling networks.. ABRE elements were extensively present in the promoters of CsPP2C genes (e.g., CsPP2C1 , CsPP2C19 , CsPP2C43 ), consistent with the well-documented role of PP2Cs in ABA signaling[ 55 ]. Additionally, this was further validated by the differential expression patterns of these genes under ABA treatment (Supplementary Table 1). MBS motifs were detected in 27 CsPP2C members, and LTR elements in 20, implying their roles in PEG and cold stress responses. For example, MBS-containing CsPP2C28 was upregulated under PEG-induced drought, while LTR-harboring CsPP2C11 exhibited marked induction under cold stress. MeJA-response elements were found in 36 CsPP2Cs , among which related genes such as CsPP2C11 and CsPP2C44 were consistently expressed at high levels under SA induction, indicating potential crosstalk between hormones. Taken together, cis -element analysis combined with qPCR data demonstrated that CsPP2Cs were transcriptionally modulated by multiple stresses and hormones, highlighting their functional versatility in citrus adaptive mechanisms. In summary, this study provides a genome-wide identification and functional characterization of the PP2C gene family in citrus. The results highlight the structural and functional diversity among CsPP2Cs , their evolutionary relationships with known stress-responsive phosphatases, and their potential roles in hormone and stress signaling. Future work should prioritize functional validation of key candidates such as CsPP2C7 and CsPP2C27 through transgenic assays to confirm their regulatory roles in stress adaptation and facilitate molecular breeding of stress-resilient citrus varieties. 4. Conclusions In this study, a total of 47 CsPP2Cs were identified in citrus and citrus-relative species, which were classified into 12 subgroups. The results indicated that CsPP2Cs were evolutionarily related to Arabidopsis and rice PP2C genes, perhaps originated from the same ancestor. Additionally, CsPP2Cs displayed comparable motif patterns and exon/intron arrangement structures. Collinearity analysis further supported evolutionary relationships of PP2Cs between citrus, rice, and Arabidopsis , with one-to-many synteny patterns suggesting lineage-specific duplication post-speciation. This study also investigated the expression patterns of CsPP2Cs in response to different abiotic stresses using qRT-PCR analyses, and found that CsPP2Cs were involved in coping with salt, PEG, low temperature and hormone(NaCl、SA、ETH) stresses. A comprehensive analysis of the cis - acting elements within the promoter regions of CsPP2C genes had unveiled their crucial roles in stress adaptation as well as in the regulation of citrus development and growth processes. In conclusion, our work may provide valuable insights into the molecular mechanisms of CsPP2Cs involved in abiotic stresses and hormone-related biological processes. 5. Materials and Methods 5.1. Identification and analysis of PP2C gene 5.1.1. Sequence Acquisition Genomic sequences, transcript sequences, and peptide sequences of Citrus clementina , Citrus sinensis and Poncirus trifoliata were download from the Phytozome database ( https://phytozome-next.jgi.doe.gov/ ), respectively. Additionally, PP2C amino acid sequences of Arabidopsis thaliana and Oryza sativa were obtained from Phytozome database ( https://phytozome-next.jgi.doe.gov/ ) based on their ID numbers. 5.1.2. Sequence alignment and NJ tree construction Multiple sequence alignment were performed using clustalx software. After removing redundant sequences across the three varieties, 47 unique PP2C members were retained for subsequent analysis. Given that PP2C members exhibit a high degree of conservation between citrus and poncirus, the one that is unique to poncirus was designated with the same naming convention as those found in citrus in this study. A neighbor-joining (NJ) phylogenetic tree was built with the combined PP2C protein sequences from the 47 members, rice and Arabidopsis employing 1000 bootstrap replicates.Beautify the NJ tree using the ITOL website ( https://itol.embl.de/z ) 5.1.3. Synthetic analysis The chromosome location information of the CsPP2Cs in Citrus clementina were obtained from Ensembl Plants ( https://plants.ensembl.org/index.html ) and diagrammed using software TBtools-Ⅱ. CsPP2Cs duplication events were examined using MCScanX via TBtools-Ⅱ. One-Step MCScanX was employed to predict synteny between genes in A.thaliana and those in O.sativa and C.clementina utilizing genome annotation and sequence files. The synteny was visualized using a dual synteny plot generated by MCScanX in the TBtools software.Beautify image results using AI software. 5.1.4. Physicochemical properties The physicochemical properties of CsPP2Cs, including the number of amino acid, theoretical isoelectric point(PI), molecular weight, instability indext, grand average of hydropathicity(GRAVY), and aliphatic index, were predicted using ExPASy ProParam tool ( https://web.expasy.org/protparam/ ). 5.1.5. Chromosome location analysis The gene IDs, gene location and chromosome length information for the PP2C gene family members were obtained from Phytozome ( https://phytozome-next.jgi.doe.gov/ ) s. The chromosome distribution map was then generated using the MG2C online tool ( http://mg2c.iask.in/mg2c_v2.1/index.html ) after data curation. 5.1.6. Gene structure and Conserved Domain The gene structure(exon-intron organization) of CsPP2C genes was visualized using the GSDS v2.0 online server( http://gsds.gaolab.org/ ). Conserved motif in the CsPP2C protein sequences were identified using the MEME suite ( https://meme-suite.org/meme/tools/meme ) with default parameters. 5.1.7. Cis -acting element prediction The 2000 bp promoter sequences upstream of the transcription start sites of CsPP2C genes were extrated from the phytozome database( https://phytozome-next.jgi.doe.gov/ ). Putative cis -acting regulatory elements within these promoter regions were predicted using the PlantCARE online website ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). 5.1.8.Expression analysis Healthy young leaves from navel orange were subjected to various abiotic stresses and phytohormones. First,put six fresh leaves into PEG (20%)[ 56 ], SA (5 mmol·L -1 ), ABA (100 µmol·L -1 )[ 57 ], ETH (500 mg·L -1 ), and NaCl (60 mmol·L -1 )[ 58 ]solutions, simmersed in the respective solutions for 10 minutes and taken out. After treatment, the leaves were blotted dry, transferred to a growth chamber maintained under constant temperature and humidity, and harvested at the designated time points (0, 6, 24 h). The harvested samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Total RNA was extracted using the Plant RNA Prep Pure Kit (Aidlab Biotechnologies Co., Ltd.), strictly following the manufacturer's protocol. RNA concentration and purity were verified spectrophotometrically. First-strand cDNA was then synthesized from 1 µg of total RNA using the PrimeScript™ RT Reagent Kit (Takara Bio Inc.), according to the manufacturer's instructions. Gene-specific primers for the PP2C gene family members were designed based on transcript sequences from the Phytozome database using Primer Premier software (primer sequences are listed in Table 2 ). qRT-PCR was performed using a quantitative PCR system with the Citrus Actin gene serving as an internal reference. Table 2 Gene-specific primers sequences for the PP2C gene family Primer name Forward primer sequence Reverse primer sequence Amplify fragment length(pb) CsPP2C1 TCGGATAGAAATGCCACC CAACAACAGCAGCAGTAA 197 CsPP2C2 GTGGATTGGGTTTCACTG AAAGCCTACCTAAATGTTTG 151 CsPP2C3 TGTTTCGGGTAGACTATGT ATCGCCTTCTGGGACATT 185 CsPP2C4 AAGCAATAAAGCGAGGAG AAGAGGTATGCCAATGAA 181 CsPP2C5 ATTGGCTGTGATGGAATC GATCAAGGGAAGTGAAGC 181 CsPP2C6 ATAGCGTTGTATTCGTTTG TTCGGAAGGATAGACTTG 180 CsPP2C7 CAAGAGGCGGTGGAGATT TTGAGGCGACGATGAAGA 162 CsPP2C8 GAAGCAGCATCAAGGAAG CGAAAGTGTAACGGGAAA 198 CsPP2C9 GCTAGAACACTGCGAACA GTGGCATTATACCACTTGAT 181 CsPP2C10 TGCTTGCGTTTAATTGTTG AGGCACTTCTGTTCCACC 192 CsPP2C11 TCTCGGTAGCAGAAAGTG GAAATCGGAGGTGAATAA 162 CsPP2C12 GGTGGTGTTCTTGCTGTT TGCTTCTGCATCGGTTAT 192 CsPP2C13 TCTCAAACAGCCAAAGTG CGTGAAGTATTGATAGAGCC 166 CsPP2C14 GGTAGTCATCAAAGGGTTG GAGCCTCTATCTATGGAAAT 176 CsPP2C15 GGGAGGTAGGAACAATACAA TGGTCCGTGCCCATCAAATA 155 CsPP2C16 CTTCCTTGCTGCATTCCC ACGCCAGATTCTTCCCTC 184 CsPP2C17 TCTTTGGGATGTAATGAGC TTTGAATCTTGGGAGGTG 182 CsPP2C18 AGAGGCTGCAAGGAAGAG TTTAGGGAGAAGATGTCG 195 CsPP2C19 GGAGCACGAGTTGAAGGC GCAACCTCACAAGCCAAG 182 CsPP2C20 AGCCAACATGGGTGAGTA GCTCTGAGCCTTTAGAGTG 155 CsPP2C21 GTTTCTGAGGCTGCAAAG ATGGGAGTTCACAACTTGAC 198 CsPP2C22 ACCGTCAAACTTTAACATTC GGTAGGAGGAGATTAGGA 189 CsPP2C23 CGGTTCCTCTGTCCAACG ACGGCTTTAAGTAATTGTCTCC 153 CsPP2C24 GGGTCATAAACTGGAACG TTCATTTGATACAACATCCC 182 CsPP2C25 GGGCTATGGGATGTTATG TAGGAGCTTTCAGGTCAA 199 CsPP2C26 GACTGTGGGAGCATCTTA AATGTCATCGTGGAAGTG 182 CsPP2C27 ATCTTCAACGCCAAACAG GAGCCACTTCCTAGCTCATT 199 CsPP2C28 GCCAAATGTTAATAACTACGAC ATGCCAAGTCTAGCAAAGCT 180 CsPP2C29 CGAACCGCTAGTGGTGAA CGCCAGCAGCCTTCTTAT 169 CsPP2C30 GGTTATGCCCATCTGTTT CTCCATCTCCTATCCCTA 170 CsPP2C31 TCACGGTTGTTACCTTAT ATTGGTTCGCTCGTCGTA 194 CsPP2C32 AAAAGAAGGAGCTGATACGG CACCAATAACGCCCAAAT 152 CsPP2C33 ATGTATAGCCACGCAGTC AAGATCAAAGTTCCCAGT 160 CsPP2C34 AAGAAGTGGTGGAGATTG AAGCCTTGCTCTTCATAG 184 CsPP2C35 GCCCAGGCATTTGCTACA TGCGGAGATTCTTCTTAGTT 190 CsPP2C36 GGCTCTGGGAACACCTAA ATGTCATCGTGGAAATGC 181 CsPP2C37 GATGTTTCTTATCGCCGCCTAA TTGCCGCCTCAACCACCT 154 CsPP2C38 AACGAATTTGCCTGTGAA GAAACGATGGCGCTTGAC 200 CsPP2C39 ATGCCTATCTTGACTGCC TTCTTGCTGCTTCTTGGA 193 CsPP2C40 AAACTGTGCTTCCCTCCC AGGTTTCCCATAACTTCG 200 CsPP2C41 CCGTAGGGTTAGTTCTGA CAGCGAAGCAACATAGAA 162 CsPP2C42 TGAAGAGGGTTCGGCTAT GGCTTTGATGTGGGTGAG 159 CsPP2C43 GTGCTGTACCGGATGTTA CACTACGTGGCCGCTTTT 180 CsPP2C44 GCAAGGGCATTTGGTGAC CAAGTGCTTTGCTGCTGA 192 CsPP2C45 CAAGCTGGACGACATCAC AAGACAGGCTAGGCAGGA 166 CsPP2C46 AGGAGCACAATGATCCAG TCAAACCCTCAGCAGAAA 171 CsPP2C47 TTTGCCTGGCTTCAATAT ATGGGTATGCACGAATGT 189 Actin CCCCATCGTTACCGTCCAG、 CGCCTTGCCAGTTGAATATCC The 10 µL qRT-PCR reaction mixture consisted of the following components: 5 µL of 2× SYBR Green Premix (Baoguang Biotechnology Research (Chongqing) Co., Ltd.), 0.25 µL of forward primer (10 µmol·L⁻¹), 0.25 µL of reverse primer (10 µmol·L⁻¹), 3 µL of diluted cDNA template, and 1.5 µL of nuclease-free water. The thermal cycling protocol was as follows: initial denaturation at 94°C for 20 seconds; followed by 40 cycles of denaturation at 94°C for 10 seconds and annealing/extension at 55°C for 10 seconds. A melt curve analysis was performed at the end of each run to confirm amplification specificity. Each sample-analysis combination was performed with three technical replicates. The relative expression levels of the target genes were calculated using the comparative 2^(–ΔΔCT) method. The resulting expression data were subjected to statistical analysis, and a heatmap was generated using MeV software to visualize the expression patterns across different treatments and time points. Declarations Author information Corresponding Authors Rangjin Xie- Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China Tingshan Yao- Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China Authors Yanfang Yang - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China Wenhua Sun - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China Zihan Wang - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China Qiao Liu - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China Author contributions Yanfang Yang: Design experiments, Investigation, Analyze the data,Write manuscripts. Wenhua Sun: Investigation, Analyze the data. Zihan Wang: Investigation, Analyze the data. Qiao Liu: Investigation, Analyze the data. Rangjin Xie: Provide ideas, Design experiments, Writing-review & editing. Tingshan Yao: Provide ideas, Design experiments, Writing-review & editing. Funding This work was supported by the National Natural Science Foundation of China (U24A20511), and the National Key R&D Program of China (2021YFD1400800). Data availability Data will be made available on request. