Characterization and Transcript Profiling of PME and PMEI Gene Families during Peach Fruit Maturation

in Journal of the American Society for Horticultural Science
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  • 1 Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, Zhengzhou 450009, P.R. China

Pectins are synthesized and secreted to the cell wall as highly methyl-esterified polymers and demethyl-esterified by pectin methylesterases (PMEs), which are regulated by pectin methylesterase inhibitors (PMEIs). PMEs and PMEIs are involved in pectin degradation during fruit softening; however, the roles of the PME and PMEI gene families during fruit softening remain unclear. Here, 71 PME and 30 PMEI genes were identified in the peach (Prunus persica) genome and shown to be unevenly distributed on all eight chromosomes. The 71 PME genes comprised 36 Type-1 PMEs and 35 Type-2 PMEs. Transcriptome analysis showed that 11 PME and 15 PMEI genes were expressed during fruit ripening in melting flesh (MF) and stony-hard (SH) peaches. Three PME and five PMEI genes were expressed at higher levels in MF than in SH fruit and exhibited softening-associated expression patterns. Upstream regulatory cis elements of these genes related to hormone response, especially naphthaleneacetic acid and ethylene, were investigated. One PME (Prupe.7G192800) and two PMEIs (Prupe.1G114500 and Prupe.2G279800), and their promoters were identified as potential targets for future studies on the biochemical metabolism and regulation of fruit ripening. The comprehensive data generated in this study will improve our understanding of the PME and PMEI gene families in peach. However, further detailed investigation is necessary to elucidate the biochemical function and regulation mechanism of the PME and PMEI genes during peach fruit ripening.

Abstract

Pectins are synthesized and secreted to the cell wall as highly methyl-esterified polymers and demethyl-esterified by pectin methylesterases (PMEs), which are regulated by pectin methylesterase inhibitors (PMEIs). PMEs and PMEIs are involved in pectin degradation during fruit softening; however, the roles of the PME and PMEI gene families during fruit softening remain unclear. Here, 71 PME and 30 PMEI genes were identified in the peach (Prunus persica) genome and shown to be unevenly distributed on all eight chromosomes. The 71 PME genes comprised 36 Type-1 PMEs and 35 Type-2 PMEs. Transcriptome analysis showed that 11 PME and 15 PMEI genes were expressed during fruit ripening in melting flesh (MF) and stony-hard (SH) peaches. Three PME and five PMEI genes were expressed at higher levels in MF than in SH fruit and exhibited softening-associated expression patterns. Upstream regulatory cis elements of these genes related to hormone response, especially naphthaleneacetic acid and ethylene, were investigated. One PME (Prupe.7G192800) and two PMEIs (Prupe.1G114500 and Prupe.2G279800), and their promoters were identified as potential targets for future studies on the biochemical metabolism and regulation of fruit ripening. The comprehensive data generated in this study will improve our understanding of the PME and PMEI gene families in peach. However, further detailed investigation is necessary to elucidate the biochemical function and regulation mechanism of the PME and PMEI genes during peach fruit ripening.

The plant cell wall is a highly organized structure composed of many different polysaccharides, proteins, and phenolic compounds. Fruit ripening involves extensive depolymerization of pectin, as well as other modifications to cell-wall components, including demethylation and removal of neutral sugar side chains (Brummell and Harpster, 2001). In current models, pectic polysaccharides are synthesized in the Golgi complex as highly methylesterified polymers [e.g., homogalacturonic acid (HGA)] that are secreted to the cell wall and partially demethylesterified by PMEs. During ripening, this methylesterification decreases dramatically, for example, the degree of pectic methylesterification drops from 90% in immature green fruit to 30% at the red-ripe stage of ripening in tomato [Solanum lycopersicum (Koch and Nevins, 1989)], and the de-esterified HGA backbone is then susceptible to cleavaged by polygalacturonase (PG) (Prasanna et al., 2007).

