Effects of Metaxenia on Stone Cell Formation in Pear (Pyrus bretschneideri) Based on Transcriptomic Analysis and Functional Characterization of the Lignin-Related Gene PbC4H2

The deposition of lignin in flesh parenchyma cells for pear stone cells, and excessive stone cells reduce the taste and quality of the fruit. The effect of metaxenia on the quality of fruit has been heavily studied, but the effect of metaxenia on stone cell formation has not been fully elucidated to date. This study used P. bretschneideri (Chinese white pear) cv. ‘Yali’ (high-stone cell content) and P. pyrifolia (Sand pear) cv. ‘Cuiguan’ (low-stone cell content) as pollination trees to pollinate P. bretschneideri cv. ‘Lianglizaosu’ separately to fill this gap in the literature. The results of quantitative determination, histochemical staining and electron microscopy indicated that the content of stone cells and lignin in YL fruit (‘Yali’ (pollen parent) × ‘Lianglizaosu’ (seed parent)) was significantly higher than that in CL fruit (‘Cuiguan’ (pollen parent) × ‘Lianglizaosu’ (seed parent)). The transcriptome sequencing results that were obtained from the three developmental stages of the two types of hybrid fruits indicated that a large number of differentially expressed genes (DEGs) related to auxin signal transduction (AUX/IAAs and ARFs), lignin biosynthesis, and lignin metabolism regulation (MYBs, LIMs, and KNOXs) between the CL and YL fruits at the early stage of fruit development. Therefore, metaxenia might change the signal transduction process of auxin in pear fruit, thereby regulating the expression of transcription factors (TFs) related to lignin metabolism, and ultimately affecting lignin deposition and stone cell development. In addition, we performed functional verification of a differentially expressed gene, PbC4H2 (cinnamate 4-hydroxylase). Heterologous expression of PbC4H2 in the c4h mutant not only restored its collapsed cell wall, but also significantly increased the lignin content in the inflorescence stem. The results of our research help to elucidate the metaxenia-mediated regulation of pear stone cell development and clarify the function of PbC4H2 in cell wall development and lignin synthesis, which establishes a foundation for subsequent molecular breeding.


Determination of Stone Cell and Lignin Content in Pear Fruit
The method for measuring the levels of stone cells and lignin was used as described previously [4]. The stone cell and lignin levels are shown as percentages (calculated stone cell content/calculated flesh weight of test sample × 100%; calculated lignin content/calculated dry weight of test sample × 100%). Three biological replicates were performed for each sample.
The Wiesner method carried out the histochemical staining of the stone cells of the pear fruit [18]. The transmission electron microscopy (TEM) observation of the SCW of stone cells was performed in accordance with the method that was described by Jin et al. [8].

RNA Extraction and Illumina Sequencing
The samples (pear fruits) that were obtained for transcriptomic analysis in this experiment were collected in equal amounts from parts of the tree facing four different directions (East, South, West, and North). Subsequently, RNA from these fruits (collected in the same period) was extracted and mixed in equal amounts for transcriptomic analysis to avoid error.
The total RNA from different fruits of pear was extracted according to the instructions of the RNAprep Pure Plant Kit (polysaccharides and polyphenolics-rich) (Tiangen, Beijing, China). The construction of the cDNA library and high-throughput sequencing (Illumina HiSeq™ 2500 sequencing platform) were commissioned by the Biomarker Technology Company (Beijing, China). Raw data (raw reads) in fastq format were first processed through in-house Perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adaptor sequences, reads containing poly-N sequences, and low-quality reads from the raw data. At the same time, the Q20, Q30, GC content, and sequence duplication level of the clean data were calculated. All of the downstream analyses were based on clean data with high quality.

Screening and Functional Annotation of Differentially Expressed Genes (DEGs)
The adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then Forests 2020, 11, 53 4 of 25 mapped to the reference genome sequence. Only reads with a perfect match or one mismatch were further analysed and annotated based on the reference genome. Tophat2 software v. 2.1.1 (Center for Bioinformatics and Computational Biology, University of Maryland, College Park, USA) was used for mapping with a reference genome. The reference genome of the pear (P. bretschneideri) was downloaded from the GigaDB Dataset (http://gigadb.org/dataset/100083).
The annotation of genes was performed while using the following method: Swiss-Port protein databases, Cluster of Orthologous Groups (COG) database, Gene Ontology (GO) database, and Kyoto Encyclopedia of Genes and Genomes (KEGG) database [6]. For the detection of DEGs, fold change (FC) ≥ 1 and false discovery rate (FDR) < 0.05 were used as screening criteria. For differential expression analysis, the well-known Benjamini-Hochberg correction method was used to correct the p-value of the original hypothesis test, and FDR was finally used as the key indicator for DEG screening [6].
The absolute transcript abundance values that were obtained for the 30 lignin biosynthetic genes were computed from fragments per kilobase of exon per million fragments mapped (FPKM) values. The FPKM values were obtained from the RNA-sequencing (RNA-seq) data. The expression level of each gene was visualized in the form of a heatmap while using TBtools software v. 0.66836 (https://github. com/CJ-Chen/TBtools/releases). The RNA-seq data have been deposited in the Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) with the access numbers SUB6384421 and SUB2967341.

Quantitative Real-Time PCR (qRT-PCR) Analysis
30 lignin biosynthetic genes were analysed using qRT-PCR to validate the expression patterns revealed by the DEG results. qRT-PCR analysis was performed while using SYBR Green Master Mix (Takara, Shiga, Japan) and detected by CFX96 Touch™ Real-Time PCR Detection System (Singapore). Three biological replicates were performed for each sample. Relative expression levels were calculated using the 2 − Ct method [31]. The pear tubulin gene (GenBank accession no. AB239680.1) was used as the reference gene [32]. Supplementary Table S1 lists all primers.

Genetic Transformation of Arabidopsis thaliana
We designed specific primers (Supplementary Table S1) for the C4H gene (Pbr017290.1) based on information in the genomic database to amplify the coding sequence. The overexpression vector pCAMBIA1304-PbC4H was constructed with the eukaryotic expression vector pCAMBIA1304 (GenBank: AF234300.1) as the backbone. Electroporation introduced the constructed vector into Agrobacterium tumefaciens EHA105.
The seeds of wild-type Arabidopsis thaliana (Columbia-0) and c4h mutants (At2g30490, SALK_070079) were purchased from the Nottingham Arabidopsis Stock Centre, UK [33]. The genetic transformation of Arabidopsis was carried out according to the floral dip method [34]. The method for the identification of Arabidopsis transgenic plants was consistent with that described by Cheng et al. [35]. β-Glucuronidase (GUS) staining was examined while using a GUS histochemical assays kit (Real-Times, Beijing, China).

Microscopic and Ultramicroscopic Observation of the Cell Wall of Arabidopsis
The inflorescence stems from Arabidopsis plants (T 3 generation transgenic plants, WT plants, c4h mutant plants) were sectioned while using a skiving machine (Leica RM2016, Wetzlar, Germany) and then stained with phloroglucinol-HCl or toluidine blue as described by Pradhan et al. [36].
The TEM observations and cell wall thickness measurements were performed using the previously described method [35,37]. Microsoft Excel 2010 (Student's t test) was used for the statistical analyses.

