Transcriptomic Analysis Reveals Genes Associated with the Regulation of Peach Fruit Softening and Senescence during Storage

Peach (Prunus persica (L.) Batsch) is a highly desirable fruit that is consumed around the world. However, the peach fruit is highly perishable after harvest, a characteristic that limits the distribution and supply to the market and causes heavy economic losses. Thus, peach fruit softening and senescence after harvest urgently need to be addressed. In the current study, transcriptomic analysis was performed to identify candidate genes associated with peach fruit softening and senescence, comparing peach fruit from cultivars with different flesh textures, namely melting and stony hard (SH) flesh textures during storage at room temperature. The mitogen-activated protein kinase signaling pathway-plant and plant hormone signal transduction pathways were associated with peach fruit softening and senescence according to the Venn diagram analysis and weighted gene co-expression network analysis. The expression levels of seven genes, including Prupe.1G034300, Prupe.2G176900, Prupe.3G024700, Prupe.3G098100, Prupe.6G226100, Prupe.7G234800, and Prupe.7G247500, were higher in melting peach fruit than in SH peach fruit during storage. Furthermore, the SH peach fruit softened rapidly after 1-naphthylacetic acid treatment, during which the levels of expression of these seven genes, determined by a quantitative reverse transcription polymerase chain reaction, were strongly induced and upregulated. Thus, these seven genes may play essential roles in regulating peach fruit softening and senescence.


Introduction
Peach (Prunus persica (L.) Batsch) is a crop of considerable economic value and has been widely cultivated around the world. Peach fruit is rich in phytonutrients, including many vitamins, amino acids, antioxidants, and proteins [1,2]. However, as a typical climacteric fruit, peach fruit is highly perishable during room temperature storage after harvest, and this feature limits its potential for long-distance transport to different markets and causes heavy economic losses, seriously restricting the development of the peach industry. Studying the physiological and molecular mechanisms behind peach fruit softening and senescence process will be important not only for developing fruit fresh-keeping techniques and achieving fruit supply at markets but also for the breeding of new peach varieties with storage tolerance.
Remodeling of the cell wall structure is regarded as the basis of changes in fruit firmness and texture [3] and is closely related to fruit softening and senescence. Numerous cell wall modifying enzymes, namely polygalacturonase [4,5], β-galactosidase [6][7][8], α-L-arabinofuranosidase [9], pectin lyase [10], and pectin methylesterase [11], are considered to play central roles in cell wall degradation, associated with fruit softening and senescence. Ethylene plays central roles in fruit softening and senescence [12][13][14]. Many tran-an orchard in Beijing, China, were used in this study. Three healthy trees of each cultivar were managed in accordance with conventional cultivation measures. All peach fruit samples were randomly collected and checked to be of uniform commercial maturity with an undamaged fruit surface. An air-conditioned room with a temperature of 25 ± 1 • C and a relative humidity of 75-85% was used to store the fruit samples for six days in 2018. Samples were collected every three days during storage, with 15 fruits being sampled at each date for RNA-seq, and five fruits served as one biological replicate. In addition, 'XC' fruit samples at commercial maturity were treated with NAA, based on the method of Tatsuki et al. [26]. Fruits were sprayed with 1mM NAA (Sangon Biotech, Shanghai, China) containing 0.01% (w/v) Tween 20 (Sangon Biotech, Shanghai, China) every 24 h for seven days, while control fruits were sprayed with 0.01% (w/v) Tween 20 without NAA. All 'XC' fruits were stored in an air-conditioned room with a temperature of 25 ± 1 • C and a relative humidity of 75-85% for eight days in 2019. Samples were collected every two days, and 15 fruits were sampled at each date to determine ethylene production and fruit firmness, with five fruits serving as each biological replicate. All experiments were carried out at the Jiangsu Academy of Agricultural Sciences. After measurements were taken, the pulp flesh was rapidly frozen in liquid nitrogen and stored in an −80 • C refrigerator until needed for further analysis.