The PME gene family was first described by Richard et al. (1996), and later classified in the Carbohydrate-Active EnZymes database as class 8 of the carbohydrate esterases (EC 3.1.1.11) (Cantarel et al., 2009). Active PME functions in two different ways: 1) a blockwise action by which blocks of de-methylesterified HGA interact with Ca2+, promoting the formation of the so-called “egg-box” structure, thereby affecting the apoplastic potential of hydrogen (pH) and the mechanical and biochemical properties of the cell wall; and 2) a random demethylesterified action on HGA in which interaction with Ca2+ is not permitted, and HGA becomes the target for pectin-degrading enzymes, such as PGs, affecting the texture and rigidity of the cell wall (Micheli, 2001; Pelloux et al., 2007). PMEs are widespread in plants and microorganisms and belong to large multigene families with different roles in cambial cell differentiation and determination of fiber length in trees (Micheli et al., 2000; Siedlecka et al., 2008), microsporogenesis (Francis et al., 2006; Lacoux et al., 2003), organ initiation (Peaucelle et al., 2011), and fruit softening and ripening (Brummell et al., 2004; Deytieux-Belleau et al., 2008; Eriksson et al., 2004). PMEs are classified as either Type-1 PMEs (i.e., those with a proregion, similar to the PMEI domain) or Type-2 PMEs (no proregion). In Type-1 PMEs, the proregion operates as an effective retention mechanism, keeping unprocessed PME in the Golgi apparatus. Consequently, proprotein processing could constitute a posttranslational mechanism regulating PME activity (Wolf et al., 2009).

The activity of PMEs is regulated by PMEIs (Balestrieri et al., 1990), which bind to the active site of the PME, generating a 1:1 complex (Di Matteo et al., 2005; Hothorn et al., 2004). PMEIs, which belong to a large family (Jolie et al., 2010; Wang et al., 2013), were originally discovered in kiwifruit [Actinidia chinensis (Balestrieri et al., 1990)] and subsequently identified in arabidopsis (Arabidopsis thaliana), pepper (Capsicum annuum), broccoli (Brassica oleracea), banana (Musa supientum), tomato, and grape (Vitis vinifera) (An et al., 2008; Lionetti et al., 2015; Peaucelle et al., 2008; Raiola et al., 2004; Reca et al., 2012; Srivastava et al., 2012; Zhang et al., 2010). Recent evidence shows the role of PMEIs in apical meristem development (Peaucelle et al., 2008), cell and organ size determination (Lionetti et al., 2007), cell growth acceleration (Pelletier et al., 2010), and pollen tube growth (Röckel et al., 2008; Zhang et al., 2010). In particular, the PMEIs from tomato are proposed to control the methylesterification of pectin during fruit softening and ripening (Reca et al., 2012).

Studies on the PME and PMEI gene families in several plant species provide a better understanding of this gene family (Pinzón-Latorre and Deyholos, 2013; Wang et al., 2013). To the best of our knowledge, no systematic investigations of the PME and PMEI gene families of peach have been reported to date. Peach cultivars can be divided into three groups based on characteristics such as fruit firmness and textural changes during ripening: MF, nonmelting flesh (NMF), and SH types (Haji et al., 2001). MF fruit exhibits sharply increasing auxin accumulation and ethylene production during the ripening process, resulting in rapid softening and a short shelf life, whereas SH fruit sustains low levels of auxin accumulation and ethylene production and barely softens on the tree or after harvest (Haji et al., 2005; Pan et al., 2015; Tatsuki et al., 2013). These two types are therefore good materials to identify the PME and PMEI genes involved in peach fruit ripening.

In the present study, the complete family of PMEs and PMEIs in peach was analyzed based on the peach genome accession no. 2.1 (Verde et al., 2013). A phylogenetic analysis was performed using PMEs and PMEIs from arabidopsis and peach. The expression profiles of all PME and PMEI genes were determined in MF and SH peaches during fruit ripening, and cis elements in the promoters of PMEs and PMEIs related to fruit ripening were analyzed.

Materials and Methods

Plant material and postharvest treatments.