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. References Pandey P, Irulappan V, Bagavathiannan MV, Senthil-Kumar M. Impact of Combined Abiotic and Biotic Stresses on Plant Growth and Avenues for Crop Improvement by Exploiting Physio-morphological Traits. 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Xu K, Zhou M, Guo Y, et al. Genome-Wide Analysis of Maize ECT Family Members and Their Expressions in Response to Abiotic Stresses. J Nuclear Agricultural Sci. 2024;38(06):1035–47. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8649733","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":582099069,"identity":"e2b45e8a-3571-4517-ab34-aa1a2588d86f","order_by":0,"name":"Yanfang Yang","email":"","orcid":"","institution":"Southwest University","correspondingAuthor":false,"prefix":"","firstName":"Yanfang","middleName":"","lastName":"Yang","suffix":""},{"id":582099071,"identity":"52358671-32b2-4016-8828-5524b1498c0e","order_by":1,"name":"Wenhua Sun","email":"","orcid":"","institution":"Southwest 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13:44:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8649733/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8649733/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101752146,"identity":"4cc8d14d-7340-44e9-9499-cd0eb5700092","added_by":"auto","created_at":"2026-02-03 10:25:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":61111,"visible":true,"origin":"","legend":"\u003cp\u003eThe distribution map of CsPP2Cs on chromosomes.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/8170b57f571a8b9b914e55ef.png"},{"id":101491747,"identity":"e14e1b81-cb7d-4835-9c03-e203f270a6ec","added_by":"auto","created_at":"2026-01-30 10:39:20","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":315269,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic analysis of PP2Cs from Rice, \u003cem\u003eArabidopsis\u003c/em\u003e and Citrus.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/547a0a01fde51108896666b6.png"},{"id":101751750,"identity":"567d3e44-08c1-4ee7-9d80-46d10b8a36a7","added_by":"auto","created_at":"2026-02-03 10:23:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":147738,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analysis of \u003cem\u003eCsPP2Cs\u003c/em\u003ebetween \u003cem\u003eA.thaliana\u003c/em\u003e, \u003cem\u003eO.sativa\u003c/em\u003e and \u003cem\u003eC.clementina. \u003c/em\u003eBackground gray lines represent collinear blocks between \u003cem\u003eC.clementina\u003c/em\u003e and other plant genomes, whereas red lines emphasize \u003cem\u003eCsPP2C\u003c/em\u003e homologous gene pairs.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/050f5f91c9572770e19d8abb.png"},{"id":101491748,"identity":"bc1e6fb3-773a-443d-a347-5803f7aca7ef","added_by":"auto","created_at":"2026-01-30 10:39:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":118087,"visible":true,"origin":"","legend":"\u003cp\u003eGene structure of CsPP2Cs members\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/9bbec9d70523ce5ae2972c03.png"},{"id":101491744,"identity":"1c1af904-7cd1-42c2-bb01-c92692394c6a","added_by":"auto","created_at":"2026-01-30 10:39:20","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":117234,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of CsPP2Cs conservative sequences\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/3f8f73a34479a3fded64ddf1.png"},{"id":101491749,"identity":"4e5bb4c3-8ce9-4471-ad1a-dec61bf41a27","added_by":"auto","created_at":"2026-01-30 10:39:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":314169,"visible":true,"origin":"","legend":"\u003cp\u003eCsPP2C cis-acting regulatory elements in the promoters of CsPP2C genes prediction diagram.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/4f52aa88d31a2205810890be.png"},{"id":101491745,"identity":"0d58e0cd-a8fd-4519-998b-e61d319ed1cf","added_by":"auto","created_at":"2026-01-30 10:39:20","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":109264,"visible":true,"origin":"","legend":"\u003cp\u003eExpression profiles of CsPP2C genes under various abiotic stress and hormone treatments.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/931b218ab35aff65628cefef.png"},{"id":104400829,"identity":"7caa0610-de51-462a-ade9-de970a988b2a","added_by":"auto","created_at":"2026-03-11 12:11:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2290799,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8649733/v1/93f86448-4325-43d7-9b32-cd356e7cc367.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Whole-genome Identification of the PP2C Gene Family in citrus and its response to Abiotic Stresses","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eCitrus represents a vital agricultural resource around the world and holds significant economic value. With the continuous development of citrus industry, increasing attention is being paid to fruit yield and quality. However, due to climate issues, citrus plants are frequently threatened by multiple abiotic stresses, which usually results in a remarkbly decrease in yield and quality, and a significant economic losses[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. To cope with adverse environmental conditions[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], plants have evolved sophisticated mechanisms to perceive and adapt to stresses through various physiological and biochemical provesses such as photosynthesis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], glucose metabolism[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], and cell growth[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], many of which involve reversible protein phosphorylation[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Protein phosphorylation and dephosphorylation, catalyzed by protein kinases and phosphatases, respectively, regulate nearly all aspects of cellular life, including the cell cycle, phytohormone signaling, and metabolic pathways[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. PP2C is a class of Mg\u003csup\u003e2+\u003c/sup\u003e- or Mn\u003csup\u003e2+\u003c/sup\u003e-dependent monomeric enzymes, belonging to the serine/threonine protein phosphatases subfamily[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In guard cells, membrane-localized PP2Cs specifically mediate the dephosphorylation of plasma membrane (PM) H⁺-ATPase ; the functional activity of this enzyme is tightly controlled by the phosphorylation status of multiple residues in its C-terminal domain[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Among these phosphorylatable sites, the penultimate threonine (Thr) residue has remained a major focus of research. Phosphorylation of this specific Thr residue activates PM H⁺-ATPase by disrupting the autoinhibitory interaction of its C-terminal domain, thereby participating in key physiological processes including light-induced stomatal opening and auxin-driven cell expansion[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, the isoforms belonging to protein phosphatase 2C (PP2C) clade D (PP2C.Ds) play a role in facilitating the dephosphorylation of PM H⁺-ATPase. This dephosphorylation process is fundamental to auxin - induced hypocotyl elongation and cell expansion events[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. PP2Cs are widely distributed across various taxa, including archaea[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], bacteria[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], fungi[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], plants[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and animals[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo date, numerous PP2C genes have been identified in various plant species including \u003cem\u003eArabidopsis\u003c/em\u003e, rice[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], paulownia[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], strawberry[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], wheat[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] and hemp[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. For instance, 80 \u003cem\u003ePP2C\u003c/em\u003e members have been reported in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], 194 in Oat (\u003cem\u003eAsaPP2C\u003c/em\u003e)[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], 87 in banana[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], 92 in tomato[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and 56 in cucumber[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Based on protein sequence aimilarity, PP2Cs can be classfied into multiple subfamilies. In \u003cem\u003eArabidopsis\u003c/em\u003e, the PP2C gene family members of can be divided into 12 subfamilies (A-L)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], each with distinct functional characteristics. Briefly, subfamily A members are involved in abscisic acid (ABA) signaling and act as negative regulators in ABA responses[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Several subfamily B proteins participate in mitogen-activated protein kinase (MAPK) signaling