Ethylene Production and Fruit Firmness
Ethylene production measurement and fruit firmness determination were based on the methods of Guo et al. [27]. Briefly, ethylene production at each stage was measured by placing peach fruits in an airtight container for 2 h at 25 • C, and a syringe was used to obtain a 1 mL gas sample from the headspace of the airtight container. Immediately, the gas sample was analyzed by a gas chromatograph (Agilent 7890A, Santa Clara, CA, USA) with an HP-Plot q column (20 m × 0.53 mm × 20 µm). The injector and detector temperatures were 220 • C, and the column temperature was 40 • C. The TA. XT. Plus Texture Analyser (Stable Micro System, Godalming, UK) was used to measure fruit firmness. The expression unit of ethylene production was µL kg −1 h −1 and fruit firmness was kg/cm 2 .

RNA Sequencing Library Preparation
Total RNA was isolated from the peach fruit pulp sample using the RNAprep Pure Plant Kit (polysaccharide-and polyphenolic-rich) (TIANGEN, Beijing, China), according to the manufacturer's instructions. Three independent biological replicates were used for RNA extraction. The total RNA quality and quantity were verified using a NanoDrop spectrophotometer and an Agilent 2100 Bioanalyzer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Briefly, the mRNA was fragmented into small pieces after purification by Oligo(dT)-attached magnetic beads. Reverse transcription was performed to obtain the first-strand cDNA using random hexamer primers, and a second-strand cDNA synthesis reaction was then undertaken. Subsequently, A-Tailing Mix and RNA Index Adapters were added to the cDNA. This was followed by polymerase chain reaction (PCR) amplification, and the Ampure XP Beads were used to purify the PCR products, and then the PCR products were eluted in an elution buffer solution. Afterward, the double-stranded PCR products underwent the reaction of heat-denatured and circularized to obtain the single-strand circle DNA as the final library. The final library was amplified using phi29 to achieve DNA nanoballs, which were loaded into the patterned nanoarray. Lastly, pair-end 100 base reads were obtained on the BGISEQ-500 platform (Beijing Genomic Institute, Shenzhen, China).

Data Filtering and Analysis
SOAPnuke (v1.4.0) [28] was used to obtain high-quality reads by filtering out the low-quality reads, reads containing the sequencing adapter, reads where the unknown base ('N' base) ratio was more than 5%, and reads with >20% nucleotides with base quality <5. Subsequently, the clean reads remaining after filtering were stored in FASTQ format. The clean reads were mapped to the reference genome Peach v2.0.a1 (v2.1) [29] using HISAT2 (v2.1.0) software [30]. Statistics of the clean reads are listed in Supplementary File S1. Differentially expressed genes (DEGs) were identified using DEGseq software [31], with a fold change ≥2 and a Q-value ≤ 0.001.
The Phyper, according to the hypergeometric test, was performed on the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of annotated DEGs [32]. And the annotated DEGs were analyzed by Gene Ontology (GO) classification [32]. Gene expression was presented as the fragments per kilobase of the exon model per million mapped fragments (FPKM) method. Screening conditions for upregulated genes were as follows: Log2 (HJML 3 d/HJML 0 d) ≥

The Expression Analysis of Candidate Genes
The PrimeScript™ RT reagent Kit with a gDNA Eraser (TaKaRa, Dalian, China) was used to synthesize cDNA from total RNA. To validate the peach transcriptome data, a quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using TB Green ® Premix Ex Taq TM II (Tli RNaseH Plus) (TaKaRa) on a 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA). The primers of qRT-PCR were designed using the NCBI/Primer-BLAST online server and listed in Supplementary File S2. Each PCR reaction was prepared in a 20 µL mixture volume containing diluted cDNA (1 µL), forward primer (0.4 µL), reverse primer (0.4 µL), TB green premix (2X, 10 µL), ROX reference dye II (0.4 µL), and RNase-free water (7.8 µL). The reaction was carried out under the following conditions: an initial denaturation step at 95 • C for 30 s, 40 cycles of 95 • C for 5 s, and 60 • C for 34 s. The relative expression level of each gene was normalized relative to the translation elongation factor 2 (TEF2) gene [34] and detected by the 2 −∆∆Ct method [35].