Representatives of four peach cultivars, Zhongyoutao 13 (CN13), Zhongyoutao 16 (CN16), Goldhoney 3, and Yumyeong, were obtained from the Institute of Zhengzhou Fruit Research, Chinese Academy of Agriculture Science, Zhengzhou, China. For mRNA extraction and analysis, ‘CN13’ (MF) fruit were collected at 81, 86, 92, and 97 d after flowering (DAF) (designated S3, S4 I, S4 II, and S4 III, respectively). ‘CN16’ (SH) samples were collected at 72, 77, 82, and 87 DAF (designated S3, S4 I, S4 II, and S4 III, respectively) (Zeng et al., 2015). For ‘Goldhoney 3’ (MF) and ‘Yumyeong’ (SH), four sampling points covering the climacteric stage at 110, 115, 122, and 127 DAF (designated S3, S4 I, S4 II, and S4 III, respectively) were selected (Pan et al., 2015). The stages indicated as S3, S4 I, S4 II, and S4 III represent fruit from the end of stage S3 (second exponential growth phase) to stage S4 (climacteric).

‘CN16’ fruit collected at the S4 III stage (87 DAF) were subjected to auxin and ethylene treatments. For auxin treatment, whole fruit were dipped in 0.5 mm 1-naphthaleneacetic acid [NAA (Sigma-Aldrich, Darmstadt, Germany)] for 5 min with 100 µL·L−1 of surfactant (Silwet L-77; Real-Times Biotechnology, Beijing, China) as a surfactant. For ethylene treatment, fruit were dipped in 1000 µL·L−1 ethephon [an ethylene releaser (Solarbio, Beijing, China)] for 1 min and were then incubated in an airtight container. After treatment, the fruit were maintained at 25 °C and sampled at 12, 24, 36, 48, 72, and 96 h. Half of the sampled fruit were used to measure ethylene production, others were used to determine flesh firmness, then immediately frozen in liquid nitrogen, and stored at −80 °C for RNA extraction. Untreated fruit were used as the control. Each experimental and control group included at least 80 fruit.

Ethylene production and flesh firmness.

Ethylene production and flesh firmness were measured as previously described (Zeng et al., 2015). The ethylene concentration was measured with a gas chromatograph (GC2010; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector. Flesh firmness was measured with a fruit-pressure tester (GY-4-J; TOP Instruments, Hangzhou, China) fitted with an 11-mm-diameter probe. Each measurement was performed in three replicates for each sample. Five to eight fruit per sample were measured.

Identification of PME and PMEI genes in peach.

Predicted proteins containing PME (PF01095), PMEI (PF04043), or both domains were identified from the peach genome (Verde et al., 2013) (accession no. 2.1) using default parameters in hmmsearch/PfamScan (Punta et al., 2011). The predicted proteins were aligned to previously described PMEs and PMEIs from arabidopsis obtained from The Arabidopsis Information Resource (Lamesch et al., 2012), using BLASTp. Peach PMEs (PpPMEs) contained PF01095 and PF04043 domains, and PMEIs contained the PF04043 domain. The genome sequence (accession no. 2.1), Identity number, and coding sequence length were downloaded from the Phytozome (Goodstein et al., 2012). The molecular weights (M) and isoelectric points (pIs) of the proteins were calculated from ExPASy (Gasteiger et al., 2003). Exon/intron structure was constructed using the Gene Structure Display Server web-based bioinformatics tool (Hu et al., 2015). For chromosome locations, genes were mapped to the chromosomes using MapInspect software (Van Berloo, 1999). A phylogenetic tree was generated with molecular evolutionary genetics analysis software (MEGA 4.0) using the neighbor-joining (NJ) method, and edge support was estimated using 1000 bootstrap replicates (Tamura et al., 2007).

Library construction, deep sequencing, and data processing.

At least 5 µg of each isolated RNA from fruit samples (‘CN13’ S3, S4 I, S4 II, S4 III, and ‘CN16’ S3, S4 I, S4 II, S4 III) was sent to Gene Denovo Co. (Guangzhou, China) for the construction of eight libraries (two cultivars four stages). Libraries were established using an Illumina kit (Illumina, San Diego, CA), and each library was sequenced on the Illumina HiSeq 2500 sequencing platform.

Expression levels were measured in fragments per kilobase of exon per million fragments mapped [FPKM (Trapnell et al., 2010)], FPKM >1 was defined as the threshold of significant gene expression and was used to analyze the differences in gene expression between the two peach flesh types. The full list of normalized PME and PMEI gene expression was shown in Supplemental Tables 1 and 2.