pathways, also serving as negative regulators[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]; Proteins in subfamily C are associated with floral organ development[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]; while subfamily D is implicated in saline-alkali stress response[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Subfamily E regulates stomatal signaling to control transiration[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], and subfamily F is involved in the induction of bacterial stress response[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePP2Cs widely participate in plant growth and development, wound response, and adaptation to biotic and abiotic stresses. Some studies have revealed that the ABA-mediated reduction in PP2C activity enhances protein kinase phosphorylation, thereby influencing plant development and stresse adaptation[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. For example, in \u003cem\u003ePoncirus trifoliate\u003c/em\u003e, 10 \u003cem\u003ePP2C\u003c/em\u003e genes exhibited notable alterations in their expression levels when subjected to low temperature, high temperature or drought stresses, and \u003cem\u003ePtrPP2C29\u003c/em\u003e was especially sensitive[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In \u003cem\u003eSaccharum spontaneum\u003c/em\u003e, 37 out of 89 \u003cem\u003ePP2Cs\u003c/em\u003e displayed distinct expression patterns under drought conditions, underscoring their importance in drought tolerance[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Among the 172 \u003cem\u003ePP2C\u003c/em\u003e members in peanut (\u003cem\u003eArachis hypogaea\u003c/em\u003e L.), \u003cem\u003eAhPP2C45\u003c/em\u003e and \u003cem\u003eAhPP2C134\u003c/em\u003e were markedly upregulated under salt stress[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Similarly, in \u003cem\u003eBroussonetia papyrifera\u003c/em\u003e, 4 out of 18 \u003cem\u003eBpPP2Cs\u003c/em\u003e genes were induced by low temperature (4\u0026deg;C), and exhibited high phosphorylation levels, indicating a role in cold stress reponse[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we obtained 47 PP2C gene members in Citrus. Bioinformatics analysis was conducted on these 47 PP2C members, including physical and chemical property, phylogenetic relationship, chromosome localization, gene structure, conserved domain, and cis-acting element prediction. In addition, the gene expression profiles under abiotic stress and hormone treatments were investigated. This study sorted out the molecular traits and stress-regulation ability of citrus PP2C members via whole-genome identification and stress tests, making the plant PP2C family research system more complete, and it also offers a base for understanding CsPP2C's roles in citrus stress responses.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Identification of \u003cem\u003ePP2Cs\u003c/em\u003e genes in citrus\u003c/h2\u003e \u003cp\u003eA total of 47 \u003cem\u003ePP2C\u003c/em\u003e members have been identified in the citrus and citrus-relative genomes, one unique to \u003cem\u003ePoncirus trifoliata\u003c/em\u003e. As shown in Fig.\u0026nbsp;1, the 47 \u003cem\u003eCsPP2C\u003c/em\u003e genes are unevenly distributed on 9 scaffolds with the detailed distribution as follows: 7 genes on scaffold_1, 3 genes on scaffold_2, 7 genes on scaffold_3, 3 genes on scaffold_4, 6 gene on scaffold_5, 4 genes on scaffold_6,5 genes on scaffold_7, 5 genes on scaffold_8, and 7 genes on scaffold_9. Scaffold_1, scaffold_3 and scaffold_9 contain the most \u003cem\u003eCsPP2C\u003c/em\u003e genes, while scaffold_4 contains the least. Most \u003cem\u003eCsPP2Cs\u003c/em\u003e are located at ends of the chromosomes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Phylogenetic analysis\u003c/h2\u003e \u003cp\u003eThe NJ tree showed the evolutionary relationship of PP2C gene families among citrus, \u003cem\u003eArabidopsis\u003c/em\u003e and rice (Fig.\u0026nbsp;2). The NJ tree divided the PP2C family members into 12 groups (Group I\u0026thinsp;~\u0026thinsp;XII), each group containing at least two PP2C members from different species; Some groups (such as Group VII and X) had a larger number of members, while others (such as Group I) were comprosed of a smaller number of members; The PP2C members of the same species were dispersed across multiple group. These results indicated that PP2C gene family is highly conserved in plants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Synteny analysis\u003c/h2\u003e \u003cp\u003eThe collinearity analysis between \u003cem\u003eC.clementina\u003c/em\u003e and two representative species including \u003cem\u003eA.thaliana\u003c/em\u003e and \u003cem\u003eO.sativa\u003c/em\u003e showed that 4 \u003cem\u003eCsPP2Cs\u003c/em\u003e were homologous to \u003cem\u003eOsPP2Cs\u003c/em\u003e found in \u003cem\u003eO.sativa\u003c/em\u003e, 7 \u003cem\u003eCsPP2Cs\u003c/em\u003e are homologous to \u003cem\u003eAtPP2Cs\u003c/em\u003e found in \u003cem\u003eA.thaliana\u003c/em\u003e (Fig.\u0026nbsp;3). In these \u003cem\u003eCsPP2Cs\u003c/em\u003e, gene demonstrated one-to-many homozygosity. For example, \u003cem\u003eC.clementina\u003c/em\u003e Chr1 corresponds to both \u003cem\u003eO.sativa\u003c/em\u003e Chr3 and Chr7, and \u003cem\u003eC.clementina\u003c/em\u003e Chr2 corresponds to both \u003cem\u003eA.thaliana\u003c/em\u003e Chr1 and Chr3.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Physical and chemical properties\u003c/h2\u003e \u003cp\u003eThe physicochemical properties of 47 CsPP2Cs were analyzed (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of amino acid ranged from 184 to 1101 amino acid residues, corresponding to relative molecular weights of 20.03 to 124.36 kDa, with an average molecular weight of approximately 47.26 kDa. The theoretical isoelectric points (pI) ranged from 4.76 to 9.41 and most of CsPP2Cs were classified as acidic proteins, due to their pI values below 7. Based on the instability index, most CsPP2C protein were predicted to be unstable. The aliphatic index ranged from 65.14 to 95.53, averaging about 83.07. Among them, CsPP2C47 had the smallest aliphatic index, and CsPP2C35 had the largest. The grand average of hydropathicity values ranged from \u0026minus;\u0026thinsp;0.795 to 0.058. With the exception of CsPP2C21, all other CsPP2C proteins had a grand average of hydropathicity below 0, indicating that most of them were hydrophilic proteins.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePhysicochemical property of PP2C proteins\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocus ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNumber of amino acid\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMolecularweight\u003c/p\u003e \u003cp\u003e(g/mol)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003epI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eInstability index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eAliphatic index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGrand average of hydropathicity\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10001476\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e384\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41661.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e81.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.246\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10005100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43771.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e51.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.092\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10018806\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e879\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e97646.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.554\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e 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align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46966.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e78.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.124\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10005158\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e383\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42358.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e86.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.248\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10020547\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41558.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e85.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.217\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10004613\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e584\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64391.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e45.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e91.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.191\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10009047\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e298\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32624.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e78.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.432\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10018605\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e124357.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e80.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.381\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10001891\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e316\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e34236.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e34.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e80.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.279\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10027924\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e704\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e78971.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e77.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.514\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10031727\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43948.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e76.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.391\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10022462\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e20031.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e82.