Statistical Analysis
Microsoft Excel 2016 was used to calculate standard errors (SEs). Origin 9.1 was used to produce graphs. SPSS 19.0 (SPSS, Chicago, IL, USA) was used to determine the significant differences between experimental data.

Identification of Candidate Genes by a Venn Diagram
Melting peach fruits have been reported to soften rapidly as a result of the release of a large amount of ethylene production after harvest during room temperature storage, whereas SH-type cultivars soften slowly with little ethylene production. Thus, Venn diagram analysis was performed at 3 d and 6 d during the storage period between one melting cultivar 'HJML' and two SH-type cultivars, 'HY' and 'XC'. The melting cultivar 'HJML' was also analyzed from 0 d to 3 d to identify genes associated with peach fruit softening and senescence. identified between the M peach 'HJML' and the SH peach cultivars 'XC' and 'HY' fro Venn analysis by group comparisons, which may be related to peach fruit softening senescence. Subsequently, 159 upregulated genes and 365 downregulated genes w identified, which are listed in Supplementary File 3 and Supplementary File 4, accor to the screening conditions.

Functional Analysis of Candidate Genes
The upregulated genes were annotated in the GO database and were categorized twenty-eight functional groups, including thirteen in biological processes, eight in cel components, and seven in molecular functions (Supplementary File 5). Furthermore 159 upregulated genes were then annotated in the KEGG pathway database, and the 20 most highly enriched pathways are shown in Figure 2a. Remarkably, 14 upregul genes were found in the mitogen-activated protein kinase (MAPK) signaling pathw plant (ko04016) and 13 genes were found in the plant hormone signal transduc pathway (ko04075); the other 18 pathways are listed in Figure 2a. The 365 downregul genes were classified into twenty-six functional groups in the GO database, inclu eleven in biological processes, ten in cellular components, and five in molecular funct (Supplementary File 5). Photosynthesis was the most highly enriched of the pathw and the top 20 most highly enriched pathways are shown in Figure 2b. The fold ch and function annotation based on the reference genome of upregulated genes in the five enriched pathways are shown in Table 1, while the corresponding downregul genes are shown in Table 2. The 14 upregulated genes in the MAPK signaling pathw plant (ko04016) and the 13 upregulated genes in the plant hormone signal transduc (ko04075) pathway were associated with ethylene biosynthesis and signal transduc which may participate in fruit softening and senescence process. The BGI_novel sequ is listed in Supplementary File 6.

Functional Analysis of Candidate Genes
The upregulated genes were annotated in the GO database and were categorized into twenty-eight functional groups, including thirteen in biological processes, eight in cellular components, and seven in molecular functions (Supplementary File S5). Furthermore, the 159 upregulated genes were then annotated in the KEGG pathway database, and the top 20 most highly enriched pathways are shown in Figure 2a. Remarkably, 14 upregulated genes were found in the mitogen-activated protein kinase (MAPK) signaling pathwayplant (ko04016) and 13 genes were found in the plant hormone signal transduction pathway (ko04075); the other 18 pathways are listed in Figure 2a. The 365 downregulated genes were classified into twenty-six functional groups in the GO database, including eleven in biological processes, ten in cellular components, and five in molecular functions (Supplementary File S5). Photosynthesis was the most highly enriched of the pathways, and the top 20 most highly enriched pathways are shown in Figure 2b. The fold change and function annotation based on the reference genome of upregulated genes in the top five enriched pathways are shown in Table 1, while the corresponding downregulated genes are shown in Table 2. The 14 upregulated genes in the MAPK signaling pathway-plant (ko04016) and the 13 upregulated genes in the plant hormone signal transduction (ko04075) pathway were associated with ethylene biosynthesis and signal transduction, which may participate in fruit softening and senescence process. The BGI_novel sequence is listed in Supplementary File S6.

