DNA methylation plays an indispensable role in genome stability, regulation of gene expression and plant stress response. It is mediated by DNA methyltransferases (MTases). Twelve putative MTases of P. betulaefolia were identified and were classified into MET1, CMT, DRM2 and Dnmt2 groups based on the organization of various characteristic domains. Three pairs of paralogous genes were identified with the Ka/Ks ratio varied from 0.232 for PbeMET1a and PbeMET1b to 0.251 for PbeCMT2 and PbeCMT3, respectively. In addition, the Ka/Ks ratio for nine pairs of orthologous gene pairs between P. betulaefolia and apple were varied from 0.053 for PbeDRM3 and MD17G1031900 to 0.278 for PbeDnmt2b and MD15G1120500, respectively. All the 12 members of MTase genes were located on nine chromosomes out of 17 P. betulaefolia chromosomes with highly conserved protein sequence structures. The isoelectric points (pI) of MTases ranged from 4.74 to 7.24, while molecular weight varied from 35.99 to 174.32. The expression profiles of MTase and other salt-responsive genes under salt stress treatment revealed their important roles involved in salt response in P. betulaefolia. Furthermore, three selected salt-responsive genes (PbeNHX2.1, PbeCBL2 and PbeAKT2) were found altered in methylation level of promoters (which contain CpG islands) under salt stress. Especially, the PbeAKT2 promoter regions showed high CHG and CHH methylation types. Our study provided a genome-wide survey of the MTase gene family and highlighted their roles in salt response. These results also provided an effective way for the breeding and improvement of salt-tolerant pear varieties.

Kiwifruit, as a recently domesticated horticultural fruit crop, has substantial economic and nutritional value, particularly its high vitamin C content. In this study, we investigated the different metabolites accumulation in the ripening fruit of two mainly cultivated kiwifruit species A. chinensis and A. eriantha. A total of 428 metabolites were identified, with low correlation coefficients found in amino acid metabolism, as well as in the metabolism of terpenoids and polyketides between two species, indicating the variations in taste, flavor, and nutritional properties. Furthermore, 115 items were clarified as differentially accumulated metabolites. Based on the Kyoto Encyclopedia of Genes and Genomes pathways enrichment analysis, differentially accumulated metabolites (DAMs) were majorly involved in tryptophan metabolism, biosynthesis of secondary metabolites, pyrimidine metabolism, flavonoid biosynthesis, vitamin B6 metabolism, phenylalanine, tyrosine, and tryptophan biosynthesis. Transcriptome analysis revealed 3,168 upregulated genes and 3,174 downregulated genes, we performed Gene Ontology (GO) enrichment analysis suggesting that differentially expressed genes were associated with the aromatic amino acids (AAAs) family catabolic process, which verified the results of metabolome analysis. Expression patterns of homologous genes of these two kiwifruits participating in AAAs metabolism pathway were compared, key structural genes including TSA1, TSB2, TAA, and TAT showed opposite expression profiles. Additionally, we built a co-expression network to identify multiple transcription factors involved in regulating the AAAs metabolism pathways, predominantly belonging to the MYB and MYB_related families, bHLH family, ERF family, and C2H2 family. Our research provides new perspectives on the accumulation of aromatic amino acids between different kiwifruit species. We proposed the potential transcription factors that regulate the AAAs metabolism pathway. This work offers theoretical support for inventing new more nutritional kiwifruit species by altering specific structural genes and corresponding regulatory elements.

SMXL genes constitute a conserved gene family that is ubiquitous in angiosperms and involved in regulating various plant processes, including branching, leaf elongation, and anthocyanin biosynthesis, but little is known about their molecular functions in pear branching. Here, we performed genome-wide identification and investigation of the SMXL genes in 16 angiosperms and analyzed their phylogenetics, structural features, conserved motifs, and expression patterns. In total, 121 SMXLs genes were identified and were classified into four groups. The number of non-redundant SMXL genes in each species varied from 3 (Amborella trichopoda Baill.) to 18 (Glycine max Merr.) and revealed clear gene expansion events over evolutionary history. All the SMXL genes showed conserved structures, containing no more than two introns. Three-dimensional protein structure prediction revealed distinct structures between but similar structures within groups. A quantitative real-time PCR analysis revealed different expressions of 10 SMXL genes from pear branching induced by fruit-thinning treatment. Overall, our study provides a comprehensive investigation of SMXL genes in the Rosaceae family, especially pear. The results offer a reference for understanding the evolutionary history of SMXL genes and provide excellent candidates for studying fruit tree branching regulation, and in facilitating pear pruning and planting strategies.