Analysis of Lignin Content in the Inflorescence Stem of Arabidopsis
The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin content of the inflorescence stem of Arabidopsis. The specific operation follows the method that was described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of the two pollination combinations to clarify the effects of different pollen parents on the content of stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key components of stone cells. Therefore, we also measured the lignin content in the 'Yali' Forests 2019, 10, x FOR PEER REVIEW The acetyl bromide method (acetyl bromide soluble lignin content) de content of the inflorescence stem of Arabidopsis. The specific operation follows t described by Anderson et al. [37]. Three biological replicates were performed fo

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pol
We compared the differences in the content of stone cells in the eight deve the two pollination combinations to clarify the effects of different pollen paren stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lian and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 D 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lign components of stone cells. Therefore, we also measured the lignin content 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone ce during fruit development, and they both showed a rise-fall tendency ( Figure 1 lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observe lignin and stone cells in the CL fruit are lower than the YL fruit in the selected e stages. In particular, the stone cells and lignin content of YL fruit were significa of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents h stone cell content and lignin synthesis in the pear fruit.

Characterization of Stone Cells in Pear Fruit After Pollination by Two Pollen Paren
We selected mature fruit for Wiesner staining (phloroglucinol-HCl) to e different pollen parents on the distribution of stone cells in pear fruits. As show observe that, regardless of whether the 'Yali' or 'Cuiguan' was used as the pol cell density in the inner flesh region of the fruit was relatively high. Howe difference in the distribution of stone cells in the middle flesh region. The dist range of the stone cell clusters in the mature fruit from 'Yali', as the pollen pa those in the mature fruit from 'Cuiguan' as the pollen parent. This finding indi Forests 2019, 10, x FOR PEER REVIEW The acetyl bromide method (ac content of the inflorescence stem of A described by Anderson et al. [37]. Thre

Differences in Stone Cell and Lignin
We compared the differences in t the two pollination combinations to c stone cells in the fruit (Figure 1a). We u and collected pollinated pear fruits at e 63 DAF, 71 DAF, 87 DAF, and the m components of stone cells. Therefore 'Lianglizaosu'♀(YL) and 'Cuiguan'♂ content of stone cells (Figure 1b).
Notably, the different pollen pare during fruit development, and they b lignin content in YL and CL fruits peak lignin and stone cells in the CL fruit a stages. In particular, the stone cells an of CL fruit from 39 DAF to fruit ripen stone cell content and lignin synthesis

Characterization of Stone Cells in Pea
We selected mature fruit for Wi different pollen parents on the distribu observe that, regardless of whether th cell density in the inner flesh region difference in the distribution of stone range of the stone cell clusters in the those in the mature fruit from 'Cuigua The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin ent of the inflorescence stem of Arabidopsis. The specific operation follows the method that was ribed by Anderson et al. [37]. Three biological replicates were performed for each sample.

sults ifferences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of wo pollination combinations to clarify the effects of different pollen parents on the content of e cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, AF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key onents of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × glizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the ent of stone cells (Figure 1b). Notably, the different pollen parents did not change the trends of stone cell and lignin content ng fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell and n content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of n and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental s. In particular, the stone cells and lignin content of YL fruit were significantly higher than that L fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the e cell content and lignin synthesis in the pear fruit.

haracterization of Stone Cells in Pear Fruit After Pollination by Two Pollen Parents
We selected mature fruit for Wiesner staining (phloroglucinol-HCl) to explore the effects of rent pollen parents on the distribution of stone cells in pear fruits. As shown in Figure 2, we can rve that, regardless of whether the 'Yali' or 'Cuiguan' was used as the pollen parent, the stone density in the inner flesh region of the fruit was relatively high. However, there is a large rence in the distribution of stone cells in the middle flesh region. The distribution density and e of the stone cell clusters in the mature fruit from 'Yali', as the pollen parent are larger than e in the mature fruit from 'Cuiguan' as the pollen parent. This finding indicates that metaxenia Forests 2019, 10, x FOR PEER REVIEW 5 of The acetyl bromide method (acetyl bromide soluble lignin content) determined the lign content of the inflorescence stem of Arabidopsis. The specific operation follows the method that w described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combination
We compared the differences in the content of stone cells in the eight developmental stages o the two pollination combinations to clarify the effects of different pollen parents on the content stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separate and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DA 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the ke components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining th content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin conten during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell an lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight development stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than th of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on th stone cell content and lignin synthesis in the pear fruit. Student's t test was used for the statistical analyses. Four fruits were selected for analysis at each period. * indicates a significant difference (p < 0.05).

Characterization of Stone Cells in Pear Fruit After Pollination by Two Pollen Parents
We selected mature fruit for Wiesner staining (phloroglucinol-HCl) to explore the effects different pollen parents on the distribution of stone cells in pear fruits. As shown in Figure 2, we ca observe that, regardless of whether the 'Yali' or 'Cuiguan' was used as the pollen parent, the ston cell density in the inner flesh region of the fruit was relatively high. However, there is a larg difference in the distribution of stone cells in the middle flesh region. The distribution density an range of the stone cell clusters in the mature fruit from 'Yali', as the pollen parent are larger tha those in the mature fruit from 'Cuiguan' as the pollen parent. This finding indicates that metaxen (CL) fruits, in addition to determining the content of stone cells ( Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell and lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the stone cell content and lignin synthesis in the pear fruit. The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin content of the inflorescence stem of Arabidopsis. The specific operation follows the method that was described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of the two pollination combinations to clarify the effects of different pollen parents on the content of stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell and lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the stone cell content and lignin synthesis in the pear fruit.

Characterization of Stone Cells in Pear Fruit After Pollination by Two Pollen Parents
We selected mature fruit for Wiesner staining (phloroglucinol-HCl) to explore the effects of different pollen parents on the distribution of stone cells in pear fruits. As shown in Figure 2, we can observe that, regardless of whether the 'Yali' or 'Cuiguan' was used as the pollen parent, the stone cell density in the inner flesh region of the fruit was relatively high. However, there is a large difference in the distribution of stone cells in the middle flesh region. The distribution density and range of the stone cell clusters in the mature fruit from 'Yali', as the pollen parent are larger than those in the mature fruit from 'Cuiguan' as the pollen parent. This finding indicates that metaxenia The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin content of the inflorescence stem of Arabidopsis. The specific operation follows the method that was described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of the two pollination combinations to clarify the effects of different pollen parents on the content of stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell and lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the stone cell content and lignin synthesis in the pear fruit.

× 'Lianglizaosu'
Forests 2019, 10, x FOR PEER REVIEW 5 The acetyl bromide method (acetyl bromide soluble lignin content) determined the li content of the inflorescence stem of Arabidopsis. The specific operation follows the method that described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combina
We compared the differences in the content of stone cells in the eight developmental stag the two pollination combinations to clarify the effects of different pollen parents on the conte stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separ and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 D 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin con during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the conte lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developme stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on stone cell content and lignin synthesis in the pear fruit. The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin content of the inflorescence stem of Arabidopsis. The specific operation follows the method that was described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of the two pollination combinations to clarify the effects of different pollen parents on the content of stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the content of stone cells ( Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell and lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the stone cell content and lignin synthesis in the pear fruit.

× 'Lianglizaosu'
Forests 2019, 10, x FOR PEER REVIEW The acetyl bromide method (acetyl bromide soluble li content of the inflorescence stem of Arabidopsis. The specific o described by Anderson et al. [37]. Three biological replicates w

Differences in Stone Cell and Lignin Content of Pear Fruit Obta
We compared the differences in the content of stone cells the two pollination combinations to clarify the effects of diffe stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan and collected pollinated pear fruits at eight developmental stag 63 DAF, 71 DAF, 87 DAF, and the mature period). It is wel components of stone cells. Therefore, we also measured th 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) content of stone cells ( Figure 1b).
Notably, the different pollen parents did not change the during fruit development, and they both showed a rise-fall te lignin content in YL and CL fruits peaked at 55 DAF. However lignin and stone cells in the CL fruit are lower than the YL fru stages. In particular, the stone cells and lignin content of YL fr of CL fruit from 39 DAF to fruit ripening. Therefore, differen stone cell content and lignin synthesis in the pear fruit.
. Student's t test was used for the statistical analyses. Four fruits were selected for analysis at each period. * indicates a significant difference (p < 0.05).