Weighted Gene Co-Expression Network Analysis of Candidate Genes
WGCNA was carried out to identify the hub genes regulating peach fruit softening and senescence. The M-type peach fruit released a larger amount of ethylene and softened noticeably after harvest, whereas the fruit from the two SH-type peach cultivars was characterized by their low ethylene production and very firm during ripening (both onand off-tree) [20,25]. In Figure 3, the green, red, and magenta modules with the highest phenotypic correlations were selected for further analysis based on fruit ethylene production after harvest. A total of 115 genes were identified in three modules and were annotated in the KEGG database. The pathways of diterpenoid biosynthesis, the MAPK signaling pathway-plant, steroid biosynthesis, plant-pathogen interaction, sesquiterpenoid and triterpenoid biosynthesis, and plant hormone signal transduction were all highly enriched ( Figure 4). Ethylene plays a central role in fruit softening and senescence, with the MAPK signaling pathway-plant also being associated with ethylene biosynthesis [36,37]. Nine genes in the MAPK signaling pathway-plant and seven genes in the plant hormone signal transduction pathways were identified for further study, while fourteen and thirteen genes were also identified by Venn diagram analysis in the MAPK signaling pathway-plant and plant hormone signal transduction pathway, respectively. We have reasons to believe that genes in the above two pathways may participate in the process of the regulation of fruit softening and senescence through ethylene biosynthesis and/or signal transduction. A total of 20 candidate genes were identified for further analysis.

Weighted Gene Co-Expression Network Analysis of Candidate Genes
WGCNA was carried out to identify the hub genes regulating peach fruit softening and senescence. The M-type peach fruit released a larger amount of ethylene and softened noticeably after harvest, whereas the fruit from the two SH-type peach cultivars was characterized by their low ethylene production and very firm during ripening (both onand off-tree) [20,25]. In Figure 3, the green, red, and magenta modules with the highest phenotypic correlations were selected for further analysis based on fruit ethylene production after harvest. A total of 115 genes were identified in three modules and were annotated in the KEGG database. The pathways of diterpenoid biosynthesis, the MAPK signaling pathway-plant, steroid biosynthesis, plant-pathogen interaction, sesquiterpenoid and triterpenoid biosynthesis, and plant hormone signal transduction were all highly enriched ( Figure 4). Ethylene plays a central role in fruit softening and senescence, with the MAPK signaling pathway-plant also being associated with ethylene biosynthesis [36,37]. Nine genes in the MAPK signaling pathway-plant and seven genes in the plant hormone signal transduction pathways were identified for further study, while fourteen and thirteen genes were also identified by Venn diagram analysis in the MAPK signaling pathway-plant and plant hormone signal transduction pathway, respectively. We have reasons to believe that genes in the above two pathways may participate in the process of the regulation of fruit softening and senescence through ethylene biosynthesis and/or signal transduction. A total of 20 candidate genes were identified for further analysis.

FPKM Analysis of the Candidate Genes
The gene expression levels of the 20 candidate genes were identified by Venn diagram analysis and the WGCNA method, and the gene expression level was analyzed by RNA-seq technology ( Figure 5). The expression levels of the 20 candidate genes were higher during room temperature storage in the M-type peach 'HJML' than in SH peach 'XC' and 'HY'. Significantly, the gene PpACS1 (Prupe.2G176900) plays a crucial role in ethylene synthesis, and its suppressed expression was associated with a low level of ethylene production, resulting in the suppression of fruit softening in SH peach cultivars [38]. Interestingly, 12 genes showed similar expression patterns to PpACS1 between the M-type peach 'HJML' and the SH-type peach cultivars ('XC' and 'HY'), namely Prupe.1G034300, Prupe.1G042500, Prupe.1G526700, Prupe.3G024700, Prupe.3G074800, Prupe.3G098100, Prupe.5G054500, Prupe.7G160600, Prupe.7G234800, Prupe.8G153100, Prupe.8G153700 and Prupe.8G153800. The expressions of Prupe.1G412400, Prupe.2G140600, Prupe.2G307400, Prupe.6G226100, Prupe.6G286000, Prupe.7G244300, and Prupe.7G247500 increased at 3 d of postharvest storage in the M-type peach 'HJML' but declined by the end of storage ( Figure 5). The FPKM and functional annotations of these 20 candidate genes related to softening and senescence are listed in Supplementary File S7.

