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Additional file 1: Fig. S1. (a) Coding sequence of PbLHY. (b) Alignment of PbLHY and AtCCA1/AtLHY protein sequences. The red box indicates the MYB domain. The blue backgrounds correspond to the percent identity of the multiple alignment. Fig. S2. Alignment of conserved domains from PbRVE proteins. (a) Alignment of MYB domains from ten PbRVEs and eleven AtRVEs. Red box indicates MYB domain, and blue box indicates SHAQK(Y/F) F sequence. (b) Alignment of LCL domains from subfamily II members. The red box indicates the MYB domain. The green box indicates the LCL domain. The blue backgrounds correspond to the percent identity of the multiple alignment. Fig. S3. The motif details of ten conserved domains identified in RVE proteins and the logos of these domains created using the MEME. Amino acids are expressed in the standard single letter code. The size of the letters at each position represents their frequency.

Background Pear fruit exhibit a single sigmoid pattern during development, while peach and strawberry fruits exhibit a double sigmoid pattern. However, little is known about the differences between these two patterns. Results In this study, fruit weights were measured and paraffin sections were made from fruitlet to maturated pear, peach, and strawberry samples. Results revealed that both single and double sigmoid patterns resulted from cell expansion, but not cell division. Comparative transcriptome analyses were conducted among pear, peach, and strawberry fruits at five fruit enlargement stages. Comparing the genes involved in these intervals among peaches and strawberries, 836 genes were found to be associated with all three fruit enlargement stages in pears (Model I). Of these genes, 25 were located within the quantitative trait locus (QTL) regions related to fruit weight and 90 were involved in cell development. Moreover, 649 genes were associated with the middle enlargement stage, but not early or late enlargement in pears (Model II). Additionally, 22 genes were located within the QTL regions related to fruit weight and 63 were involved in cell development. Lastly, dual-luciferase assays revealed that the screened bHLH transcription factors induced the expression of cell expansion-related genes, suggesting that the two models explain the single sigmoid pattern. Conclusions Single sigmoid patterns are coordinately mediated by Models I and II, thus, a potential gene regulation network for the single sigmoid pattern was proposed. These results enhance our understanding of the molecular regulation of fruit size in Rosaceae.

Supplemental Document 1

Peach is a typical climacteric fruit that releases ethylene during fruit ripening. Several studies have been conducted on the transcriptional regulation of ethylene biosynthesis in peach fruit. Herein, an ethylene response factor, PpERF.A16, which was induced by exogenous ethylene, could enhance ethylene biosynthesis by directly inducing the expression of 1-aminocyclopropane-1-carboxylic acid synthase (PpACS1) and 1-aminocyclopropane-1-carboxylic acid oxidase (PpACO1) genes. Moreover, the NAM/ATAF1/2/CUC2 (NAC) transcription factor (TF) PpNAC.A59 was coexpressed with PpERF.A16 in all tested peach cultivars. Interestingly, PpNAC.A59 can directly interact with the promoter of PpERF.A16 to induce its expression but not enhance LUC activity driven by any promoter of PpACS1 or PpACO1. Thus, PpNAC.A59 can indirectly mediate ethylene biosynthesis via the NAC-ERF signaling cascade to induce the expression of both PpACS1 and PpACO1. These results enrich the genetic network of fruit ripening in peach and provide new insight into the ripening mechanism of other perennial fruits.

Background: Pear is one of the most abundant fruit crops and has been cultivated world-wide. However, the salt injury events caused by increased salinity limited the distribution and sustainable production of pear crops. Therefore, it is needed to take further efforts to understand the genetics and mechanisms of salt tolerance to improved salt resistance and productivity.Results: In this work, we analyzed the dynamic transcriptome of pear (Pyrus ussuriensis Maxim) under salt stress by using RNA-Seq and WGCNA. A total of 3540, 3831, 8374, 6267 and 5381 genes were identified that were differentially expressed after exposure to 200mM NaCl for 4, 6, 12, 24 and 48 hours, respectively, and 1163 genes were shared among the five comparisons. KEGG enrichment analysis of these DEGs (differentially expressed genes) revealed that “MAPK signaling” and “Plant hormone signal transduction” pathways were highly enriched. Meanwhile, 622 DEGs identified from WGCNA were highly correlated with these pathways, and some of them were able to indicate the salt tolerance of pear varieties. In addition, we provide a network to demonstrate the time-sequence of these co-expressed MAPK and hormone related genes.Conclusion: A comprehensive analysis about salt-responsive pear transcriptome were performed by using RNA-Seq and WGCNA. We demonstrated that “MAPK signaling” and “Plant hormone signal transduction” pathways were highly recruited during salt stress, and provided new insights into the metabolism of plant hormones related signaling at transcriptome level underlying salt resistance in pear. The dynamic transcriptome data obtained from this study and these salt-sensitive DEGs may provide potential genes as suitable targets for further biotechnological manipulation to improve pear salt tolerance.