Characterization of Stone Cells in Pear Fruit after Pollination by Two Pollen Parents
We selected mature fruit for Wiesner staining (phloroglucinol-HCl) to explore the effects of different pollen parents on the distribution of stone cells in pear fruits. As shown in Figure 2, we can observe that, regardless of whether the 'Yali' or 'Cuiguan' was used as the pollen parent, the stone cell density in the inner flesh region of the fruit was relatively high. However, there is a large difference in the distribution of stone cells in the middle flesh region. The distribution density and range of the stone cell clusters in the mature fruit from 'Yali', as the pollen parent are larger than those in the mature fruit from 'Cuiguan' as the pollen parent. This finding indicates that metaxenia plays a regulatory role in the development of stone cells in fruits. This further suggests that the stone cell content of the pollen parents might determine the stone cell content of the hybrid fruits.  We further observed differences in the ultramicrostructure of the SCW of stone cells in YL and CL fruits ( Figure 3). The SCW of stone cells in the CL and YL fruits are composed of light (cellulose microfibrils) and dark stripes (lignin) ( Figure 3). Notably, the dark stripes of the SCWs of the stone cells in the YL fruit are significantly darker than the CL fruit. This finding indicates that the degree of lignification of stone cells in YL fruit is higher than that of CL fruit (Figure 3c,f). In addition, we also found that the stripes that were composed of lignin and cellulose microfibrils in the SCW of stone cells in CL fruit were loose at three developmental stages (Figure 3a-c). The light and dark stripes in the stone cells in the YL fruit are clearly separated and tightly bound, which results in a high density (Figure 3d-f). We hypothesize that the higher degree of lignification and density of the SCW structure may give the stone cells greater rigidity. This finding also reflects the effect of different pollination on the deposition of lignin in the SCW of stone cells. The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin content of the inflorescence stem of Arabidopsis. The specific operation follows the method that was described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of the two pollination combinations to clarify the effects of different pollen parents on the content of stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell and lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the stone cell content and lignin synthesis in the pear fruit.

Characterization of Stone Cells in Pear Fruit After Pollination by Two Pollen Parents
We selected mature fruit for Wiesner staining (phloroglucinol-HCl) to explore the effects of different pollen parents on the distribution of stone cells in pear fruits. As shown in Figure 2, we can observe that, regardless of whether the 'Yali' or 'Cuiguan' was used as the pollen parent, the stone cell density in the inner flesh region of the fruit was relatively high. However, there is a large difference in the distribution of stone cells in the middle flesh region. The distribution density and range of the stone cell clusters in the mature fruit from 'Yali', as the pollen parent are larger than those in the mature fruit from 'Cuiguan' as the pollen parent. This finding indicates that metaxenia Forests 2019, 10, x FOR PEER REVIEW The acetyl bromide method (acetyl bromide soluble l content of the inflorescence stem of Arabidopsis. The specific o described by Anderson et al. [37]. Three biological replicates w

Differences in Stone Cell and Lignin Content of Pear Fruit Obt
We compared the differences in the content of stone cell the two pollination combinations to clarify the effects of diff stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan and collected pollinated pear fruits at eight developmental sta 63 DAF, 71 DAF, 87 DAF, and the mature period). It is wel components of stone cells. Therefore, we also measured t 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the during fruit development, and they both showed a rise-fall t lignin content in YL and CL fruits peaked at 55 DAF. Howeve lignin and stone cells in the CL fruit are lower than the YL fru stages. In particular, the stone cells and lignin content of YL fr of CL fruit from 39 DAF to fruit ripening. Therefore, differen stone cell content and lignin synthesis in the pear fruit.

Characterization of Stone Cells in Pear Fruit After Pollination
We selected mature fruit for Wiesner staining (phlorog different pollen parents on the distribution of stone cells in pe observe that, regardless of whether the 'Yali' or 'Cuiguan' wa cell density in the inner flesh region of the fruit was relati difference in the distribution of stone cells in the middle fles range of the stone cell clusters in the mature fruit from 'Yali those in the mature fruit from 'Cuiguan' as the pollen parent The acetyl bromide method (acetyl bromide soluble lig content of the inflorescence stem of Arabidopsis. The specific op described by Anderson et al. [37]. Three biological replicates w

Differences in Stone Cell and Lignin Content of Pear Fruit Obtai
We compared the differences in the content of stone cells the two pollination combinations to clarify the effects of differ stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' and collected pollinated pear fruits at eight developmental stag 63 DAF, 71 DAF, 87 DAF, and the mature period). It is wellcomponents of stone cells. Therefore, we also measured th 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) f content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the tr during fruit development, and they both showed a rise-fall ten lignin content in YL and CL fruits peaked at 55 DAF. However, lignin and stone cells in the CL fruit are lower than the YL frui stages. In particular, the stone cells and lignin content of YL fru of CL fruit from 39 DAF to fruit ripening. Therefore, different stone cell content and lignin synthesis in the pear fruit.

Characterization of Stone Cells in Pear Fruit After Pollination by
We selected mature fruit for Wiesner staining (phloroglu different pollen parents on the distribution of stone cells in pea observe that, regardless of whether the 'Yali' or 'Cuiguan' was cell density in the inner flesh region of the fruit was relativ difference in the distribution of stone cells in the middle flesh range of the stone cell clusters in the mature fruit from 'Yali', those in the mature fruit from 'Cuiguan' as the pollen parent. T The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin content of the inflorescence stem of Arabidopsis. The specific operation follows the method that was described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of the two pollination combinations to clarify the effects of different pollen parents on the content of stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content during fruit development, and they both showed a rise-fall tendency ( Figure 1). The stone cell and lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the stone cell content and lignin synthesis in the pear fruit.

Characterization of Stone Cells in Pear Fruit After Pollination by Two Pollen Parents
We selected mature fruit for Wiesner staining (phloroglucinol-HCl) to explore the effects of different pollen parents on the distribution of stone cells in pear fruits. As shown in Figure 2, we can observe that, regardless of whether the 'Yali' or 'Cuiguan' was used as the pollen parent, the stone cell density in the inner flesh region of the fruit was relatively high. However, there is a large difference in the distribution of stone cells in the middle flesh region. The distribution density and range of the stone cell clusters in the mature fruit from 'Yali', as the pollen parent are larger than those in the mature fruit from 'Cuiguan' as the pollen parent. This finding indicates that metaxenia (CL) fruit: (a) YL fruit; and, (b) CL fruit. The blue marked area is the inner flesh region, and the green marked area is the middle flesh region.
We further observed differences in the ultramicrostructure of the SCW of stone cells in YL and CL fruits ( Figure 3). The SCW of stone cells in the CL and YL fruits are composed of light (cellulose microfibrils) and dark stripes (lignin) (Figure 3). Notably, the dark stripes of the SCWs of the stone cells in the YL fruit are significantly darker than the CL fruit. This finding indicates that the degree of lignification of stone cells in YL fruit is higher than that of CL fruit (Figure 3c,f). In addition, we also found that the stripes that were composed of lignin and cellulose microfibrils in the SCW of stone cells in CL fruit were loose at three developmental stages (Figure 3a-c). The light and dark stripes in the stone cells in the YL fruit are clearly separated and tightly bound, which results in a high density (Figure 3d-f). We hypothesize that the higher degree of lignification and density of the SCW structure may give the stone cells greater rigidity. This finding also reflects the effect of different pollination on the deposition of lignin in the SCW of stone cells.