Quantitative real-time PCR validation.

RNA isolation and complementary DNA synthesis were performed as previously described (Zeng et al., 2015). Quantitative reverse-transcription PCR (qRT-PCR) was performed to investigate the expression of PME and PMEI genes in peach (Zeng et al., 2015). The relative gene expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen, 2001). All gene-expression analyses were performed with three independent biological replicates. Primers used for qRT-PCR were designed using PrimerExpress 3.0 software (Applied Biosystems, Foster City, CA) (Table 1).

Table 1.

Primers of five pectin methylesterase inhibitor (PMEI) and three pectin methylesterase (PME) genes used for real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis.

Table 1.

Promoter region analysis of eight peach PME and PMEI genes.

To investigate the motifs or cis elements in the promoter sequences of the eight peach PME and PMEI genes related to hormone treatment, 1.5 kb of genomic DNA sequences upstream of the initiation codon (ATG) were obtained from the peach database. The motifs or elements in the promoter sequences were analyzed in the plant cis-acting regulatory DNA elements (PLACE) database (Higo et al., 1999) and the PlantCARE database (Lescot et al., 2002). The fruit-specific cis elements, the motifs TCCAAAA and TGTCACA, were searched manually in the promoter sequence (Yamagata et al., 2002; Yu et al., 2014).

Results

Identification of the peach PME and PMEI family genes.

A search of predicted transcripts of the peach whole-genome assembly (accession no. 2.1) for the programmed frequency amplitude modulation (PFAM) domains pectinesterase (PF01095) and PMEI (PF04043) identified 71 putative PMEs and 30 putative PMEIs (Tables 2 and 3). Independent alignment of 66 arabidopsis PME and 71 PMEI protein sequences in the TBLASTN program to the peach genome did not identify any additional peach genes other than those identified by the PFAM domain alignment. Among the predicted 71 PpPMEs, 36 were Type-1 [i.e., encoding both a PMEI (PF04043) and PME (PF01095) domains] and 35 were Type-2 (i.e., encoding a PME domain, but no PMEI domain). Only one of the genes (Prupe.3G031000) contained two PME and PMEI domains. Detailed information on the PpPME and PpPMEI genes is showed in Tables 2 and 3, respectively, including accession no. 1.0, accession no. 2.1, the PFAM domain, the open reading frame (ORF), the theoretical pI and M.

Table 2.

A summary of accessions numbers, programmed frequency amplitude modulation (PFAM), open reading frame (ORF) length, position of chromosome, theoretical isoelectric point (pI), and molecular weight (M) of peach pectin methylesterase (PME) genes.

Table 2.
Table 3.

A summary of accessions numbers, programmed frequency amplitude modulation (PFAM), open reading frame (ORF) length, position of chromosome, theoretical isoelectric point (pI), and molecular weight (M) of peach pectin methylesterase inhibitor (PMEI) genes.

Table 3.

The ORF lengths of the Type-1 PME family members except Prupe.3G031000 ranged from 1473 bp (Prupe.6G344600) to 1827 bp (Prupe.2G310600), encoding peptides of 409–608 aa. The predicted M of these genes varied from 54.16 to 67.66 kDa. The ORF lengths of the Type-2 PME family members ranged from 477 bp (Prupe.2G210900) to 1443 bp (Prupe.1G377100), encoding peptides of 158–480 aa. The predicted M of these genes varied from 17.26 to 51.48 kDa, and the theoretical pIs of the PME family ranged from 4.93 (Prupe.6G312200) to 9.63 (Prupe.2G278300). The ORF lengths of the PMEI family members ranged from 297 bp (Prupe.3G008000) to 1356 bp (Prupe.8G267500), encoding peptides of 98–451 aa. The predicted M of these genes varied from 11.06 to 49.98 kDa, and the theoretical pIs of the PMEI family ranged from 4.44 (Prupe.5G112600) to 10.02 (Prupe.5G076900).

Genomic distribution and sequence analysis.