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10011223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e679\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75471.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e36.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e75.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.543\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10025810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42217.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e76.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.400\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10028649\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e382\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42561.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e91.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.259\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e 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\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29650.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e9.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e76.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.457\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10028878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e312\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33856.41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e88.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e0.058\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10011931\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e390\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e43954.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e53.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.207\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10031682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e411\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44810.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e56.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e80.39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.367\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10005200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e372\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40744.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e81.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.403\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10027412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e261\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e29000.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e54.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e91.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.312\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10025781\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e397\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44283.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e87.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.283\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10001565\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40842.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e77.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.216\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10007997\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e523\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57186.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e78.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.364\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10011576\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e53862.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e75.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.438\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10007908\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e550\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e59725.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e46.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.111\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10009076\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e292\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31361.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e79.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.279\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10013751\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e57718.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e83.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.372\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10031798\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e386\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42841.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e39.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e90.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.237\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10015132\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e470\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e51912.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e44.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e68.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.497\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10020542\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e42761.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e95.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.060\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10028600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e397\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e44096.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e88.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.286\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10010157\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e329\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36032.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e34.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e92.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10023343\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e32406.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e6.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e48.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e89.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.092\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10005185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e375\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41865.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e91.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.226\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10001232\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46747.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e80.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.369\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10025314\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e544\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e58862.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e43.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e94.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10001248\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e427\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e45785.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e30.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e88.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.122\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10028495\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e429\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e46985.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e75.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.425\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10016165\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e284\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31368.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e34.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e88.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.312\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10000910\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e511\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e54983.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e7.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e83.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.099\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCiclev10008648\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e41376.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e5.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e58.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e81.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.234\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePtrif.0002s2428\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e259\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e28567.47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e49.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e65.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.795\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Gene structure and conserved domains\u003c/h2\u003e \u003cp\u003eTo determine the quantity and positions of introns and exons, gene structure analysis was performed by comparing the full-length cDNA sequences with their corresponding genomic DNA sequences. As showed in Fig.\u0026nbsp;4, the number of exon varied considerably ranged from 1 (\u003cem\u003eCsPP2C8\u003c/em\u003e) to 20 (\u003cem\u003eCsPP2C11\u003c/em\u003e), revealing diverse intron-exon architectures. Among the 47 \u003cem\u003eCsPP2Cs\u003c/em\u003e, approximately 78.7% (37 genes) contained 3 to 6 exons, indicating that most family members possess a moderate number of exons. \u003cem\u003eCsPP2C9\u003c/em\u003e, \u003cem\u003eCsPP2C11\u003c/em\u003e, \u003cem\u003eCsPP2C21\u003c/em\u003e, \u003cem\u003eCsPP2C35\u003c/em\u003e had a considerably higher number of exons than most of the other genes in the family. It was worthy to note that \u003cem\u003eCsPP2C8\u003c/em\u003e had only a single exon (intronless genes), whereas \u003cem\u003eCsPP2C11\u003c/em\u003e contained the highest number of exons.