Analysis of Transcriptomic Data and Mapping to Reference Genome Sequences
We performed transcriptome sequencing of the two types of pear fruit to reveal the molecular mechanism underlying the differences in stone cell development in CL and YL fruits. The SCW of the stone cells began to thicken and lignify at 23 DAF [11]. The stone cell and lignin content peaked at 55 DAF and then reached a steady level as the fruit matured ( Figure 1). Therefore, we selected the initial phase (23 DAF), the vigorous phase (55 DAF), and the stable phase (mature period) of stone cell formation for comparative transcriptome analysis. We named the YL fruits that were collected at 23 DAF, 55 DAF, and the mature period (MP) as YL-23, YL-55, and YL-MP, respectively. Similarly, CL-23, CL-55, and CL-MP represent the CL fruits that were collected at 23 DAF, 55 DAF, and MP, respectively. Whole-genome sequencing of Chinese white pear (P. bretschneideri Rehd.) has been completed as a reference genome for this study [1].
It can be observed from Supplementary Table S2 that a total of 47.67 Gb of Clean Data was obtained after sequencing quality control, the Q30 base percentage of each sample was not less than 96.60%, and the GC content was not less than 48.09%. This result indicates that the sequencing yield and sequence quality were high. Mapping efficiency refers to the percentage of Mapped Reads in Clean Reads. The mapping efficiency between the reads and the reference genome of each sample ranged from 71.40% to 72.99%. These results indicate that we obtained transcriptome data that could be used for subsequent analysis and that the selected reference genome assembly was sufficient for information analysis.

Analysis of Transcriptomic Data and Mapping to Reference Genome Sequences
We performed transcriptome sequencing of the two types of pear fruit to reveal the molecular mechanism underlying the differences in stone cell development in CL and YL fruits. The SCW of the stone cells began to thicken and lignify at 23 DAF [11]. The stone cell and lignin content peaked at 55 DAF and then reached a steady level as the fruit matured ( Figure 1). Therefore, we selected the initial phase (23 DAF), the vigorous phase (55 DAF), and the stable phase (mature period) of stone cell formation for comparative transcriptome analysis. We named the YL fruits that were collected at 23 DAF, 55 DAF, and the mature period (MP) as YL-23, YL-55, and YL-MP, respectively. Similarly, CL-23, CL-55, and CL-MP represent the CL fruits that were collected at 23 DAF, 55 DAF, and MP, respectively. Whole-genome sequencing of Chinese white pear (P. bretschneideri Rehd.) has been completed as a reference genome for this study [1].
It can be observed from Supplementary Table S2 that a total of 47.67 Gb of Clean Data was obtained after sequencing quality control, the Q30 base percentage of each sample was not less than 96.60%, and the GC content was not less than 48.09%. This result indicates that the sequencing yield and sequence quality were high. Mapping efficiency refers to the percentage of Mapped Reads in Clean Reads. The mapping efficiency between the reads and the reference genome of each sample ranged from 71.40% to 72.99%. These results indicate that we obtained transcriptome data that could be used for subsequent analysis and that the selected reference genome assembly was sufficient for information analysis.

Identification of DEGs in Pear Fruits Pollinated by Two Pollen Parents Based on Transcriptome Sequencing
We identified a total of 9018 DEGs between the fruits of YL and CL at three different developmental stages, according to the results of transcriptome sequencing (Figure 4 and Supplementary Table S3). Among these DEGs, as compared with YL fruit, there were 4245 upregulated genes and 4773 downregulated genes in CL fruits. As shown in Figure 4a, YL and CL fruits had the highest number of DEGs at 23 DAF, followed by 55 DAF, with the lowest number of DEGs between the two types of pear fruits being observed at maturity. In each library, YL-23 vs_CL-23, YL-55 vs_CL-55, and YL-MP vs_CL-MP had 5454, 2583, and 981 DEGs, respectively (Figure 4b). These results indicate that pollination with different pollen parents resulted in significant changes in the transcriptional levels of a large number of genes in early fruit development. However, the effect on gene expression in fruits in the middle and late stages of development was relatively small. These results indicate that early fruit development in pears is a highly active process, while the metabolic processes in mature fruits are slowed. We identified a total of 9018 DEGs between the fruits of YL and CL at three different developmental stages, according to the results of transcriptome sequencing (Figure 4 and Supplementary Table S3). Among these DEGs, as compared with YL fruit, there were 4245 upregulated genes and 4773 downregulated genes in CL fruits. As shown in Figure 4a, YL and CL fruits had the highest number of DEGs at 23 DAF, followed by 55 DAF, with the lowest number of DEGs between the two types of pear fruits being observed at maturity. In each library, YL-23 vs_CL-23, YL-55 vs_CL-55, and YL-MP vs_CL-MP had 5454, 2583, and 981 DEGs, respectively (Figure 4b). These results indicate that pollination with different pollen parents resulted in significant changes in the transcriptional levels of a large number of genes in early fruit development. However, the effect on gene expression in fruits in the middle and late stages of development was relatively small. These results indicate that early fruit development in pears is a highly active process, while the metabolic processes in mature fruits are slowed. We used multiple databases to annotate the DEGs to predict the functions of the DEGs (Supplementary Table S4 and Supplementary Figure S2). According to the KEGG annotation, most of the DEGs were related to plant hormone signal transduction, phenylpropanoid biosynthesis, phenylalanine metabolism, starch and sucrose metabolism, biosynthesis of amino acids, and carbon metabolism ( Figure 5). Notably, at 23 DAF, there were 98 DEGs that were associated with phenylalanine metabolism and phenylpropanoid metabolism between YL and CL fruits. However, the number of these DEGs between YL and CL fruits was only 30 and 13 at 55 DAF and at maturity. In particular, we used the KEGG method to annotate upregulated genes and downregulated genes at different developmental stages between YL and CL fruits. Most genes that were related to phenylpropane metabolism in CL fruits showed a downward trend during the early stages of fruit development ( Figure 5). We used multiple databases to annotate the DEGs to predict the functions of the DEGs (Supplementary Table S4 and Figure S2). According to the KEGG annotation, most of the DEGs were related to plant hormone signal transduction, phenylpropanoid biosynthesis, phenylalanine metabolism, starch and sucrose metabolism, biosynthesis of amino acids, and carbon metabolism ( Figure 5). Notably, at 23 DAF, there were 98 DEGs that were associated with phenylalanine metabolism and phenylpropanoid metabolism between YL and CL fruits. However, the number of these DEGs between YL and CL fruits was only 30 and 13 at 55 DAF and at maturity. In particular, we used the KEGG method to annotate upregulated genes and downregulated genes at different developmental stages between YL and CL fruits. Most genes that were related to phenylpropane metabolism in CL fruits showed a downward trend during the early stages of fruit development ( Figure 5).    Phenylpropanoid biosynthesis and phenylalanine metabolism are marked with an asterisk because they are closely related to lignin metabolism. Up-regulated genes in A_vs._B: genes whose expression level in sample B is higher than that in sample A; otherwise they are down-regulated genes.
We further performed KEGG pathway enrichment analysis with the DEGs in YL-23 vs_CL-23, YL-55 vs_CL-55, and YL-MP vs_CL-MP ( Figure 6). The results indicate that plant hormone signal transduction was highly enriched throughout the three key developmental stages (23 DAF, 55 DAF, and mature period). This result suggests that metaxenia has a significant effect on hormone metabolism in fruits. In addition, phenylpropanoid biosynthesis and phenylalanine metabolism were enriched during the early stages of fruit development. When combined with the results of KEGG annotation, it was shown that metaxenia significantly affected the phenylpropanoid metabolic pathway (including lignin biosynthesis) in the early stage of fruit development. At 55 DAF and at maturity, the number of DEGs that were associated with phenylpropanoid metabolism between YL and CL fruits gradually decreased. We speculate that the difference in transcription of phenylpropanoid/phenylalanine metabolism-related genes in the early stage of fruit development might be the cause of the difference in the content of stone cell clusters in YL and CL fruits. The abscissa represents the proportion of genes in a metabolic pathway to the total number of annotated genes. Phenylpropanoid biosynthesis and phenylalanine metabolism are marked with an asterisk because they are closely related to lignin metabolism. Up-regulated genes in A_vs._B: genes whose expression level in sample B is higher than that in sample A; otherwise they are down-regulated genes.
We further performed KEGG pathway enrichment analysis with the DEGs in YL-23 vs_CL-23, YL-55 vs_CL-55, and YL-MP vs_CL-MP ( Figure 6). The results indicate that plant hormone signal transduction was highly enriched throughout the three key developmental stages (23 DAF, 55 DAF, and mature period). This result suggests that metaxenia has a significant effect on hormone metabolism in fruits. In addition, phenylpropanoid biosynthesis and phenylalanine metabolism were enriched during the early stages of fruit development. When combined with the results of KEGG annotation, it was shown that metaxenia significantly affected the phenylpropanoid metabolic pathway (including lignin biosynthesis) in the early stage of fruit development. At 55 DAF and at maturity, the number of DEGs that were associated with phenylpropanoid metabolism between YL and CL fruits gradually decreased. We speculate that the difference in transcription of phenylpropanoid/phenylalanine metabolism-related genes in the early stage of fruit development might be the cause of the difference in the content of stone cell clusters in YL and CL fruits.