Here, cytosine methylation in the whole genome of pear flower buds was mapped at a single-base resolution. There was 19.4% methylation across all sequenced C sites in the Pyrus pyrifolia cultivar ‘Sucui 1’ flower bud genome. Meantime, the CG, CHG, and CHH sequence contexts (where H = A, T or C) exhibited 47.4%, 33.3%, and 11.9% methylation, respectively. Methylation in different gene regions was revealed through combining methylome and transcriptome analysis, which presented various transcription trends. Genes with methylated promoters exhibited lower expression levels than genes with non-methylated promoters, while body-methylated genes displayed an obvious negative correlation with their transcription levels. The methylation profiles of auxin- and cytokinin-related genes were estimated. And some of them proved to be hypomethylated, with increased transcription levels, in wizened buds. More specifically, the expression of the genes PRXP73, CYP749A22, and CYP82A3 was upregulated as a result of methylation changes in their promoters. Finally, auxin and cytokinin concentrations were higher in wizened flower buds than in normal buds. The exogenous application of paclobutrazol (PP333) in the field influenced the DNA methylation status of some genes and changed their expression level, reducing the proportion of wizened flower buds in a concentration-dependent manner. Overall, our results demonstrated the relationship between DNA methylation and gene expression in wizened flower buds of P. pyrifolia cultivar ‘Sucui 1’, which was associated with changes in auxin and cytokinin concentrations.

Soil salinization severely restricts the sustainable development of the pear industry. Pyrus betulifolia, a vital native salt-tolerant rootstock in China, holds great significance for investigating stress resistance mechanisms. Plant-specific DNA-binding One Zinc Finger (Dof) transcription factors act as pivotal regulators in stress adaptation. However, their functions in P. betulifolia remain largely unexplored. In this study, we identified 43 PbeDof members within the P. betulifolia genome and classified them into eight subfamilies via phylogenetic analysis. Gene structure and conserved motif analyses revealed that PbeDof members within the same subfamily share similar exon-intron organizations and protein architecture, suggesting evolutionary conservation. Promoter analysis indicated that PbeDof genes are rich in cis-acting elements related to light, phytohormones (especially ABA and MeJA), and stress responses, implying their potential roles in diverse biological processes. Chromosomal localization and collinearity analyses revealed that segmental duplication was the primary driver of this family’s expansion. Combined transcriptomic profiling and qRT-PCR assays demonstrated that PbeDof9.1 is predominantly expressed in roots and is strongly induced by salt stress. Subcellular localization confirmed that PbeDof9.1 targets the nucleus. Functional characterization indicated that heterologous overexpression of PbeDof9.1 in Arabidopsis thaliana significantly enhances salt tolerance at germination and seedling stages. Notably, under 175 mM NaCl stress, the transgenic lines exhibited a superior root system architecture, with primary root length and lateral root numbers being approximately 1.5-fold higher than those of the wild type. Furthermore, homologous overexpression in pear calli confirmed that PbeDof9.1 mitigates oxidative damage by boosting the activities of peroxidase (POD) and catalase (CAT) to scavenge reactive oxygen species (ROS), thereby reducing malondialdehyde (MDA) accumulation. Collectively, this study characterizes the PbeDof family and establishes PbeDof9.1 as a key candidate gene for the genetic improvement of salt tolerance in pear rootstocks.

Salt stress severely restricts the growth and development of pear trees, and DNA methylation may potentially modulate the salt tolerance of Pyrus betulaefolia by regulating ion transporter genes. Cyclic nucleotide-gated channel (CNGC) family genes are essential for plant ion transport and salt stress adaptation; however, the epigenetic regulatory relationship between DNA methylation and CNGC genes underlying pear salt tolerance remains unclear. In this study, 26 PbCNGC genes were systematically identified from P. betulaefolia, which possess the conserved motifs characteristic of the CNGC family and can be classified into five subclades. PbCNGC members exhibit distinct spatiotemporal expression patterns in ordinary and salt-tolerant genotypes. Under salt stress, core members PbCNGC3, PbCNGC4, PbCNGC10, and PbCNGC14 displayed typical fluctuating up-and-down expression patterns across roots, stems, and leaves, with distinct spatiotemporal specificity in their expression peaks. In contrast, PbCNGC19 and PbCNGC20;1 were exclusively induced in roots under salt stress. Quantitative results showed marked genotypic differences in salt responsiveness. At 4 h of salt treatment, the root transcript levels of PbCNGC4 and PbCNGC14 were upregulated by 8.27-fold and 6.76-fold in the salt-tolerant genotype, respectively, which were considerably higher than those in the ordinary genotype (2.08-fold and 4.50-fold). Obvious genotypic differences in mCHH methylation modifications of PbCNGC4 and PbCNGC14 were detected after salt treatment, and pharmacological experiments confirmed that DNA methylation negatively modulates the transcription of these two genes. Yeast functional complementation assays further demonstrated that PbCNGC4 and PbCNGC14 act as functional Na⁺ and K⁺ permeable cation influx channels. Combined with methylation analysis results, this study reveals a potential regulatory cascade in which DNA methylation may inhibit the transcription of PbCNGC4 and PbCNGC14, modulating their ion-transport capacity, effectively reduces Na⁺ overaccumulation, sustains cellular K⁺ retention to alleviate salt-induced ionic toxicity, and may ultimately shape the salt tolerance of P. betulaefolia. These findings enrich the understanding of epigenetic regulation of plant salt tolerance and provide valuable candidate genes and theoretical references for salt-tolerant molecular breeding in pear trees.