\u003c/p\u003e \u003cp\u003eThe conserved motifs in 47 CsPP2Cs were analysized, which revealed that all 47 CsPP2C proteins contained conserved motifs with high sequence similarity (Fig.\u0026nbsp;5). The number of motifs varied considerably among different CsPP2C proteins, with the majority harboring either 6 or 7 motifs. Motif 3 and motif 1 were present in nearly all CsPP2C proteins, and motif 1, motif 2, motif 4 and motif 5 were also widely distributed. Notably, the positions of motif 1 and motif 3 were highly conserved. 45 members (95.7%) contained motif 1 and 44 members (93.6%) contained motif 3. In contrast, some motifs (such us motif 8) exhibited restricted distribution patterns, occurring only in specific subsets of CsPP2C proteins. The substantial variation in motif composition and copy number suggested significant functional diversification within the CsPP2C family.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. \u003cem\u003eCis\u003c/em\u003e-acting elements in the promoters of \u003cem\u003eCsPP2Cs\u003c/em\u003e\u003c/h2\u003e \u003cp\u003ePutative \u003cem\u003ecis\u003c/em\u003e-acting regulatory elements in the promoter of \u003cem\u003eCsPP2C\u003c/em\u003e genes were predicted using PlantCARE (Fig.\u0026nbsp;6). These \u003cem\u003ecis\u003c/em\u003e-acting elements were classified into three primary categories: hormone response, stress response, and plant growth/photoregulatory elements. Hormone-responsive elements included the CGTCA-motif and TGACG-motif (involved in MeJA-responsiveness), ABRE (abscisic acid responsiveness), auxin-responsive element, the TCA-element (salicylic acid responsiveness), and gibberellin-responsive element (P-box and GARE-motif). Stress-responsive elements encompassed LTR (low-temperature responsiveness), MBS (drought-inducibility), TC-rich repeats (defense and stress responsiveness), and the GC-motif (involved in anaerobic induction). Photoregulatory and circadian-related elements included the G-box, ACE, GT1-motif, AE-box, TCT-motif (all involved in light responsiveness),and the circadian control element.\u003c/p\u003e \u003cp\u003eNotably, a significant abundance of ABA-responsive elements (ABRE) was detected within the promoter regions of the majority of \u003cem\u003eCsPP2C\u003c/em\u003e genes, indicating the extensive participation of this gene family in the ABA signaling pathway.. MeJA-responsive elements (CGTCA-motif and TGACG-motif) were also widely distributed among members, such as \u003cem\u003eCsPP2C1\u003c/em\u003e, \u003cem\u003eCsPP2C15\u003c/em\u003e, \u003cem\u003eCsPP2C20\u003c/em\u003e, \u003cem\u003eCsPP2C29\u003c/em\u003e, and \u003cem\u003eCsPP2C38\u003c/em\u003e, indicating their potential roles in JA-mediated stress responses. Furthermore, a significant number of elements related to light responsiveness (e.g., G-box, ACE) were detected in the promoters of \u003cem\u003eCsPP2C5\u003c/em\u003e, \u003cem\u003eCsPP2C13\u003c/em\u003e, \u003cem\u003eCsPP2C27\u003c/em\u003e, and \u003cem\u003eCsPP2C44\u003c/em\u003e, implying possible regulation by light signals. Elements involved in low-temperature (LTR) and drought (MBS) responsiveness were identified in several members, including \u003cem\u003eCsPP2C3\u003c/em\u003e, \u003cem\u003eCsPP2C9\u003c/em\u003e, \u003cem\u003eCsPP2C24\u003c/em\u003e, and \u003cem\u003eCsPP2C41\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eCollectively, these results suggested that most \u003cem\u003eCsPP2C\u003c/em\u003es were involved in diverse biological processes, responding to various phytohormone (e.g., ABA, MeJA,SA, Auxin, GA) and environmental stimuli (e.g., low temperature, drought, light, and anaerobic stress).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Expression analysis\u003c/h2\u003e \u003cp\u003eExpression profiling of \u003cem\u003eCsPP2C\u003c/em\u003e genes under various treatments (including NaCl, SA, ABA, low temperature, ETH, and PEG) revealed distinct response patterns among members (Fig.\u0026nbsp;7). Genes including \u003cem\u003eCsPP2C1, CsPP2C5, CsPP2C9, CsPP2C13, CsPP2C15, CsPP2C17, CsPP2C21, CsPP2C25, CsPP2C29, CsPP2C33, CsPP2C37, CsPP2C41, and CsPP2C45\u003c/em\u003e showed low or negligible expression changes across most conditions, suggesting limited involvement in the tested stress or hormone responses. In contrast, a subset of genes exhibited notably treatment-specific expression dynamics. Under NaCl treatment, \u003cem\u003eCsPP2C3\u003c/em\u003e expression increased sharply at the beginning of treatment (1h) but returned to baseline levels thereafter; \u003cem\u003eCsPP2C7\u003c/em\u003e was strongly induced by ABA, peaking at 6h, and showed continuously increasing expression under low temperature; \u003cem\u003eCsPP2C11\u003c/em\u003e expression was suppressed by ETH treatment for 24h. \u003cem\u003eCsPP2C19\u003c/em\u003e was up-regulated under both NaCl and low temperature but down-regulated by ETH; \u003cem\u003eCsPP2C27\u003c/em\u003e was steadily up-regulated during low temperature exposure but significantly down regulated by ETH; \u003cem\u003eCsPP2C31\u003c/em\u003e was significantly induced under ABA and low temperature treatments, thouth it exhibited no clear trend under SA; \u003cem\u003eCsPP2C35\u003c/em\u003e expression increased under multiple treatments (such as SA, low temperature, NaCl), particularly under low temperature. \u003cem\u003eCsPP2C39\u003c/em\u003e was induced by low temperature and showed transient up-regulation followed by a decline under NaCl and SA; \u003cem\u003eCsPP2C43\u003c/em\u003e expression rose then fell under ABA, SA and low temperature conditions; \u003cem\u003eCsPP2C47\u003c/em\u003e was down-regulated in response to SA and PEG.\u003c/p\u003e \u003cp\u003eThese results indicated that \u003cem\u003eCsPP2C3, CsPP2C7, CsPP2C11, CsPP2C19, CsPP2C23, CsPP2C27, CsPP2C31, CsPP2C35, CsPP2C39, CsPP2C43\u003c/em\u003e, and \u003cem\u003eCsPP2C47\u003c/em\u003e were likely involved in biological processes related to hormones (e.g., ABA, ETH) and abiotic stresses (e.g., low temperature, PEG, and NaCl stress), thereby highlighting their potential roles in citrus growth, development, and stress adaptation.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eIn this study, a comprehensive genome-wide analysis obtained a total of 47 PP2C genes across three citrus species: \u003cem\u003eCitrus clementina\u003c/em\u003e (46), \u003cem\u003eCitrus sinensis\u003c/em\u003e (46), and \u003cem\u003ePoncirus trifoliate\u003c/em\u003e (46). After removing redundancies, 47 unique members (\u003cem\u003eCsPP2C1\u003c/em\u003e\u0026ndash;\u003cem\u003eCsPP2C47\u003c/em\u003e) were retained for further characterization. The number of PP2C genes in citrus is relatively small compared to \u003cem\u003eA.thaliana\u003c/em\u003e (80)[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], rice (87)[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and oat (194)[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This variation may result from differences in genome size, lineage-specific evolutionary events, or gene family expansion/contraction.\u003c/p\u003e \u003cp\u003eMembers clustered within the same subgroup exhibit shorter branch lengths, indicative of closer genetic affinities. By contrast, longer branch lengths between distinct subgroups reflect substantial genetic divergence among them. The PP2C members from \u003cem\u003eOryza sativa\u003c/em\u003e (\u003cem\u003eOsPP2C\u003c/em\u003e), \u003cem\u003eCitrus\u003c/em\u003e (\u003cem\u003eCsPP2C\u003c/em\u003e), and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eAtPP2C\u003c/em\u003e) were consistently categorized into 12 subgroups. This result suggests that the PP2C family had already established a conserved classification framework prior to the speciation and differentiation of these three plant species. Each subgroup encompasses members derived from three plant species, providing evidence of the presence of cross-species homologous genes within the PP2C family. Specifically, PP2C homologs that possess conserved sequence characteristics and likely similar biological functions are preserved across these distinct taxonomic groups. In addition, PP2C members from the same species are scattered across multiple subgroups; this distribution pattern implies that the PP2C family has undergone extensive gene duplication events accompanied by functional divergence during the evolutionary process of individual species, and that family expansion is primarily mediated via a mechanism of dispersed clustering.\u003c/p\u003e \u003cp\u003eTo verify the evolutionary relationships inferred from the phylogenetic tree, collinearity analysis was performed. The results revealed a notable collinearity between the PP2C genes in citrus and those in \u003cem\u003eO.sativa\u003c/em\u003e and \u003cem\u003eA.thaliana\u003c/em\u003e, thereby corroborating the evolutionary homology among the PP2C families of these three species. Collinearity was predominantly manifested in a one-to-many pattern, implying that lineage-specific duplication events occurred in the PP2C gene family after speciation. This evolutionary process would have resulted in a single ancestral PP2C gene corresponding to multiple homologous genes in derived species.\u003c/p\u003e \u003cp\u003eTo further contextualize the exon-intron architectural characteristics of CsPP2Cs (Fig.\u0026nbsp;1), we compared their structural profiles with those of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AtPP2Cs) and \u003cem\u003eOryza sativa\u003c/em\u003e (OsPP2Cs). Our findings unveiled a considerable degree of diversity in the gene structures of CsPP2Cs, with exon numbers spanning from 1 to 20. This range surpassed that observed in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AtPP2Cs), where exons ranged from 0 to 13[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and in \u003cem\u003eOryza sativa\u003c/em\u003e (OsPP2Cs), where exons ranged from 0 to 18[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Such an expanded structural variation suggested that the citrus PP2C family exhibited greater evolutionary plasticity compared to these two model species.\u003c/p\u003e \u003cp\u003eNotably, one CsPP2C member (\u003cem\u003eCsPP2C8\u003c/em\u003e) was identified as intronless (Fig.\u0026nbsp;1)\u0026mdash;a feature also reported in a subset of AtPP2C-A subfamily members (e.g., \u003cem\u003eABI1\u003c/em\u003e, \u003cem\u003eABI2\u003c/em\u003e), but rare in OsPP2Cs. Intronless genes are widely recognized to enable rapid transcriptional activation in stress responses, suggesting that \u003cem\u003eCsPP2C8\u003c/em\u003e may function as fast-acting regulators in abiotic stress signaling[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApproximately 78.7% of CsPP2Cs contained 3\u0026ndash;6 exons (Fig.