FPKM Analysis of the Candidate Genes
The gene expression levels of the 20 candidate genes were identified by Venn diagram analysis and the WGCNA method, and the gene expression level was analyzed by RNA-seq technology ( Figure 5). The expression levels of the 20 candidate genes were higher during room temperature storage in the M-type peach 'HJML' than in SH peach 'XC' and 'HY'. Significantly, the gene PpACS1 (Prupe.2G176900) plays a crucial role in ethylene synthesis, and its suppressed expression was associated with a low level of ethylene production, resulting in the suppression of fruit softening in SH peach cultivars [38]. Interestingly, 12 genes showed similar expression patterns to PpACS1 between the M-type peach 'HJML' and the SH-type peach cultivars ('XC' and 'HY'), namely Prupe.1G034300, Prupe.1G042500, Prupe.1G526700, Prupe.3G024700, Prupe.3G074800, Prupe.3G098100, Prupe.5G054500, Prupe.7G160600, Prupe.7G234800, Prupe.8G153100, Prupe.8G153700 and Prupe.8G153800. The expressions of Prupe.1G412400, Prupe.2G140600, Prupe.2G307400, Prupe.6G226100, Prupe.6G286000, Prupe.7G244300, and Prupe.7G247500 increased at 3 d of postharvest storage in the M-type peach 'HJML' but declined by the end of storage ( Figure 5). The FPKM and functional annotations of these 20 candidate genes related to softening and senescence are listed in Supplementary File 7.

The Effect of NAA Treatment on Candidate Gene Expression
SH-type peach fruit can be induced to release a large amount of ethylene and achieve rapid softening in response to NAA treatment. In the present study, the SH-type peach cultivar 'XC' was used to analyze the candidate gene expression after NAA treatment. The control 'XC' fruit maintained fruit firmness (0-4 d), and hardly synthesized any ethylene during room temperature storage (Figure 6), whereas the firmness of fruit treated with NAA declined markedly after two days and retained a significantly lower firmness than that of the control fruit during storage. Ethylene production of 'XC' fruit was induced by NAA treatment after two days, with a pattern of increased production during storage ( Figure 6). Generally, after NAA treatment, the SH-type cultivar 'XC' exhibited softening characteristics similar to those of M-type peach; thus, these materials would be extremely suitable for the identification of candidate genes associated with fruit softening and senescence. The expression levels of the 20 candidate genes identified earlier as being related to softening and senescence were analyzed by RNA-Seq using SH-type peach 'XC' with NAA treatment (FPKM data were listed in Supplementary File S8). Expression levels of seventeen of the twenty genes in 'XC' fruit during room temperature storage were altered in response to NAA treatment, whereas those of the remaining three genes (Prupe.2G140600, Prupe.2G307400, Prupe.6G286000) were not affected by NAA treatment (Figure 7). Of the seventeen genes, six genes, namely Prupe.1G042500, Prupe.1G412400, Prupe.7G160600, Prupe.7G244300, Prupe.8G153100, and Prupe.8G153700, exhibited lower expression levels, whereas four genes (Prupe.1G526700, Prupe.3G074800, Prupe.5G054500, and Prupe.7G244300) in 'XC' peach fruit were slightly upregulated by NAA treatment. On the other hand, the expression levels of seven genes (Prupe.1G034300, PpACS1, Prupe.3G024700, Prupe.3G098100 (PpWRKY13), Prupe.6G226100, Prupe.7G234800, and Prupe.7G247500) were strongly induced and significantly upregulated in 'XC' peach fruit treated with NAA in comparison with control 'XC' fruit. Furthermore, qRT-PCR of these seven genes showed that they are strong candidates for genes positively associated with peach fruit softening and senescence (Figure 8).

