In the previous decades, numerous investigations have been done that the Alfin-like (AL) transcription factors (TFs) are participating in several physiological and development processes in plants including root hair elongation, development of root and meristem, etc. Although the AL-TFs had been comprehensively evaluated in several species, the little finding is reported on the AL-TFs in Chinese pear (Pyrus bretschenedri). In the current study, 15 PbAL genes were systemically characterized and nomenclature as PbAL1 to PbAL15 corresponding to their chromosomal localization. Phylogeny analysis and structural features revealed that the 15 genes could be classified into six subfamilies (S-I to S-VI).Whole-genome duplication (WGD) events are the most common mechanism and may play a crucial role in the expansion of the AL gene family. Microsynteny analysis among Pyrus bretschenedri, Fragaria vesca, Prunus avium, Prunus mume, and Prunus persica showed that AL duplicated regions were more conserved and undergo purifying selection during the evolutionary process. Further analysis of introns-exons, physicochemical properties, and conserved motif showed their functional divergence, diversification in quantity, and structure. The expression pattern of PbAL gene family in various organs and different fruit development stages were conducted through transcriptomic data. Moreover, stress-related cis-acting elements of PbAL genes were found on the promoters regions to be associated with hormonal and environmental stress responses. Noticeably, the expression patterns evaluated by qRT-PCR demonstrated that PbAL genes were differentially expressed under indole acetic acid (IAA), Gibberellic acid (GA), Melatonin (ME) and abscisic acid (ABA) treatment, which response to abiotic stress and fruit growth development. Current work explored the potential function and evolution of PbAL genes in Pyrus bretschenedri. PbAL were investigated as candidate genes for further research and important clues for improving fruit quality through molecular breeding.

Cystathionine γ-synthase (CGS), S-adenosyl-L-homocysteine hydrolase (SAHH), and S-adenosy-L-methionine synthetase (SAMS) play an important role in the regulation of plant growth, development, and secondary metabolism. In this study, a total of 6 CGS, 6 SAHH, and 28 SAMS genes were identified from five Rosaceae species (Pyrus bretschneideri, Prunus persica, Prunus mume, Fragaria vesca, and Malus domestica). The evolutionary relationship and microsynteny analysis in five Rosaceae species revealed that duplicated regions were conserved between three gene families (CGS, SAHH, SAMS). Moreover, the chromosomal locations, gene structures, conserved motifs, cis-elements, physicochemical properties, and Ka/Ks analysis were performed by using numerous bioinformatics tools. The expression of different organs showed that the CGS, SAHH and SAMS genes of pear have relatively high expression patterns in flowers and stems, except for PbCGS1. RNA-seq and qRT-PCR combined analysis showed that PbSAMS1 may be involved in the regulation of pear stone cell development. In summary, this study provides the basic information of CGS, SAHH and SAMS genes in five Rosaceae species, further revealing the expression patterns in the pear fruit, which provides the theoretical basis for the regulation of pear stone cells.

GRAS is a transcription regulator factor, which plays an important role in plant growth and development. Previous analyses found that several GRAS functions have been identified, such as axillary bud meristem formation, radial root elongation, gibberellin signaling, light signaling, and abiotic stress. The GRAS family has been comprehensively evaluated in several species. However, little finding is on the GRAS transcription factors (TFs) in Chinese white pear. In this study, 99 PbGRAS were systemically characterized and renamed PbGRAS1 to PbGRAS99 according to their chromosomal localizations. Phylogenetic analysis and structural features revealed that could be classified into eight subfamilies (LISCL, Ls, SHR, HAM, SCL, PAT, SCR, and DELLA). Further analysis of introns/exons and conserved motifs revealed that they are diverse and functionally differentiated in number and structure. Synteny analysis among Pyrus bretschenedri, Prunus mume, Prunus avium, Fragaria vesca, and Prunus persica showed that GRAS duplicated regions were more conserved. Dispersed duplication events are the most common mechanism and may play a crucial role in the expansion of the GRAS gene family. In addition, cis-acting elements of the PbGRAS gene were found in promoter regions associated with hormone and environmental stress responses. Notably, the expression pattern detected by qRT-PCR indicated that PbGRAS genes were differentially expressed under gibberellin (GA), abscisic acid (ABA), and auxin (IAA) conditions, which are responsive to abiotic stress. PbGRAS89 and PbGRAS99 were highly expressed at different stages of hormone treatment and may play important role in leaf development. Therefore, we selected PbGRAS89 and PbGRAS99 to clone and construct pCAMBIA1301-PbGRAS89, 99 and transferred them into Arabidopsis thaliana. Finally, we observed and compared the changes of overexpressed plants and wild-type plants during regeneration. This method was used to analyze their roles in leaf regeneration of Chinese white pear. In addition, we also constructed pCAMBIA1305-PbGRAS89, 99, and transferred them into onion cells to determine the subcellular localization. Subcellular localization experiments showed that PbGRAS89 and PbGRAS99 were localized in the nucleus. In summary, the results of this study indicate that PbGRAS89 and PbGRAS99 are mainly responsible for leaf regeneration of Chinese white pear, which plays a positive role in callus formation and provides rich resources for studying GRAS gene functions.