\u0026nbsp;1), a proportion consistent with that of AtPP2Cs but higher than that of OsPP2Cs. This enrichment in moderate exon range was hypothesized to hypothesized to represent an evolutionary trade-off between functional versatility and transcriptional efficiency[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], potentially explaining its conservation of this exon range in eudicots (e.g., citrus and \u003cem\u003eArabidopsis\u003c/em\u003e) and its relative depletion in monocots such as rice. The broader exon count range of CsPP2Cs likely reflected lineage-specific gene duplication and intron gain/loss events during citrus evolution, processes typically associated with functional divergence. Overall, the structural diversity of CsPP2Cs reflected conservative functional roles and lineage specific specialization compared to AtPP2Cs and OsPP2Cs[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePhysicochemical characterization revealed that approximately 78.7% CsPP2C proteins were acidic, with isoelectric points (pI) ranging from 4.76 to 6.97. All CsPP2C members exhibited inherent instability (instability index\u0026thinsp;\u0026gt;\u0026thinsp;40) and hydrophilicity (grand average of hydropathicity, GRAVY\u0026thinsp;\u0026lt;\u0026thinsp;0), These characteristics were also evolutionarily conserved in \u003cem\u003eArabodopsis\u003c/em\u003e AtPP2Cs and rice OsPP2Cs[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In terms of molecular weight, CsPP2Cs displayed a broader range (20.03-124.36 kDa) compared to OsPP2Cs (35\u0026ndash;62 kDa) and was comparable to the size diversity of AtPP2Cs. Additionally, CsPP2Cs showed significant changes in the fatty index (65.14\u0026ndash;95.53) which is related to protein thermal stability[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Specifically, CsPP2C35 had the highest aliphatic index (95.53), indicating a greater degree of thermal stability, while CsPP2C47 presented the lowest lipid index (65.14), corresponding to the lowest level of thermal stability among the tested proteins.\u003c/p\u003e \u003cp\u003eTo investigate the functional divergence of \u003cem\u003eCsPP2C\u003c/em\u003e genes in response to stresses, their expression profiles were analyzed under multiple abiotic stresses (NaCl, PEG, low temperature) and hormone treatments (ABA, ETH, SA). The results showed that a suite of \u003cem\u003eCsPP2C\u003c/em\u003e genes, specifically \u003cem\u003eCsPP2C3\u003c/em\u003e, \u003cem\u003eCsPP2C7\u003c/em\u003e, \u003cem\u003eCsPP2C19\u003c/em\u003e, \u003cem\u003eCsPP2C23\u003c/em\u003e, \u003cem\u003eCsPP2C27\u003c/em\u003e, \u003cem\u003eCsPP2C31\u003c/em\u003e, \u003cem\u003eCsPP2C35\u003c/em\u003e, \u003cem\u003eCsPP2C39\u003c/em\u003e, \u003cem\u003eCsPP2C43\u003c/em\u003e, and \u003cem\u003eCsPP2C47\u003c/em\u003e, exhibited responsiveness to a range of stress conditions and hormone treatments, including NaCl, PEG-simulated drought, low temperature, as well as abscisic acid (ABA), ethylene (ETH), and salicylic acid (SA). Notably, these genes displayed distinct temporal expression patterns, suggesting potential functional specialization in their roles during stress responses.. For instance, \u003cem\u003eCsPP2C7\u003c/em\u003e was strongly induced by both ABA and low temperature\u0026mdash;consistent with the expression behavior of its \u003cem\u003eArabidopsis\u003c/em\u003e homologs (e.g., \u003cem\u003eAHG3/AtPP2CA\u003c/em\u003e of Arabidopsis plays a major role among \u003cem\u003ePP2Cs\u003c/em\u003e in the ABA response in seeds[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. \u003cem\u003eCsPP2C27\u003c/em\u003e showed a sustained up-regulation under cold stress but was suppressed by ETH, suggesting a specific regulatory role in low-temperaure adaptation. Such stress-responsive expression patterns of \u003cem\u003eCsPP2Cs\u003c/em\u003e aligned with observations in other plant species: In \u003cem\u003eBrachypodium distachyon\u003c/em\u003e, nearly all \u003cem\u003eBdPP2C\u003c/em\u003e genes were up-regulated under abiotic stresses such as cold, heat, PEG and NaCl treatments[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]; in maize, most \u003cem\u003eZmPP2C-A\u003c/em\u003e clade genes (e.g., \u003cem\u003eZmPP2C-A2\u003c/em\u003e, \u003cem\u003eZmPP2C-A6\u003c/em\u003e) were dramatically induced by drought, salt, and ABA, and further functional analysis confirmed their negative regulation of drought responses[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Similarly, \u003cem\u003eBnPP2C1\u003c/em\u003e, \u003cem\u003eBnPP2C26\u003c/em\u003e, and \u003cem\u003eBnPP2C27\u003c/em\u003e in ramie showed strong responses to drought, high salt, and ABA[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], while \u003cem\u003eZoPP2Cs\u003c/em\u003e in ginger exhibited diverse expression dynamics during maturation and under drought/salt stresses[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. These cross-species similarities indicated the conserved functions of PP2Cs in orchestrating responses to abiotic stress and modulating hormone signaling pathways.\u003c/p\u003e \u003cp\u003ePromoter analysis of \u003cem\u003eCsPP2C\u003c/em\u003e genes uncovered a wealth of \u003cem\u003ecis\u003c/em\u003e-acting elements intricately linked to stress responses and hormone signaling, such as ABRE, MBS, LTR, and MeJA-responsive motifs, which suggested the involvement of CsPP2C genes in a diverse array of signaling networks.. ABRE elements were extensively present in the promoters of \u003cem\u003eCsPP2C\u003c/em\u003e genes (e.g., \u003cem\u003eCsPP2C1\u003c/em\u003e, \u003cem\u003eCsPP2C19\u003c/em\u003e, \u003cem\u003eCsPP2C43\u003c/em\u003e), consistent with the well-documented role of \u003cem\u003ePP2Cs\u003c/em\u003e in ABA signaling[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Additionally, this was further validated by the differential expression patterns of these genes under ABA treatment (Supplementary Table\u0026nbsp;1). MBS motifs were detected in 27 \u003cem\u003eCsPP2C\u003c/em\u003e members, and LTR elements in 20, implying their roles in PEG and cold stress responses. For example, MBS-containing \u003cem\u003eCsPP2C28\u003c/em\u003e was upregulated under PEG-induced drought, while LTR-harboring \u003cem\u003eCsPP2C11\u003c/em\u003e exhibited marked induction under cold stress. MeJA-response elements were found in 36 \u003cem\u003eCsPP2Cs\u003c/em\u003e, among which related genes such as \u003cem\u003eCsPP2C11\u003c/em\u003e and \u003cem\u003eCsPP2C44\u003c/em\u003e were consistently expressed at high levels under SA induction, indicating potential crosstalk between hormones. Taken together, \u003cem\u003ecis\u003c/em\u003e-element analysis combined with qPCR data demonstrated that \u003cem\u003eCsPP2Cs\u003c/em\u003e were transcriptionally modulated by multiple stresses and hormones, highlighting their functional versatility in citrus adaptive mechanisms.\u003c/p\u003e \u003cp\u003eIn summary, this study provides a genome-wide identification and functional characterization of the PP2C gene family in citrus. The results highlight the structural and functional diversity among \u003cem\u003eCsPP2Cs\u003c/em\u003e, their evolutionary relationships with known stress-responsive phosphatases, and their potential roles in hormone and stress signaling. Future work should prioritize functional validation of key candidates such as \u003cem\u003eCsPP2C7\u003c/em\u003e and \u003cem\u003eCsPP2C27\u003c/em\u003e through transgenic assays to confirm their regulatory roles in stress adaptation and facilitate molecular breeding of stress-resilient citrus varieties.\u003c/p\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this study, a total of 47 \u003cem\u003eCsPP2Cs\u003c/em\u003e were identified in citrus and citrus-relative species, which were classified into 12 subgroups. The results indicated that \u003cem\u003eCsPP2Cs\u003c/em\u003e were evolutionarily related to \u003cem\u003eArabidopsis\u003c/em\u003e and rice \u003cem\u003ePP2C\u003c/em\u003e genes, perhaps originated from the same ancestor. Additionally, CsPP2Cs displayed comparable motif patterns and exon/intron arrangement structures. Collinearity analysis further supported evolutionary relationships of PP2Cs between citrus, rice, and \u003cem\u003eArabidopsis\u003c/em\u003e, with one-to-many synteny patterns suggesting lineage-specific duplication post-speciation. This study also investigated the expression patterns of \u003cem\u003eCsPP2Cs\u003c/em\u003e in response to different abiotic stresses using qRT-PCR analyses, and found that \u003cem\u003eCsPP2Cs\u003c/em\u003e were involved in coping with salt, PEG, low temperature and hormone(NaCl、SA、ETH) stresses. A comprehensive analysis of the cis - acting elements within the promoter regions of \u003cem\u003eCsPP2C\u003c/em\u003e genes had unveiled their crucial roles in stress adaptation as well as in the regulation of citrus development and growth processes. In conclusion, our work may provide valuable insights into the molecular mechanisms of \u003cem\u003eCsPP2Cs\u003c/em\u003e involved in abiotic stresses and hormone-related biological processes.