The Effect of NAA Treatment on Candidate Gene Expression
SH-type peach fruit can be induced to release a large amount of ethylene and achieve rapid softening in response to NAA treatment. In the present study, the SH-type peach cultivar 'XC' was used to analyze the candidate gene expression after NAA treatment. The control 'XC' fruit maintained fruit firmness (0-4 d), and hardly synthesized any ethylene during room temperature storage (Figure 6), whereas the firmness of fruit treated with NAA declined markedly after two days and retained a significantly lower firmness than that of the control fruit during storage. Ethylene production of 'XC' fruit was induced by NAA treatment after two days, with a pattern of increased production during storage ( Figure 6). Generally, after NAA treatment, the SH-type cultivar 'XC' exhibited softening characteristics similar to those of M-type peach; thus, these materials would be extremely suitable for the identification of candidate genes associated with fruit softening and senescence. The expression levels of the 20 candidate genes identified earlier as being related to softening and senescence were analyzed by RNA-Seq using SH-type peach 'XC' with NAA treatment (FPKM data were listed in Supplementary File 8). Expression levels of seventeen of the twenty genes in 'XC' fruit during room temperature storage were altered in response to NAA treatment, whereas those of the remaining three genes (Prupe.2G140600, Prupe.2G307400, Prupe.6G286000) were not affected by NAA treatment (Figure 7). Of the seventeen genes, six genes, namely Prupe.1G042500, Prupe.1G412400, Prupe.7G160600, Prupe.7G244300, Prupe.8G153100, and Prupe.8G153700, exhibited lower expression levels, whereas four genes (Prupe.1G526700, Prupe.3G074800, Prupe.5G054500, and Prupe.7G244300) in 'XC' peach fruit were slightly upregulated by NAA treatment. On the other hand, the expression levels of seven genes (Prupe.1G034300, PpACS1, Prupe.3G024700, Prupe.3G098100 (PpWRKY13), Prupe.6G226100, Prupe.7G234800, and Prupe.7G247500) were strongly induced and significantly upregulated in 'XC' peach fruit treated with NAA in comparison with control 'XC' fruit. Furthermore, qRT-PCR of these seven genes showed that they are strong candidates for genes positively associated with peach fruit softening and senescence (Figure 8).

Discussion
M-type peach fruit and SH-type peach are good models for investigating the effect of ethylene on fruit softening and senescence. Ethylene is a plant hormone with diverse functions, which involve various plant biology processes including germination, plant growth, organ senescence, and fruit ripening [39][40][41]. MAPK modules play essential roles in the transduction of developmental and environmental signals through the phosphorylation of other kinases, other enzymes, transcription factors, or other downstream signaling targets [42]. Studies have shown that MAPKs are also involved in ethylene biosynthesis and signaling [37,42,43]. In the present study, seven genes identified from the MAPK signaling pathway-plant and plant hormone signal transduction pathways may be associated with peach fruit softening and senescence, according to the Venn diagram and WGCNA analyses by the RNA-Seq technology, with the differential softening and senescence characteristics of peach fruit (M-type peach fruit and SH-type peach fruit) during room temperature storage.
Seven genes were identified from the MAPK signaling pathway-plant and plant hormone signal transduction pathways, in which expression levels were strongly induced and upregulated in 'XC' peach fruit after NAA treatment. PpACS1 encodes the ACS, one