Differentially Expressed Transcription Factors (TFs) between YL and CL fruits
Lignin is one of the main components of stone cells, and lignin metabolism is an important branch of phenylpropanoid metabolism. Therefore, we screened for differentially expressed phenylpropanoid pathway-related TFs between YL and CL fruits (Supplementary Tables S5 and S6). As shown in Supplementary Table S5, several MYBs (V-myb myeloblastosis viral oncogene homolog proteins), WRKYs, and KNOXs (KNOTTED1-like homeobox) were differentially expressed between the two types of pear fruits.
The KEGG annotation showed that the number of DEGs associated with phenylpropanoid metabolism was the highest in the fruit at 23 DAF. Therefore, we focused on the types of TFs that were differentially expressed at 23 DAF. Previous studies have shown that PbMYB169 (Pbr012624, LOC103959908) and PbKNOX1 (Pbr019805, LOC103951709) positively and negatively regulate the lignification of pear stone cells, respectively [35,38]. The expression level of PbMYB169 in YL fruits of 23 DAF was significantly higher than that of CL fruits, while PbKNOX1 showed the opposite pattern ( Figure 7). The differential expression of these genes might result in altered transcriptional levels of the regulated target genes (structural genes for lignin synthesis).
In addition, we analysed the expression profiles of five putative positive regulatory TFs (PbWLIM1a, PbWLIM1b, PbMYB4, PbMYB30, and PbMYB171) that were related to lignin metabolism in pear fruit [32,39]. The expression levels of these TFs in YL fruits were higher than those in the CL fruits (Figure 7), which suggested that the differential expression of these TFs might be one of the reasons for the difference in phenylpropanoid metabolism between the two types of pear fruits. Figure 6. KEGG pathway enrichment analysis of differentially expressed genes. Each circle in the figure represents a metabolic pathway of KEGG. The ordinate indicates the name of the metabolic pathway. The abscissa represents the enrichment factor. Phenylpropanoid biosynthesis (ko00940) and phenylalanine metabolism (ko00360) were enriched in the early stage (23 DAF) of pear fruit development.

Differentially Expressed Transcription Factors (TFs) between YL and CL fruits
Lignin is one of the main components of stone cells, and lignin metabolism is an important branch of phenylpropanoid metabolism. Therefore, we screened for differentially expressed phenylpropanoid pathway-related TFs between YL and CL fruits (Supplementary Table S5 and  Supplementary Table S6). As shown in Supplementary Table S5, several MYBs (V-myb myeloblastosis viral oncogene homolog proteins), WRKYs, and KNOXs (KNOTTED1-like homeobox) were differentially expressed between the two types of pear fruits.
The KEGG annotation showed that the number of DEGs associated with phenylpropanoid metabolism was the highest in the fruit at 23 DAF. Therefore, we focused on the types of TFs that were differentially expressed at 23 DAF. Previous studies have shown that PbMYB169 (Pbr012624, LOC103959908) and PbKNOX1 (Pbr019805, LOC103951709) positively and negatively regulate the lignification of pear stone cells, respectively [35,38]. The expression level of PbMYB169 in YL fruits of 23 DAF was significantly higher than that of CL fruits, while PbKNOX1 showed the opposite pattern (Figure 7). The differential expression of these genes might result in altered transcriptional levels of the regulated target genes (structural genes for lignin synthesis).
In addition, we analysed the expression profiles of five putative positive regulatory TFs (PbWLIM1a, PbWLIM1b, PbMYB4, PbMYB30, and PbMYB171) that were related to lignin metabolism in pear fruit [32,39]. The expression levels of these TFs in YL fruits were higher than those in the CL fruits (Figure 7), which suggested that the differential expression of these TFs might be one of the reasons for the difference in phenylpropanoid metabolism between the two types of pear fruits.  The acetyl bromide method (acetyl bromide soluble lignin content) d content of the inflorescence stem of Arabidopsis. The specific operation follows described by Anderson et al. [37]. Three biological replicates were performed f

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Po
We compared the differences in the content of stone cells in the eight dev the two pollination combinations to clarify the effects of different pollen pare stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lia and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 D 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lig components of stone cells. Therefore, we also measured the lignin conten 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in additio content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone c during fruit development, and they both showed a rise-fall tendency (Figure lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observ lignin and stone cells in the CL fruit are lower than the YL fruit in the selected stages. In particular, the stone cells and lignin content of YL fruit were signific of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents stone cell content and lignin synthesis in the pear fruit. The acetyl bromide method (ace content of the inflorescence stem of Ar described by Anderson et al. [37]. Thre

Differences in Stone Cell and Lignin C
We compared the differences in th the two pollination combinations to cl stone cells in the fruit (Figure 1a). We u and collected pollinated pear fruits at e 63 DAF, 71 DAF, 87 DAF, and the m components of stone cells. Therefore 'Lianglizaosu'♀(YL) and 'Cuiguan'♂ content of stone cells (Figure 1b).
Notably, the different pollen pare during fruit development, and they bo lignin content in YL and CL fruits peak lignin and stone cells in the CL fruit ar stages. In particular, the stone cells and of CL fruit from 39 DAF to fruit ripen stone cell content and lignin synthesis i The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin ntent of the inflorescence stem of Arabidopsis. The specific operation follows the method that was scribed by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of e two pollination combinations to clarify the effects of different pollen parents on the content of one cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately d collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key mponents of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × ianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the ntent of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content ring fruit development, and they both showed a rise-fall tendency (Figure 1). The stone cell and nin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of nin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental ages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the one cell content and lignin synthesis in the pear fruit. The acetyl bromide method (acetyl bromide soluble lignin content) determined the lig content of the inflorescence stem of Arabidopsis. The specific operation follows the method that w described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinati
We compared the differences in the content of stone cells in the eight developmental stages the two pollination combinations to clarify the effects of different pollen parents on the content stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separat and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DA 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the k components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin cont during fruit development, and they both showed a rise-fall tendency (Figure 1). The stone cell a lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the conten lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmen stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than t of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on stone cell content and lignin synthesis in the pear fruit.