Of 71 peach PME genes, 69 were unevenly distributed among the eight peach chromosomes varying from 1 to 18. Chromosome (Chr) 6, Chr 4, and Chr 8 were anchored by 1, 2, and 3 PpPME genes, respectively; Chr 3 × 6; Chr 1 and Chr 2 × 18; Chr 7 × 10; and Chr 6 × 11 genes. Prupe.I005000 and Prupe.I005200 belonged to the Type-2 PME family and were not mapped to the peach chromosome. Thirty-six PpPME genes were distributed in 15 clusters on peach chromosomes, including tandem-duplicated genes, such as Prupe.6G128000, Prupe.6G128100, Prupe.6G128200, and Prupe.6G128300 (Fig. 1). Similarly, 30 PMEI genes were nonrandomly distributed on eight peach chromosomes (Fig. 1). One PMEI gene was distributed on Chr 7; two on Chr 4 and Chr 8; three on Chr 6; five each on Chr 1, Chr 2, and Chr 3; and six on Chr 5. A total of 13 PpPMEI genes were located in five clusters on four peach chromosomes, including four clusters of tandem-duplicated genes, such as Prupe.3G007700, Prupe.3G007800, Prupe.3G007900, and Prupe.3G008000. Several PME and PMEI genes were located in four clusters on peach chromosomes, such as Prupe.1G330800 (PME family member) and Prupe.1G330900 (PMEI family member).

Fig. 1.
Fig. 1.

Genomic distribution of pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes on chromosomes of peach. PMEI genes were shown by triangle, others were PME genes. The chromosome numbers are demonstrated at the top of each chromosome.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

Phylogenetic and gene structure analysis.

To classify the predicted PpPMEs and PpPMEIs on the basis of amino acid sequence similarity and inferred evolutionary relationships, their amino acid sequences were aligned with predicted PMEs and PMEIs from arabidopsis. Following alignment, NJ phylogenetic trees for PMEs (Fig. 2A) and PMEIs (Fig. 2B) were constructed. Four major monophyletic groups of PMEs were defined based on the arabidopsis PME groups. Group 1 included 24 PMEs, including two Type-2 PMEs. There were 11 and 3 PMEs in groups 2 and 3, respectively, and they were all Type-1 PMEs. Group 4 was composed of 33 Type-2 PMEs. In the PMEI phylogenetic tree (Fig. 2B), groups were distinguished by very low bootstrap values in the base nodes, making subclassification of PMEIs ambiguous. Furthermore, we did not find any common sequence features that distinguished subgroups of PMEIs from each other. PMEs contained one to five exons, and PMEI genes mostly contained only one exon (Fig. 3).

Fig. 2.
Fig. 2.

Phylogenetic analysis of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) protein sequences from peach and arabidopsis. (A) Phylogenetic relationships among 71 peach PMEs and 66 arabidopsis PMEs determined based on amino acid sequences. (B) Phylogenetic relationships among 30 peach PMEIs and 71 arabidopsis PMEIs determined based on amino acid sequences. Black circle = arabidopsis PMEs and PMEIs, square frame = peach PMEs and PMEIs.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

Fig. 3.
Fig. 3.

Gene structure of pectin methylesterase (PME) (A) and pectin methylesterase inhibitor (PMEI) (B) genes in peach. Exons and introns are indicated by filled boxes and single lines, respectively. Untranslated regions (UTRs) are indicated by thick lines at both ends of the diagrams. CDS = coding sequence.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

Expression of the PME and PMEI family genes during fruit ripening in MF and SH peaches.

To elucidate the possible functions of the 71 PME and 30 PMEI genes, their expression profiles were investigated by qRT-PCR during fruit ripening in MF and SH peaches. Based on our transcript data, 11 PME and 15 PMEI genes showed a high level of expression (FPKM >1) in the two types of peach fruit during ripening (Fig. 4A and B). Among the 11 PME and 15 PMEI genes, three PME genes (including Prupe.7G190300, Prupe.7G190400, and Prupe.7G192800) showed obvious differences in expression (log2|Fold Change|>1) between MF peaches (‘CN13’ and ‘Goldhoney 3’) and SH peaches (‘CN16’ and ‘Yumyeong’) during fruit ripening, and five PMEI genes (including Prupe.1G113800, Prupe.1G114500, Prupe.1G330900, Prupe.2G279700, and Prupe.2G279800) showed obvious differences in expression (log2|Fold Change| >1).