\u003c/p\u003e"},{"header":"5. Materials and Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e5.1. Identification and analysis of \u003cem\u003ePP2C\u003c/em\u003e gene\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e5.1.1. Sequence Acquisition\u003c/h2\u003e \u003cp\u003eGenomic sequences, transcript sequences, and peptide sequences of \u003cem\u003eCitrus clementina\u003c/em\u003e, \u003cem\u003eCitrus sinensis\u003c/em\u003e and \u003cem\u003ePoncirus trifoliata\u003c/em\u003e were download from the Phytozome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively. Additionally, PP2C amino acid sequences of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e were obtained from Phytozome database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) based on their ID numbers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e5.1.2. Sequence alignment and NJ tree construction\u003c/h2\u003e \u003cp\u003eMultiple sequence alignment were performed using clustalx software. After removing redundant sequences across the three varieties, 47 unique PP2C members were retained for subsequent analysis. Given that PP2C members exhibit a high degree of conservation between citrus and poncirus, the one that is unique to poncirus was designated with the same naming convention as those found in citrus in this study. A neighbor-joining (NJ) phylogenetic tree was built with the combined PP2C protein sequences from the 47 members, rice and \u003cem\u003eArabidopsis\u003c/em\u003e employing 1000 bootstrap replicates.Beautify the NJ tree using the ITOL website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://itol.embl.de/z\u003c/span\u003e\u003cspan address=\"https://itol.embl.de/z\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e5.1.3. Synthetic analysis\u003c/h2\u003e \u003cp\u003eThe chromosome location information of the CsPP2Cs in \u003cem\u003eCitrus clementina\u003c/em\u003e were obtained from Ensembl Plants (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/index.html\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and diagrammed using software TBtools-Ⅱ. CsPP2Cs duplication events were examined using MCScanX via TBtools-Ⅱ. One-Step MCScanX was employed to predict synteny between genes in \u003cem\u003eA.thaliana\u003c/em\u003e and those in \u003cem\u003eO.sativa\u003c/em\u003e and \u003cem\u003eC.clementina\u003c/em\u003e utilizing genome annotation and sequence files. The synteny was visualized using a dual synteny plot generated by MCScanX in the TBtools software.Beautify image results using AI software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e5.1.4. Physicochemical properties\u003c/h2\u003e \u003cp\u003eThe physicochemical properties of CsPP2Cs, including the number of amino acid, theoretical isoelectric point(PI), molecular weight, instability indext, grand average of hydropathicity(GRAVY), and aliphatic index, were predicted using ExPASy ProParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e5.1.5. Chromosome location analysis\u003c/h2\u003e \u003cp\u003eThe gene IDs, gene location and chromosome length information for the PP2C gene family members were obtained from Phytozome (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ) s. The chromosome distribution map was then generated using the MG2C online tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mg2c.iask.in/mg2c_v2.1/index.html\u003c/span\u003e\u003cspan address=\"http://mg2c.iask.in/mg2c_v2.1/index.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) after data curation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e5.1.6. Gene structure and Conserved Domain\u003c/h2\u003e \u003cp\u003eThe gene structure(exon-intron organization) of \u003cem\u003eCsPP2C\u003c/em\u003e genes was visualized using the GSDS v2.0 online server(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.gaolab.org/\u003c/span\u003e\u003cspan address=\"http://gsds.gaolab.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Conserved motif in the CsPP2C protein sequences were identified using the MEME suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default parameters.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e5.1.7. \u003cem\u003eCis\u003c/em\u003e-acting element prediction\u003c/h2\u003e \u003cp\u003eThe 2000 bp promoter sequences upstream of the transcription start sites of \u003cem\u003eCsPP2C\u003c/em\u003e genes were extrated from the phytozome database(\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e ). Putative \u003cem\u003ecis\u003c/em\u003e-acting regulatory elements within these promoter regions were predicted using the PlantCARE online website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e5.1.8.Expression analysis\u003c/h2\u003e \u003cp\u003eHealthy young leaves from navel orange were subjected to various abiotic stresses and phytohormones. First,put six fresh leaves into PEG (20%)[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e], SA (5 mmol\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e), ABA (100 \u0026micro;mol\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e)[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], ETH (500 mg\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e), and NaCl (60 mmol\u0026middot;L\u003csup\u003e-1\u003c/sup\u003e)[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]solutions, simmersed in the respective solutions for 10 minutes and taken out. After treatment, the leaves were blotted dry, transferred to a growth chamber maintained under constant temperature and humidity, and harvested at the designated time points (0, 6, 24 h). The harvested samples were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C until RNA extraction.\u003c/p\u003e \u003cp\u003eTotal RNA was extracted using the Plant RNA Prep Pure Kit (Aidlab Biotechnologies Co., Ltd.), strictly following the manufacturer's protocol. RNA concentration and purity were verified spectrophotometrically. First-strand cDNA was then synthesized from 1 \u0026micro;g of total RNA using the PrimeScript\u0026trade; RT Reagent Kit (Takara Bio Inc.), according to the manufacturer's instructions.\u003c/p\u003e \u003cp\u003eGene-specific primers for the PP2C gene family members were designed based on transcript sequences from the Phytozome database using Primer Premier software (primer sequences are listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). qRT-PCR was performed using a quantitative PCR system with the \u003cem\u003eCitrus Actin\u003c/em\u003e gene serving as an internal reference.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGene-specific primers sequences for the PP2C gene family\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePrimer name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward primer sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eReverse primer sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAmplify fragment length(pb)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCGGATAGAAATGCCACC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAACAACAGCAGCAGTAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e197\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTGGATTGGGTTTCACTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAAGCCTACCTAAATGTTTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e151\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGTTTCGGGTAGACTATGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATCGCCTTCTGGGACATT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e185\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAGCAATAAAGCGAGGAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAGAGGTATGCCAATGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATTGGCTGTGATGGAATC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGATCAAGGGAAGTGAAGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATAGCGTTGTATTCGTTTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTCGGAAGGATAGACTTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAAGAGGCGGTGGAGATT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTGAGGCGACGATGAAGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGAAGCAGCATCAAGGAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGAAAGTGTAACGGGAAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCTAGAACACTGCGAACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGTGGCATTATACCACTTGAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGCTTGCGTTTAATTGTTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAGGCACTTCTGTTCCACC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCTCGGTAGCAGAAAGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAAATCGGAGGTGAATAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGTGGTGTTCTTGCTGTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGCTTCTGCATCGGTTAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCTCAAACAGCCAAAGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGTGAAGTATTGATAGAGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGTAGTCATCAAAGGGTTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGCCTCTATCTATGGAAAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e176\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGAGGTAGGAACAATACAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGGTCCGTGCCCATCAAATA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCTTCCTTGCTGCATTCCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACGCCAGATTCTTCCCTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e184\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCTTTGGGATGTAATGAGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTTGAATCTTGGGAGGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e182\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGAGGCTGCAAGGAAGAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTTAGGGAGAAGATGTCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e195\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGAGCACGAGTTGAAGGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCAACCTCACAAGCCAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e182\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGCCAACATGGGTGAGTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGCTCTGAGCCTTTAGAGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e155\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTTTCTGAGGCTGCAAAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATGGGAGTTCACAACTTGAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eACCGTCAAACTTTAACATTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGTAGGAGGAGATTAGGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCGGTTCCTCTGTCCAACG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eACGGCTTTAAGTAATTGTCTCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e153\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGTCATAAACTGGAACG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTCATTTGATACAACATCCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e182\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGGCTATGGGATGTTATG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTAGGAGCTTTCAGGTCAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGACTGTGGGAGCATCTTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAATGTCATCGTGGAAGTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e182\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATCTTCAACGCCAAACAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAGCCACTTCCTAGCTCATT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e199\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCCAAATGTTAATAACTACGAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATGCCAAGTCTAGCAAAGCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCGAACCGCTAGTGGTGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGCCAGCAGCCTTCTTAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e169\