Differences in Monolignol Metabolism between YL and CL Fruits
We found that multiple TFs that regulate phenylpropanoid or lignin metabolism had significant differences in the transcription levels between the two types of pear fruit, especially at 23 DAF. Therefore, we systematically compared the structural gene expression profiles in the lignin biosynthesis, transport, and polymerization pathways of pear fruit (Supplementary Table S7 and Figure S3). We previously analysed members of the 4CL (4-coumarate: CoA ligase), OMT (O-methyltransferase), CCR (cinnamoyl CoA reductase), CAD (cinnamyl alcohol dehydrogenase), UGT (family-1 uridine diphosphate-glycosyltransferases), and DIR (dirigent) gene families [11,17,18,40]. Therefore, members of these gene families have a fixed nomenclature. The naming of members of the shikimate O-hydroxycinnamoyltransferase (HCT) and laccase (LAC) gene family was performed according to Xue et al. [19] and Ma et al. [41].
It can be observed from Figure 8 that the transcript abundance of other lignin synthesis genes in YL-23 (YL fruit at 23 DAF) was higher than that in CL-23 (CL fruit at 23 DAF), except for PbC4H1 and PbCAD3. The transcriptional levels of TFs or structural genes were significantly different at 23 DAF, which further indicated that stone cell formation and lignin deposition occurred during the early stages of pear fruit development.  The acetyl bromide method (acetyl bromide soluble lignin content) determined the lignin content of the inflorescence stem of Arabidopsis. The specific operation follows the method that was described by Anderson et al. [37]. Three biological replicates were performed for each sample.

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination Combinations
We compared the differences in the content of stone cells in the eight developmental stages of the two pollination combinations to clarify the effects of different pollen parents on the content of stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu' separately and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 DAF, 55 DAF, 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is one of the key components of stone cells. Therefore, we also measured the lignin content in the 'Yali'♂ × 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to determining the content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lignin content during fruit development, and they both showed a rise-fall tendency (Figure 1). The stone cell and lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that the content of lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight developmental stages. In particular, the stone cells and lignin content of YL fruit were significantly higher than that of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an effect on the stone cell content and lignin synthesis in the pear fruit. The acetyl bromide method (acetyl bromide soluble lignin content) deter content of the inflorescence stem of Arabidopsis. The specific operation follows the described by Anderson et al. [37]. Three biological replicates were performed for e

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollin
We compared the differences in the content of stone cells in the eight develo the two pollination combinations to clarify the effects of different pollen parents stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Liangl and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin components of stone cells. Therefore, we also measured the lignin content in 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition t content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell during fruit development, and they both showed a rise-fall tendency (Figure 1). lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eig stages. In particular, the stone cells and lignin content of YL fruit were significantl of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents hav stone cell content and lignin synthesis in the pear fruit. The acetyl bromide method (acetyl bromide soluble lignin content) determined content of the inflorescence stem of Arabidopsis. The specific operation follows the meth described by Anderson et al. [37]. Three biological replicates were performed for each sa

Differences in Stone Cell and Lignin Content of Pear Fruit Obtained from Two Pollination C
We compared the differences in the content of stone cells in the eight developmen the two pollination combinations to clarify the effects of different pollen parents on th stone cells in the fruit (Figure 1a). We used 'Yali' and 'Cuiguan' to pollinate 'Lianglizaosu and collected pollinated pear fruits at eight developmental stages (23 DAF, 39 DAF, 47 D 63 DAF, 71 DAF, 87 DAF, and the mature period). It is well-known that lignin is on components of stone cells. Therefore, we also measured the lignin content in the 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Lianglizaosu'♀(CL) fruits, in addition to dete content of stone cells (Figure 1b).
Notably, the different pollen parents did not change the trends of stone cell and lig during fruit development, and they both showed a rise-fall tendency (Figure 1). The st lignin content in YL and CL fruits peaked at 55 DAF. However, it can be observed that th lignin and stone cells in the CL fruit are lower than the YL fruit in the selected eight dev stages. In particular, the stone cells and lignin content of YL fruit were significantly high of CL fruit from 39 DAF to fruit ripening. Therefore, different pollen parents have an e stone cell content and lignin synthesis in the pear fruit. The acetyl bromide method (acetyl brom content of the inflorescence stem of Arabidopsis. described by Anderson et al. [37]. Three biologic

Differences in Stone Cell and Lignin Content of P
We compared the differences in the content the two pollination combinations to clarify the e stone cells in the fruit (Figure 1a). We used 'Yali' and collected pollinated pear fruits at eight devel 63 DAF, 71 DAF, 87 DAF, and the mature peri components of stone cells. Therefore, we also 'Lianglizaosu'♀(YL) and 'Cuiguan'♂× 'Liangli content of stone cells (Figure 1b).
We selected 30 key genes involved in lignin synthesis for qRT-PCR validation to verify the accuracy and reproducibility of the RNA-seq results (Figure 8). The results showed that the transcript abundance and FPKM values of most genes were largely consistent during the three developmental stages of pear fruit, which indicated that the results of transcriptome sequencing were reliable.

Cloning and Phylogenetic Analysis of Putative Lignin-Related PbC4H2
The results of transcriptome sequencing and qRT-PCR showed that the transcript abundance of PbC4H2 was significantly different between the YL and CL fruits, and the expression pattern of PbC4H2 at the three developmental stages of the fruit was consistent with the changes in stone cells and lignin content (Figure 1 and Supplementary Figure S3). In addition, the function of C4H in pears, as a second key enzyme in lignin synthesis [42], has not been studied. Therefore, we selected this gene for functional verification to demonstrate whether PbC4H2 plays a role in stone cell lignification at different stages of pear fruit development.
We designed specific primers to clone the full-length sequences of the coding regions (CDS) of PbC4H2 (Pbr017290.1) based on information from the pear genome database. The results of maximum likelihood (ML) phylogenetic tree clustering indicated that PbC4H2 and dicotyledon-derived C4Hs clustered into a clade and had close genetic relationships with known lignin-related C4Hs, such as AtC4H, AaC4H, LrC4H, GbC4H, CsC4Ha, CsC4Hb, and CsC4Hc ( Figure 9).
We designed specific primers to clone the full-length sequences of the coding regions (CDS) of PbC4H2 (Pbr017290.1) based on information from the pear genome database. The results of maximum likelihood (ML) phylogenetic tree clustering indicated that PbC4H2 and dicotyledon-derived C4Hs clustered into a clade and had close genetic relationships with known lignin-related C4Hs, such as AtC4H, AaC4H, LrC4H, GbC4H, CsC4Ha, CsC4Hb, and CsC4Hc ( Figure 9).