Discussion
M-type peach fruit and SH-type peach are good models for investigating the effect of ethylene on fruit softening and senescence. Ethylene is a plant hormone with diverse functions, which involve various plant biology processes including germination, plant growth, organ senescence, and fruit ripening [39][40][41]. MAPK modules play essential roles in the transduction of developmental and environmental signals through the phosphorylation of other kinases, other enzymes, transcription factors, or other downstream signaling targets [42]. Studies have shown that MAPKs are also involved in ethylene biosynthesis and signaling [37,42,43]. In the present study, seven genes identified from the MAPK signaling pathway-plant and plant hormone signal transduction pathways may be associated with peach fruit softening and senescence, according to the Venn diagram and WGCNA analyses by the RNA-Seq technology, with the differential softening and senescence characteristics of peach fruit (M-type peach fruit and SH-type peach fruit) during room temperature storage.
Seven genes were identified from the MAPK signaling pathway-plant and plant hormone signal transduction pathways, in which expression levels were strongly induced and upregulated in 'XC' peach fruit after NAA treatment. PpACS1 encodes the ACS, one Figure 8. The relative expression analysis by qRT-PCR of candidate genes in 'XC' fruit treated with NAA. The X-axis represents the number of days after harvest, whereas the Y-axis represents the relative gene expression levels. Data are means ± SEs (n = 3). 'XC' represents 'Xia Cui'; significant differences (p < 0.01) by Student's t-test between means are indicated by the symbol **.