Fig. 4.
Fig. 4.

Hierarchical cluster analysis of the expression levels of pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes in melting flesh (MF) and stony hard (SH) peaches during fruit ripening. (A) 11 PME genes and 15 PMEI genes in ‘Goldhoney 3’ and ‘Yumyeong’. (B) 11 PME genes and 15 PMEI genes in ‘Zhongyoutao 13’ (‘CN13’) and ‘Zhongyoutao 16’ (‘CN16’). The log2 ratios and scale bars are shown in the figure, which were obtained using Java TreeView (Saldanha, 2004).

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

To validate the gene expression changes determined by transcript data, quantitative PCR analysis was performed on a selection of differentially expressed PME and PMEI genes. Overall, the quantification of three PME genes and five PMEI genes by qRT-PCR exhibited close agreement with transcript data (Fig. 5). One of the three PME genes (Prupe.7G192800) was upregulated during fruit development, and its expression peaked at S4 III in MF peaches (‘CN13’ and ‘Goldhoney 3’), whereas its expression level was low in SH peaches (‘CN16’ and ‘Yumyeong’). Similarly, two of the five PMEI genes (Prupe.1G114500 and Prupe.2G279800) were upregulated during fruit development, and their expression peaked at S4 III in MF peaches (‘CN13’ and ‘Goldhoney 3’), whereas their expression was almost undetectable in SH peaches (‘CN16’ and ‘Yumyeong’). One PME (Prupe.7G190400) was upregulated in ‘Goldhoney 3’ during fruit development, but not in ‘CN13’. One PMEI gene (Prupe.2G279700) was upregulated in ‘CN13’, but not in ‘Goldhoney 3’.

Fig. 5.
Fig. 5.

Expression of eight pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes during peach fruit ripening. (A) Expression of eight PME and PMEI genes in ‘Goldhoney 3’ (‘GH3’) and ‘Yumyeong’ (‘YM’). (B) Expression of eight PME and PMEI genes in ‘Zhongyoutao 13’ (‘CN13’) and ‘Zhongyoutao 16’ (‘CN16’). Expression levels were normalized to input RNA, and the value of ‘CN13’ and ‘GH3’ at stage S3 was set to 1. Data are means ± sd of at least three individual experiments.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

Fruit firmness and ethylene production changes in response to auxin and ethylene treatment.

The effects of auxin and ethylene on peach fruit softening were determined by evaluating ethylene production and flesh firmness. In this study, the SH type ‘CN16’ was used to explore the effect of NAA and ethylene treatment. Ethylene was not detected without treatment, and ethylene treatment did not stimulate the production of endogenous ethylene; however, NAA treatment resulted in the production of abundant endogenous ethylene in SH peaches, with a rapid increase in ethylene production beginning at 36 h that peaked at 96 h [8.34 μL·g−1·h−1 (Fig. 6A)]. Fruit firmness decreased moderately in ‘CN16’ without treatment or with NAA and ethylene treatment; however, fruit firmness decreased to a greater extent with NAA than with ethylene treatment at the same time point, and to a greater extent with ethylene than without treatment at the same time point. ‘CN16’ fruit hardly softened, and flesh firmness was maintained over 20 N/cm2 without treatment or with NAA and ethylene treatment (Fig. 6B).

Fig. 6.
Fig. 6.

Effects of ethylene (C2H4) and 1-naphthaleneacetic acid (NAA) treatment on postharvest ‘Zhongyoutao 16’ (‘CN16’) fruit. Values are means ± sd of three biological replicates. CK = control.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

Promoter analysis of the PMEs and PMEIs related to plant hormone responses during fruit ripening.