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGTTATGCCCATCTGTTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCTCCATCTCCTATCCCTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTCACGGTTGTTACCTTAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATTGGTTCGCTCGTCGTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e194\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAAAGAAGGAGCTGATACGG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCACCAATAACGCCCAAAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e152\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATGTATAGCCACGCAGTC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAGATCAAAGTTCCCAGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAGAAGTGGTGGAGATTG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAGCCTTGCTCTTCATAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e184\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCCCAGGCATTTGCTACA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTGCGGAGATTCTTCTTAGTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e190\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGGCTCTGGGAACACCTAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATGTCATCGTGGAAATGC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e181\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGATGTTTCTTATCGCCGCCTAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTGCCGCCTCAACCACCT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e154\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAACGAATTTGCCTGTGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGAAACGATGGCGCTTGAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C39\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eATGCCTATCTTGACTGCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTTCTTGCTGCTTCTTGGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e193\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAAACTGTGCTTCCCTCCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAGGTTTCCCATAACTTCG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C41\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCGTAGGGTTAGTTCTGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAGCGAAGCAACATAGAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e162\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTGAAGAGGGTTCGGCTAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGGCTTTGATGTGGGTGAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e159\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGTGCTGTACCGGATGTTA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCACTACGTGGCCGCTTTT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e180\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGCAAGGGCATTTGGTGAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCAAGTGCTTTGCTGCTGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCAAGCTGGACGACATCAC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAAGACAGGCTAGGCAGGA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e166\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGGAGCACAATGATCCAG\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTCAAACCCTCAGCAGAAA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e171\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCsPP2C47\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTTTGCCTGGCTTCAATAT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eATGGGTATGCACGAATGT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e189\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCCCCATCGTTACCGTCCAG、\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCGCCTTGCCAGTTGAATATCC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe 10 \u0026micro;L qRT-PCR reaction mixture consisted of the following components: 5 \u0026micro;L of 2\u0026times; SYBR Green Premix (Baoguang Biotechnology Research (Chongqing) Co., Ltd.), 0.25 \u0026micro;L of forward primer (10 \u0026micro;mol\u0026middot;L⁻\u0026sup1;), 0.25 \u0026micro;L of reverse primer (10 \u0026micro;mol\u0026middot;L⁻\u0026sup1;), 3 \u0026micro;L of diluted cDNA template, and 1.5 \u0026micro;L of nuclease-free water.\u003c/p\u003e \u003cp\u003eThe thermal cycling protocol was as follows: initial denaturation at 94\u0026deg;C for 20 seconds; followed by 40 cycles of denaturation at 94\u0026deg;C for 10 seconds and annealing/extension at 55\u0026deg;C for 10 seconds. A melt curve analysis was performed at the end of each run to confirm amplification specificity. Each sample-analysis combination was performed with three technical replicates.\u003c/p\u003e \u003cp\u003eThe relative expression levels of the target genes were calculated using the comparative 2^(\u0026ndash;ΔΔCT) method. The resulting expression data were subjected to statistical analysis, and a heatmap was generated using MeV software to visualize the expression patterns across different treatments and time points.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Authors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRangjin Xie- Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China\u003c/p\u003e\n\u003cp\u003eTingshan Yao- Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYanfang Yang - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China\u003c/p\u003e\n\u003cp\u003eWenhua Sun - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China\u003c/p\u003e\n\u003cp\u003eZihan Wang - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China\u003c/p\u003e\n\u003cp\u003eQiao Liu - Citrus Research Institute, Southwest University, Chongqing 400712, China /National Citrus Engineering Research Center, Chongqing 410712, China\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYanfang Yang: Design experiments, Investigation, Analyze the data,Write manuscripts.\u003c/p\u003e\n\u003cp\u003eWenhua Sun: Investigation, Analyze the data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eZihan Wang: Investigation, Analyze the data.\u003c/p\u003e\n\u003cp\u003eQiao Liu: Investigation, Analyze the data.\u003c/p\u003e\n\u003cp\u003eRangjin Xie: Provide ideas, Design experiments, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eTingshan Yao: Provide ideas, Design experiments, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (U24A20511), and the National Key R\u0026amp;D Program of China (2021YFD1400800).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePandey P, Irulappan V, Bagavathiannan MV, Senthil-Kumar M. 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Plant Physiol. 2006;140(1):115\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBernau VM, Jard\u0026oacute;n Barbolla L. McHale LK,Mercer KL.Germination response of diverse wild and landrace chile peppers (Capsicum spp.) under drought stress simulated with polyethylene glycol. PLoS ONE. 2020;15(11):e0236001.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu GQ, Wang JL, Li SJ. Identification of Na+/H\u0026thinsp;+\u0026thinsp;Antiporter (NHX) Genes in Sugar Beet (Beta vulgaris L.) and Their Regulated Expression under Salt Stress. Genes (Basel). 2019;10(5):401.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu K, Zhou M, Guo Y, et al. Genome-Wide Analysis of Maize ECT Family Members and Their Expressions in Response to Abiotic Stresses. J Nuclear Agricultural Sci. 2024;38(06):1035\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"citrus, Protein Phosphatase 2Cs, bioinformatics, stresses, expression analysis","lastPublishedDoi":"10.21203/rs.3.rs-8649733/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8649733/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eIn plants, Protein Phosphatase 2C (PP2C) functions as a key protease in responses to various environmental stresses. Although the PP2C gene family has been extensively characterized in plants such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e, its structural features and stress-responsive patterns remain rarely unexplored in citrus. This study aims to address this gap by conducting a genome-wide identification and functional characterization of \u003cem\u003ePP2C\u003c/em\u003e genes in citrus, with a focus on their expression profiles under abiotic stress conditions.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA comprehensive genome-wide analysis was performed in citrtus, and a total of 47 \u003cem\u003ePP2C\u003c/em\u003e genes (\u003cem\u003eCsPP2C1\u003c/em\u003e\u0026ndash;\u003cem\u003eCsPP2C47\u003c/em\u003e) were identified. All CsPP2C members possess conserved PP2C catalytic domains. Physicochemical characterization revealed that CsPP2C proteins exhibited a molecular weight range of 20.03\u0026ndash;124.36 kDa, with the majority being acidic (pI\u0026thinsp;\u0026lt;\u0026thinsp;7), inherently unstable (instability index\u0026thinsp;\u0026gt;\u0026thinsp;40), and hydrophilic (grand average of hydropathicity\u0026thinsp;\u0026lt;\u0026thinsp;0). Phylogenetic analysis clustered them into 12 subgroups consistent with model species, indicating functional conservation of the gene family. These genes are unevenly distributed across 9 citrus chromosomes, and promoter cis-acting element analysis uncovered enrichment of stress-related motifs (MBS, LTR) and hormone-responsive elements (ABRE). Quantitative real-time PCR analysis revealed distinct expression patterns of \u003cem\u003eCsPP2Cs\u003c/em\u003e in response to PEG, low temperature, salt, and hormone treatments at 0, 12, and 24h, suggesting the important roles in citrus growth and development as well as abiotic stresses.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThis study presented a comprehensive characterization of the PP2C gene family in citrus, clarifying its evolutionary relationships, structural diversity, and stress-responsive expression profiles. The findings established a foundational framework for elucidating the molecular mechanisms underlying CsPP2C-mediated stress adaptation, and provided candidate genes for molecular breeding of stress-resilient citrus varieties.\u003c/p\u003e","manuscriptTitle":"Whole-genome Identification of the PP2C Gene Family in citrus and its response to Abiotic Stresses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-30 10:39:11","doi":"10.21203/rs.3.rs-8649733/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ace0c52-d22a-4f0e-b4a9-0106aaea6f81","owner":[],"postedDate":"January 30th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-03T09:42:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-30 10:39:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8649733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8649733","identity":"rs-8649733","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}