Complementation Analysis of the c4h Mutant with PbC4H2
A total of five rescued lines were obtained from the transformation of the Arabidopsis c4h mutant. We performed further analysis by selecting three transgenic lines (rescued line-1, rescued line-4, and rescued line-5) with higher transcript levels of PbC4H2 (Supplementary Figure S4). The cross-sections of the inflorescence stems of the three genotypes (WT, c4h mutants, and rescued plants) of Arabidopsis were used for toluidine blue staining, Wiesner staining, and TEM observation ( Figure 10). The results of Wiesner and toluidine blue staining showed that the degree of staining of the xylem of the c4h mutant was weaker than that of WT and rescued plants, which suggested that the lignin deposition in the cell wall of the c4h mutant xylem cells was decreased. In addition, the xylem cells of the c4h mutant show some wall irregularities and even collapsed, which is consistent with previous report [43]. However, the heterologous expression of PbC4H2 in the c4h mutant resulted in xylem cells recovering the phenotype of WT plants.
( Figure 10). The results of Wiesner and toluidine blue staining showed that the degree of staining of the xylem of the c4h mutant was weaker than that of WT and rescued plants, which suggested that the lignin deposition in the cell wall of the c4h mutant xylem cells was decreased. In addition, the xylem cells of the c4h mutant show some wall irregularities and even collapsed, which is consistent with previous report [43]. However, the heterologous expression of PbC4H2 in the c4h mutant resulted in xylem cells recovering the phenotype of WT plants. We found that the AcBr lignin content in the inflorescence stem of the c4h mutant was reduced by approximately 24% s compared with that of the WT plants, and the difference reached a significant level, through the detection of acetyl bromide (AcBr) lignin (Figure 11a). It is worth noting that the heterologous expression of PbC4H2 in the c4h mutant can almost restore the AcBr lignin content of the c4h mutant to the level of WT plants.
In addition, the TEM observations revealed that the PbC4H2 gene also promoted the development of SCW in vessel cells ( Figure 10). Although the heterologous expression of PbC4H2 did not completely restore the cell wall thickness of the mutant to the level of WT plants, it still significantly promoted the SCW formation of xylem cells of mutant plants ( Figure 10 and Figure  11b). These results indicate that PbC4H2 plays an important role in the development and lignification of SCWs. We found that the AcBr lignin content in the inflorescence stem of the c4h mutant was reduced by approximately 24% s compared with that of the WT plants, and the difference reached a significant level, through the detection of acetyl bromide (AcBr) lignin (Figure 11a). It is worth noting that the heterologous expression of PbC4H2 in the c4h mutant can almost restore the AcBr lignin content of the c4h mutant to the level of WT plants.

Discussion
The deposition of lignin in fruits has a certain positive effect [44], but it also has some negative effects, such as lignification in stone cells of pears and in citrus juice sacs [19,45]. At present, there are few studies regarding the regulation of lignification of pear stone cells by metaxenia, and the related molecular mechanisms have not been elucidated. In this study, varieties with high and low stone cell content were used as the pollen parent to analyse its effect on stone cell formation in the pear fruit after pollination.
Through transcriptome sequencing, we found that PbPAL2, PbC4H2, and Pb4CL1, which were In addition, the TEM observations revealed that the PbC4H2 gene also promoted the development of SCW in vessel cells ( Figure 10). Although the heterologous expression of PbC4H2 did not completely restore the cell wall thickness of the mutant to the level of WT plants, it still significantly promoted the SCW formation of xylem cells of mutant plants (Figures 10 and 11b). These results indicate that PbC4H2 plays an important role in the development and lignification of SCWs.

Discussion
The deposition of lignin in fruits has a certain positive effect [44], but it also has some negative effects, such as lignification in stone cells of pears and in citrus juice sacs [19,45]. At present, there are few studies regarding the regulation of lignification of pear stone cells by metaxenia, and the related molecular mechanisms have not been elucidated. In this study, varieties with high and low stone cell content were used as the pollen parent to analyse its effect on stone cell formation in the pear fruit after pollination.
Through transcriptome sequencing, we found that PbPAL2, PbC4H2, and Pb4CL1, which were involved in the general phenylpropanoid pathway, were upregulated in YL fruits (especially at 23 and 55 DAF) (Figure 8). This upregulation not only significantly affects the monolignol metabolic flux, but also provides sufficient anabolic intermediates for monolignol synthesis. In addition, the transcript abundance of PbC3H, PbHCT2, PbHCT50, PbCCoAOMT1, and PbCCoAOMT2 in YL fruit was also significantly higher than that of CL fruit, which promoted the conversion of hydroxycinnamic acid to hydroxy-CoA thioester (Supplementary Table S7 and Figure S3). Previous studies have shown that the PbCCoAOMT-catalysed reaction is a rate-limiting step in the metabolism of pear lignin [1]. Therefore, high levels of PbCCoAOMT expression may significantly increase the accumulation of the lignin monomer.
Our analysis revealed that the key genes in the monolignol-specific biosynthesis pathway (PbCCR1, PbCCR2, PbF5H1, PbF5H2, PbF5H3, and PbCOMT), the transport pathway (PbUGT72E and PbBGLU), and the polymerization pathway (PbPOD1, PbPOD2, PbPOD3, PbLAC1, PbLAC2, PbLAC3, PbLAC15, PbLAC18, PbLAC20, and PbDIR4) were significantly higher in the YL fruit of 23 DAF than in the CL fruit of the same developmental stage (Supplementary Figure S3). This finding suggests that the biosynthesis of lignin monomer in YL fruit is stronger than that of CL fruit during the development of pear fruit, especially in the early stage of fruit development, which might be one of the main reasons for the high content of stone cells in YL fruit. Interestingly, we noticed that the transcript abundance of lignin-specific LACs in YL fruit was higher than that of CL fruit. Xue et al. [19] demonstrated that salicylic acid (SA) can induce PbrmiR397a expression and inhibit LAC transcription [19]. It has been clarified that the catalytic reaction of C4H and synthesis of SA compete for the same precursor (cinnamic acid) [14]. The high level of transcription of PbC4H2 in YL fruit causes an increase in the amount of cinnamic acid participating in lignin synthesis, which might reduce the SA level in the fruit to some extent. This results in a decrease in the transcriptional level of PbrmiR397a, resulting in an up-regulation of lignin-specific LACs ( Figure 12). Cinnamate 4-hydroxylase (EC 1.14.13.11) belongs to the CYP73A subfamily of the cytochrome monooxygenase superfamily [49]. This protein is not only the second key enzyme in the general phenylpropanoid pathway but it also plays a highly important role in the entire pathway [14,42]. Previous studies have shown that the product of C4H, p-coumaric acid, is a key substance affecting the metabolism of pear lignin and the development of stone cells [4,13]. Therefore, we investigated the function of the DEG PbC4H2 in SCW development and lignification in this study. After the heterologous expression of PbC4H2 in the c4h mutant, the lignin content of the inflorescence stems of the rescued plants increased by approximately 15% and the SCW thickness increased by approximately 2% (Figure 11). In particular, PbC4H2 can restore the collapsed phenotype of the cell wall of the c4h mutant ( Figure 10). This result strongly indicates that PbC4H2 plays an important role in pear cell wall development and lignin synthesis. Interestingly, we found that PbC4H1 and PbC4H2 showed distinct expression patterns, which are similar to those that were observed in tea plants [49]. PbC4H1 exhibits high transcript abundance during fruit ripening (Figure 8). The synthesis of lignin and the development of stone cells have largely stopped in this stage [7]. Therefore, PbC4H1 might not be a lignin-specific C4H and it may be responsible for the biosynthesis of other phenylpropanoids. Since studies have shown that phenylpropanoid metabolism plays an important role in fruit ripening, its function in pear fruit warrants further research.