Identification and analysis of the regulatory motifs in the promoters of genes expressed during fruit development and ripening is beneficial to improve our understanding of the molecular regulation of these complex developmental processes mediated by numerous transcription factors and various plant hormones. Here, the cis elements in the promoter sequences involved in plant hormone (especially NAA and ethylene) response, were surveyed using PlantCARE, PLACE, and a manual search to understand the transcriptional regulation and potential functions of the fruit ripening–related PME and PMEI genes. Seven types of cis elements were involved in plant hormone response, and one fruit-specific cis element was present in the 1.5 kb upstream sequences of the eight PpPME and PpPMEI genes, representing their predicted promoter regions (Supplemental Table 3; Fig. 7). All members of these cis elements in the promoter regions of PpPME and PpPMEI genes are shown in Supplemental Table 4 and Fig. 7. The fruit-specific cis elements, TCCAAAA-motif and TGTCACA-motif, were detected in the promoter regions of two PpPMEI genes (Prupe.1G114500 and Prupe.2G279800) and two PpPME genes (Prupe.7G190400 and Prupe.7G192800). The ethylene-responsive elements (EREs) (C-repeat/drought-responsive element, ERE, and GCC-box), which play important roles in the regulation of many ethylene-related genes, were detected in the upstream promoter regions of Prupe.1G113800, Prupe.1G330900, Prupe.2G279800, and Prupe.7G190300. The auxin-responsive elements (TGA-element, TGA-box, and S000270), which play important roles in the regulation of auxin-related genes, were detected in the upstream promoter regions of Prupe.1G113800, Prupe.1G114500, Prupe.1G330900, and Prupe.7G192800. Eight promoters contained at least one cis element for plant hormone responses.

Fig. 7.
Fig. 7.

Cis elements in the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes promoter predicted by database analysis. Cylinder represents fruit specific element including TGTCACA-motif and TCCAAAA-motif; parallelogram represents gibberellic acid (GA) response element including TATC-box, gibberellin-responsive element (GARE-motif) and P-box. Triangle represents ethylene response element including ethylene responsive element (ERE), GCC-box and C-repeat/drought-responsive element (CRT/DRE). Rectangle represents auxin response element including TGA-element, TGA-box, S000270; sun represents abscisic acid (ABA) response element including abscisic acid-responsive element (ABRE). Rhombus represents salicylic acid (SA) response element including TCA-element; smiling face represents methyl jasmonate (MeJA) response element including TGACG-motif.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

Expression of the eight PME and PMEI family genes in SH fruit (‘CN16’) in response to NAA and ethylene.

To further analyze the cis elements involved in auxin and ethylene response in the promoter regions of the eight PME and PMEI family genes identified, their expression in response to NAA and ethylene treatment was investigated in ‘CN16’ fruit (Fig. 8). NAA induced the expression of one PME gene (Prupe.7G192800) and four PMEI genes (Prupe.1G113800, Prupe.1G114500, Prupe.1G330900, and Prupe.2G279800) in ‘CN16’ fruit, whereas ethylene induced the expression of one PME gene (Prupe.7G192800) and two PMEI genes (Prupe.1G113800 and Prupe.1G114500). Prupe.1G114500 showed the highest upregulation in response to ethylene treatment, whereas Prupe.7G192800 showed the highest upregulation in response to NAA. Other genes were downregulated by NAA or ethylene treatment.

Fig. 8.
Fig. 8.

Effects of 1-naphthaleneacetic acid (NAA) and ethylene (Eth) treatment on expression of eight pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes in ‘Zhongyoutao 16’ (‘CN16’) peach fruit. Expression levels were normalized to the amount of input RNA, and values at 0 h were set to 1. Data are means ± sd of at least three individual experiments. CK = control.

Citation: Journal of the American Society for Horticultural Science J. Amer. Soc. Hort. Sci. 142, 4; 10.21273/JASHS04039-17