Class III peroxidases (PRXs) are plant-specific enzymes that play key roles in the responses to biotic and abiotic stress during plant growth and development. In addition, some peroxidases also play roles in plant lignification. In this study, a total of 114 PRX (designated PbPRXs) genes were identified in the pear (Pyrus bretschneideri Rehd) genome based on systematic analysis. These PRX genes were divided into 12 groups based on their phylogenetic relationships. We performed systematic bioinformatics analysis of the PRX genes, including analysis of gene structures, conserved motifs, phylogenetic relationships, and gene expression patterns during pear fruit growth. The PbPRXs are unevenly distributed on the 17 pear chromosomes and some of them on other scaffolds. Gene duplication event analysis indicated that whole-genome duplication (WGD) and segmental duplication play key roles in PRX gene amplification. Ka/Ks analysis suggested that most duplicated PbPRXs experienced purifying selection, with limited functional divergence during the duplication events. Furthermore, the analysis indicated that those highly expressed genes might play significant roles in the lignification of cells to form stone cells in pear fruit. We examined the expression of those highly expressed genes during fruit growth using quantitative real-time PCR (qRT-PCR), verifying differential expression patterns at different stages of fruit. This study provides useful information for further functional analysis of the PRX gene family in pears.

PbrKAT1, which is inhibited by external Na+ in Xenopus laevis oocytes, is characterized as encoding a typical inward rectifying channel that is mainly expressed in guard cells. Potassium (K+) is the most abundant cation in plant cells necessary for plant growth and development. The uptake and transport of K+ are mainly completed through transporters and channels, and the Shaker family genes are the most studied K+ channels in plants. However, there is far less information about this family in Rosaceae species. We performed a genome-wide analysis and identified Shaker K+ channel gene family members in Rosaceae. We cloned and characterized a Shaker K+ channel KAT1 from pear (Pyrus × bretschneideri). In total, 36 Shaker K+ channel genes were identified from Rosaceae species and were classified into five subgroups based on structural characteristics and a phylogenetic analysis. Whole-genome and dispersed duplications were the primary forces underlying Shaker K+ channel gene family expansion in Rosaceae, and purifying selection played a key role in the evolution of Shaker K+ channel genes. β-Glucuronidase and qRT-PCR assays revealed that PbrKAT1 was mainly expressed in leaves, especially in guard cells. PbrKAT1 displayed a typical inward-rectifying current when expressed in Xenopus laevis oocytes. The activity of PbrKAT1 was inhibited by external sodium ions, possibly playing an important role in the regulation of salt tolerance in pear. These results provide valuable information on evolution, expression and functions of the Shaker K+ channel gene family in plants.

Seed coat-induced dormancy protects embryos from environmental conditions, and delays seed germination. In this study, we developed a simple method to accelerate embryo germination. That is, pear s...

With-No-Lysine kinases (WNK) have been reported to be associated with plant growth regulation in Arabidopsis, soybean, and rice, but little is known of their roles in fruits. In this study, a total of 114 WNK genes were identified from 8 fruit trees species, and these WNK genes belonged to 2 classes (I and II) that respectively contain 4 (A, B, C, D) and 2 groups (E, F). The WNK genes had variable exon-intron structures and were randomly distributed among most chromosomes of each genus. The expression levels of six of the 18 WNK genes in peach were almost undetectable in fruits, suggesting that they may not be associated with fruit development and ripening. Of the other 12 WNK genes, PpWNK.B1 exhibited stronger stability of expression levels than the best reference genes reported previously in fruits from two peach cultivars. These results suggested that PpWNK.B1 could be a reliable reference gene for gene expression studies of peach fruit. Moreover, qRT-PCR assays yielded evidence that when using single reference gene as internal control, mistakes were more possible, indicating that the use of three reference genes is necessary for gene expression studies. Based on the gene expression profiles validated by qRT-PCR, PpWNK.A1 is predicted to likely be involved in fruit ripening, while PpWNK.A2 and PpWNK.E3.1 are likely associated with early fruit development. The present study represented the WNK genes in fruits, and it will be a valuable resource in continuing investigation of gene regulation network during fruit development and ripening. Keywords: peach, With-No-Lysine kinase, phylogenetic tree, reference gene, gene expression

Background The content of stone cells in pears has a great influence on taste. Stone cells are formed by the accumulation of lignin. The treatment of exogenous calcium can affect the lignin synthesis, but this Ca-mediated mechanism is still unclear. In this study, the author performed a comparative transcriptomic analysis of callus of pears (Pyrus x bretschneideri) treated with calcium nitrate Ca (NO3)2 to investigate the role of calcium in lignin synthesis. Results There were 2889 differentially expressed genes (DEGs) detected between the Control and Ca (NO3)2 treatment in total. Among these 2889 DEGs, not only a large number of genes related to Ca single were found, but also many genes were enriched in secondary metabolic pathway, especially in lignin synthesis. Most of them were up-regulated during the development of callus after Ca (NO3)2 treatment. In order to further explore how calcium nitrate treatment affects lignin synthesis, the author screened genes associated with transduction of calcium signal in DEGs, and finally found CAM, CML, CDPK, CBL and CIPK. Then the author identified the PbCML3 in pears and conducted relevant experiments finding the overexpression of PbCML3 would increase the content of pear stone cells, providing potential insights into how Ca treatment enhances the stone cell in pears. Conclusions Our deep analysis reveals the effects of exogenous calcium on calcium signal and lignin biosynthesis pathway. The function of PbCML3 on stone cells formation was verified in pear.