COBRA-Like (COBL) genes encode a glycosylphosphatidylinositol (GPI) anchoring protein unique to plants. In current study, 87 COBRA genes were identified in 6 Rosaceae species, including Pyrus bretschneideri (16 genes), Malus domestica (22 genes), Fragaria vesca (13 genes), Prunus mume (11 genes), Rubus occidentalis (13 genes) and Prunus avium (12 genes). We revealed the evolution of the COBRA gene in six Rosaceae species by phylogeny, gene structure, conservative sequence, hydrophobicity analysis, gene replication events and sliding window analysis. In addition, based on the analysis of expression patterns in pear fruit combined with bioinformatics, we identified PbCOBL12 and PbCOBL13 as potential genes regulating secondary cell wall (SCW) formation during pear stone cell development. This study aimed to understand the evolutionary relationship of the COBRA gene in Rosaceae species, clarify the potential function of COBRA in pear fruit development, and provide essential theoretical basis and gene resources for improving pear fruit quality through genetical modification mechanism.

The SAUR (small auxin-up RNA) gene family is the biggest family of early auxin response genes in higher plants and has been associated with the control of a variety of biological processes. Although SAUR genes had been identified in several genomes, no systematic analysis of the SAUR gene family has been reported in Chinese white pear. In this study, comparative and systematic genomic analysis has been performed in the SAUR gene family and identified a total of 116 genes from the Chinese white pear. A phylogeny analysis revealed that the SAUR family could be classified into four groups. Further analysis of gene structure (introns/exons) and conserved motifs showed that they are diverse functions and SAUR-specific domains. The most frequent mechanisms are whole-genome duplication (WGD) and dispersed duplication (DSD), both of which may be important in the growth of the SAUR gene family in Chinese white pear. Moreover, cis-acting elements of the PbrSAUR genes were found in promoter regions associated with the auxin-responsive elements that existed in most of the upstream sequences. Remarkably, the qRT-PCR and transcriptomic data indicated that PbrSAUR13 and PbrSAUR52 were significantly expressed in fruit ripening. Subsequently, subcellular localization experiments revealed that PbrSAUR13 and PbrSAUR52 were localized in the nucleus. Moreover, PbrSAUR13 and PbrSAUR52 were screened for functional verification, and Dangshan pear and frandi strawberry were transiently transformed. Finally, the effects of these two genes on stone cells and lignin were analyzed by phloroglucinol staining, Fourier infrared spectroscopy, and qRT-PCR. It was found that PbrSAUR13 promoted the synthesis and accumulation of stone cells and lignin, PbrSAUR52 inhibited the synthesis and accumulation of stone cells and lignin. In conclusion, these results indicate that PbrSAUR13 and PbrSAUR52 are predominantly responsible for lignin inhibit synthesis, which provides a basic mechanism for further study of PbrSAUR gene functions.

TCP is a plant-specific transcription factor that plays an important role in flowering, leaf development and other physiological processes. In this study, we identified a total of 155 TCP genes: 34 in Pyrus bretschneideri, 19 in Fragaria vesca, 52 in Malus domestica, 19 in Prunus mume, 17 in Rubus occidentalis and 14 in Prunus avium. The evolutionary relationship of the TCP gene family was examined by constructing a phylogenetic tree, tracking gene duplication events, performing a sliding window analysis. The expression profile analysis and qRT-PCR results of different tissues showed that PbTCP10 were highly expressed in the flowers. These results indicated that PbTCP10 might participated in flowering induction in pear. Expression pattern analysis of different developmental stages showed that PbTCP14 and PbTCP15 were similar to the accumulation pattern of fruit lignin and the stone cell content. These two genes might participate in the thickening of the secondary wall during the formation of stone cells in pear. Subcellular localization showed that PbTCPs worked in the nucleus. This study explored the evolution of TCP genes in six Rosaceae species, and the expression pattern of TCP genes in different tissues of “Dangshan Su” pear. Candidate genes related to flower induction and stone cell formation were identified. In summary, our research provided an important theoretical basis for improving pear fruit quality and increasing fruit yield by molecular breeding.

The Multidrug and Toxic Compound Extrusion (MATE) protein belongs to a secondary transporter gene family, which plays a primary role in transporting many kinds of substrates such as organic compounds, secondary metabolites, and phytohormones. MATE protein members exist in both prokaryotes and eukaryotes. However, evolution and comprehensive analysis of the MATE genes has not been performed in Rosaceae species. In the present study, a total of 404 MATEs genes were identified from six Rosaceae genomes (Prunus avium, Pyrus bretschneideri, Prunus persica, Fragaria vesca, Prunus mume, and Malus domestica) and classified into eight main subfamilies (I-VII) based on structural and phylogenetic analysis. Microcollinearity analysis showed that whole-genome duplication events might play a vital role in the expansion of the MATE genes family. The Ka/Ks analysis, chromosomal localization, subcellular localization, and molecular characteristics (length, weight, and pI) were performed using various bioinformatics tools. Furthermore, different subfamilies have different introns-exons structures, cis-acting elements, and conserved motifs analysis, indicating functional divergence in the MATE family. Subsequently, RNA-seq analysis and real-time qRT-PCR were conducted during Chinese pear fruit development. Moreover, PbMATE genes were significantly expressed under hormonal treatments of MeJA (methyl jasmonate), SA (salicylic acid), and ABA (abscisic acid). Overall, our results provide helpful insights into the functions, expansion complexity, and evolutions of the MATE genes in Chinese pear and five Rosaceae species.