Conclusion
In summary, this study used 'Cuiguan' (pollen parent) × 'Lianglizaosu' (seed parent) and 'Yali' (pollen parent) × 'Lianglizaosu' (seed parent) as the materials to demonstrate that metaxenia can significantly affect stone cell development and lignin metabolism in pear fruit. RNA-seq showed that the pollen parents with different stone cell contents had a significant effect on the expression of structural genes (PbC4H, Pb4CL, PbC3H, PbHCT, PbCCoAOMT, PbCCR, PbF5H, PbCOMT, PbUGT72E, PbBGLU, PbPOD, PbLAC, and PbDIR) and TFs (PbKNOX1, PbWLIM1a, PbWLIM1b, PbMYB4, PbMYB30, PbMYB169, and PbMYB171) that were related to lignin metabolism in the early Notably, the difference in lignin and stone cell contents between YL and CL fruits was not significant, although the expression levels of most lignin synthesis-related structural genes and TFs in YL fruits were higher than those in CL fruits at 23 DAF (Figure 1). This result might be due to the formation of stone cells and lignin deposition with hysteresis when compared to gene expression. Xue et al. [19] also found that most lignin-specific LACs reached the peak of transcription level at 35 DAF, but the stone cell content in the flesh reached a high level at 49 DAF [19].
Importantly, the results of transcriptome sequencing and qRT-PCR suggest that metaxenia has a significant effect on lignin metabolism in the early stage of pear fruit development (Supplementary Figure S3). It has been clarified that the parenchyma cells of the flesh first differentiate into sclereid primordium cells in the early stage of pear fruit development, and then further lignified into stone cells [7,11,28]. Therefore, we speculate that lignin synthesis in the early stage of pear fruit development determines the number of sclereid primordium cells. As stone cells and lignin are not degraded after they are formed, they remain in the flesh; therefore, the absolute content of stone cells and lignin in the fruit remains constant, even if the stone cell development and lignification process stop in the late stage of fruit development. It is possible that the growth of stone cell clusters represents mainly volume expansion and the number of newly formed sclereid primordium cells is limited in the middle and late stages of pear fruit development. Therefore, the number of sclereid primordium cells in the early stage of fruit development directly determines the content of stone cells in mature pear fruit.
More strikingly, we found that plant hormone signal transduction-related genes are differentially expressed in YL and CL fruits ( Figure 6 and Supplementary Table S8), which suggests that metaxenia has a significant effect on hormone levels in hybrid fruits. There is a hypothesis that seeds in the fruit are heterozygous for the parental genotype. Metaxenia might alter the synthesis of hormones in heterozygous seeds, thereby affecting the levels of hormones secreted into the fruit and ultimately regulating the relevant metabolic processes [26,46]. Therefore, we speculate that the effect of metaxenia on lignin biosynthesis in pear fruit might be achieved by regulating hormone metabolism. In the early stage of fruit development, a large number of auxin signal transduction-related genes have significant differences in transcript abundance between YL and CL fruits, such as Aux/IAA (auxin/indoleacetic acids) family genes, auxin response factors (ARFs), auxin-responsive proteins, and indole-3-acetic acid-amido synthetase GH3s (Supplementary Table S8) [47,48]. This phenomenon might have a large effect on the content of auxin and its signal transduction in pear fruit. Previous studies have shown that auxin can inhibit the transcription of the lignin biosynthesis negative regulator BREVIPEDICELLUS (a member of the KNOX gene family) [35], thereby regulating the synthesis of lignin. In addition, Aux/IAAs and ARFs have also been shown to function to regulate lignin synthesis and SCW development [47]. Therefore, metaxenia might change the signal transduction process of auxin in pear fruit, thereby regulating the expression of TFs that are related to lignin metabolism and ultimately affecting lignin deposition and stone cell development ( Figure 12).
Cinnamate 4-hydroxylase (EC 1.14.13.11) belongs to the CYP73A subfamily of the cytochrome monooxygenase superfamily [49]. This protein is not only the second key enzyme in the general phenylpropanoid pathway but it also plays a highly important role in the entire pathway [14,42]. Previous studies have shown that the product of C4H, p-coumaric acid, is a key substance affecting the metabolism of pear lignin and the development of stone cells [4,13]. Therefore, we investigated the function of the DEG PbC4H2 in SCW development and lignification in this study. After the heterologous expression of PbC4H2 in the c4h mutant, the lignin content of the inflorescence stems of the rescued plants increased by approximately 15% and the SCW thickness increased by approximately 2% (Figure 11). In particular, PbC4H2 can restore the collapsed phenotype of the cell wall of the c4h mutant ( Figure 10). This result strongly indicates that PbC4H2 plays an important role in pear cell wall development and lignin synthesis. Interestingly, we found that PbC4H1 and PbC4H2 showed distinct expression patterns, which are similar to those that were observed in tea plants [49]. PbC4H1 exhibits high transcript abundance during fruit ripening (Figure 8). The synthesis of lignin and the development of stone cells have largely stopped in this stage [7]. Therefore, PbC4H1 might not be a lignin-specific C4H and it may be responsible for the biosynthesis of other phenylpropanoids. Since studies have shown that phenylpropanoid metabolism plays an important role in fruit ripening, its function in pear fruit warrants further research.

Conclusions
In summary, this study used 'Cuiguan' (pollen parent) × 'Lianglizaosu' (seed parent) and 'Yali' (pollen parent) × 'Lianglizaosu' (seed parent) as the materials to demonstrate that metaxenia can significantly affect stone cell development and lignin metabolism in pear fruit. RNA-seq showed that the pollen parents with different stone cell contents had a significant effect on the expression of structural genes (PbC4H, Pb4CL, PbC3H, PbHCT, PbCCoAOMT, PbCCR, PbF5H, PbCOMT, PbUGT72E, PbBGLU, PbPOD, PbLAC, and PbDIR) and TFs (PbKNOX1, PbWLIM1a, PbWLIM1b, PbMYB4, PbMYB30, PbMYB169, and PbMYB171) that were related to lignin metabolism in the early stage of fruit development. By heterologous expression of PbC4H2 in the c4h mutant, it was demonstrated that this gene has functions responsible for cell wall development and lignin biosynthesis. Our findings not only establish a foundation for regulating the intrinsic quality of pears through metaxenia in the future, but also provide a target gene for the molecular regulation of pear stone cell development.
Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4907/11/1/53/s1, Figure S1: Comparison of stone cell contents in mature fruits of two pollen parents, Figure S2: GO (top line) and COG (bottom line) classification of putative functions of differentially expressed genes, Figure S3: Comparison of transcription levels of lignin synthesis-related genes between YL and CL fruits, Figure S4: Detection of positive plants from transgenic Arabidopsis seedlings, Table S1: Primers used in this study, Table S2: Summary statistics for pear genes based on RNA-seq data, Table S3: Statistics on the number of differentially expressed genes, Table S4: Number of differentially expressed genes annotated, Table S5: Changes of phenylpropanoid metabolism-related TFs in YL and CL fruits, Table S6: Changes of phenylpropanoid pathway-related TFs in YL and CL fruits at 23 DAF, Table S7: DEGs related to lignin metabolism in YL and CL fruits, Table S8: Changes of plant hormone signal transduction-related TFs in YL and CL fruits at 23 DAF.