Discussion
M-type peach fruit and SH-type peach are good models for investigating the effect of ethylene on fruit softening and senescence. Ethylene is a plant hormone with diverse functions, which involve various plant biology processes including germination, plant growth, organ senescence, and fruit ripening [39][40][41]. MAPK modules play essential roles in the transduction of developmental and environmental signals through the phosphorylation of other kinases, other enzymes, transcription factors, or other downstream signaling targets [42]. Studies have shown that MAPKs are also involved in ethylene biosynthesis and signaling [37,42,43]. In the present study, seven genes identified from the MAPK signaling pathway-plant and plant hormone signal transduction pathways may be associated with peach fruit softening and senescence, according to the Venn diagram and WGCNA analyses by the RNA-Seq technology, with the differential softening and senescence characteristics of peach fruit (M-type peach fruit and SH-type peach fruit) during room temperature storage.
Seven genes were identified from the MAPK signaling pathway-plant and plant hormone signal transduction pathways, in which expression levels were strongly induced and upregulated in 'XC' peach fruit after NAA treatment. PpACS1 encodes the ACS, one of the enzymes which catalyze the synthesis of ethylene and is responsible for ethylene production by climacteric fruit [38,44]. Interestingly, the high expression level of PpACS1 in M-type peach fruit results in a large amount of ethylene, followed by the rapid softening of the fruit [38]. The very low ethylene production in SH-type peach fruit may contribute to the maintenance of the fruit hardness resulting from the suppressed transcription of PpACS1 [12,38,45,46]. Thus, the PpACS1 was confirmed to play a central role in regulating peach fruit softening and senescence through the control of ethylene synthesis.
Additionally, the Prupe.1G034300 gene was functionally annotated as ethylene response sensor 2-related (Supplementary File S7), and Prupe.1G034300 showed expression patterns similar to PpACS1 between the M-type peach 'HJML' and the SH-type peach ('XC' or 'HY'). The increased expression level with fruit softening and senescence was consistent with a report examining pears and persimmons [47,48]. The high level of gene expression in M-type peaches may be due to the large amount of ethylene required for the ethylene receptor, which needs time to dissociate after binding with ethylene [49]. The low expression level in SH-type peach fruit, in association with low ethylene production, may result from insufficient ethylene being available with which to bind. Thus, the ethylene receptor gene Prupe.1G034300 may participate in the process of peach fruit softening and senescence through ethylene signal transduction.
Three genes (Prupe.6G226100, Prupe.7G234800, and Prupe.7G247500) were functionally annotated as being associated with auxin response (Supplementary File S7). The gene Prupe.6G226100 is functionally annotated as Gretchen Hagen 3 (GH3) and is associated with auxin homeostasis, whereas the other two genes (Prupe.7G234800 and Prupe.7G247500) are functionally annotated as being associated with auxin/indole-3-acetic acid (Aux/IAA). The early or primary auxin-response genes consist of three major classes of genes, known as GH3, Aux/IAA, and the small auxin-up RNAs (SAURs) [50][51][52]. Generally, free auxin has biological activity, and it can be conjugated with sugars, amino acids, or peptides to generate auxin storage forms in plants [53]. The GH3 family gene members encode GH3 enzymes that catalyze the conjugation of amino acids to IAA, which may maintain the balance of free auxin to regulate the various life processes of plants [51,54]. Actually, the IAA concentration is higher in M-type peach fruit than in SH-type peach fruit, which is consistent with the system 2 ethylene production during the ripening period [26]. Additionally, the expression of Prupe.6G226100 is higher in M-type peach than in SH-type peach fruit, suggesting that Prupe.6G226100 may have an influence on regulating fruit softening and senescence by maintaining the balance of free and bound auxin. Degradation of Aux/IAA proteins by way of the ubiquitin/26S proteasome and the ARF activity is derepressed; thus, ARF proteins regulate the subsequent expression of auxin-responsive genes in the auxinmediated transcription activation [55,56]. Overexpression of an Aux/IAA gene PpIAA19 (prupe.3G074800) in tomato cultivar 'Micro-Tom' suggested that this gene was not related to the ripening of tomato fruit [57]. However, an Aux/IAA gene, Prupe.7G234800 (PpIAA1), enhanced the transcription of the PpACS1 gene and the cell wall degradation gene PpPG1 in peach fruit, with overexpression of PpIAA1 in tomatoes resulting in transgenic fruit softening more rapidly than the wild type [46]. Thus, the Aux/IAA gene Prupe.7G247500 may have a similar function, and the function of this gene needs further analysis.
Three WRKY transcription factor genes (Prupe.2G307400, PpWRKY13, and Prupe.6G286000) were identified from the MAPK signaling pathway-plant and plant hormone signal transduction pathways. Significantly, the expression of PpWRKY13 was strongly upregulated in 'XC' peach fruit in response to NAA treatment during the subsequent rapid softening process. WRKY transcription factors, as activators and inhibitors of transcription, are widely involved in the regulation of stress response, hormone signal transduction, and fruit ripening and senescence [24,[58][59][60]. Significantly, WRKY transcription factors can bind to the promoter of key genes in the ethylene biosynthesis pathway to mediate ethylene biosynthesis. The gene VvWRKY13 from Vitis vinifera was found to mediate the expression of genes ACS2 and ACS8 in ethylene biosynthesis [61]. Chromatin-immunoprecipitation assays revealed that WRKY33 regulated the expression of ACS2 and ACS6 by binding to the promoter regions of these genes in Arabidopsis [62]. Furthermore, WRKY transcription factors may affect fruit softening and senescence by regulating enzymes related to cell wall degradation. Based on one report, the wild strawberry WRKY transcription factor FvWRKY48 binds to the promoter region of the corresponding pectate lyase gene FvPLA to control the pectin degradation and fruit softening in Fragaria vesca [24]. Thus, PpWRKY13 may regulate the peach fruit softening and senescence by mediating the expression of key enzymes in ethylene biosynthesis and/or the enzymes related to cell wall degradation.

Conclusions
The expression of seven genes, including Prupe.1G034300, Prupe.2G176900, Prupe.3G024700, Prupe.3G098100, Prupe.6G226100, Prupe.7G234800, and Prupe.7G247500, were upregulated in M-type peach fruit relative to SH-type peach fruit and were strongly induced and upregulated in SH-type peach fruit after NAA treatment, which stimulated fruit flesh softening during room temperature storage. The transcriptomic analysis revealed that these seven genes are considered to be candidate genes associated with peach fruit softening and senescence.