Discussion

PME is the first enzyme acting on pectin, a major component of the plant cell wall. PME produces pectin with different structural and functional properties and therefore plays an important role in plant physiology (Giovane et al., 2004). Recent data indicate that several PME isoforms detected in the cell walls are encoded by a multigene family (Pinzón-Latorre and Deyholos, 2013; Wang et al., 2013). In the present study, 71 PMEs were identified in the peach genome, of which 36 were distributed in 15 clusters on peach chromosomes, suggesting that whole-genome duplication and tandem duplication contribute to the expansion of the large family of PME genes (Wang et al., 2013). The 71 PMEs were divided into 36 Type-1 PMEs and 35 Type-2 PMEs, and both types of PME genes function in demethylesterification. Further study should be aimed at clarifying the relationship between gene family expansion and the function of the two types of PME genes. PME activity is controlled at the posttranscriptional level by PMEIs, and the PMEI family appeared later than Type-1 PME and Type-2 PME genes (Wang et al., 2013). Only 30 PMEIs were identified in the peach genome. Phylogenetic analysis with arabidopsis PMEIs showed no common sequence features that distinguished subgroups of PMEIs from each other. This may be because peach has not undergone recent whole-genome duplication (Verde et al., 2013), and the expansion of subclasses is lower than that of other different plants.

Peach fruit are classified into three groups according to the character of textural changes during ripening: MF, NMF, and SH types (Haji et al., 2001). The MF and NMF characters were expressed in response to ethylene treatment in SH fruit, indicating that the SH trait was epistatic to the MF/NMF trait (Haji et al., 2005). In the present study, ‘CN16’ fruit treated with ethylene and NAA did not soften considerably during fruit storage, suggesting that ‘CN16’ fruit belong to SH NMF type.

PMEs are involved in peach fruit ripening (Brummell et al., 2004). In the present study, 11 PME genes showed significant expression in two peach texture types during fruit ripening, and three PME genes (including Prupe.7G190300, Prupe.7G190400, and Prupe.7G192800) showed obvious difference in expression between MF peaches and SH peaches during fruit ripening. Previous research showed that PME1 (AB231903) and PME2 (X95991) are upregulated in MF type peaches compared with SH type peaches (Murayama et al., 2009); the accession no. 2.1 of these two genes are Prupe.7G190300 and Prupe.7G190400, respectively. PMEI is present in the aqueous phase of fully ripe fruit, and is probably synthesized as a large precursor that undergoes proteolytic cleavage during the fruit ripening process (Giovane et al., 2004). Of 30 PMEI genes identified, 15 were expressed in two types of peach fruit during fruit ripening; five PMEI genes were upregulated in MF type peaches compared with their expression in SH type peaches, suggesting that these genes are involved in fruit ripening.

Although many PME genes were upregulated during peach fruit ripening, the timing and extent of the increase in the PME activity were not necessarily related to ethylene or fruit ripening (Brummell et al., 2004; Hayama et al., 2006). Overexpression of PMEIs in arabidopsis and tobacco (Nicotiana tabacum) results in a lower level of PME activity and a higher degree of pectin esterification (Lionetti et al., 2007, 2012). Therefore, the high expression of PMEIs may lead to decrease of PME activity in peach fruit during ripening or in response to ethylene treatment. Prupe.7G190300 and Prupe.7G190400 are Type-1 PMEs, and Prupe.7G192800 is a Type-2 PME. The proregion of Type-1 PMEs may be involved in the autoinhibition of activity, preventing premature de-esterification during transport in pectin-containing secretory vesicles (Wolf et al., 2009). Because Type-2 PMEs lack the PMEI-like proregion, the specific five PMEIs may be coexpressed during fruit ripening to block activity within secretory vesicles. However, the specific PMEI that regulates PME activity, and the effect of PMEs on pectin during peach fruit ripening remain unclear. Further analysis of the biological functions of these genes (including PME and PMEI) during fruit ripening is necessary.

In addition to PMEI, PME activity is also regulated by hormones. Auxin-induced PME activity increases cell wall extension, and consequently water absorption by the cell (Micheli, 2001). In the present study, NAA induced the expression of one PME and four PMEI genes, whereas ethylene induced the expression of one PME gene and two PMEI genes. The plant hormone response and fruit-specific cis elements in the promoter sequences of the PME and PMEI genes were also investigated. The promoter of a pepper PMEI contains crucial EREs and ethylene-response factors (An et al., 2009). EREs were also identified in the promoter of the E4 gene, which is controlled by an increase in ethylene concentration during tomato fruit ripening (Montgomery et al., 1993), suggesting a common regulatory mechanism during ripening. Most of the promoter regions of PME and PMEI genes contain more than one cis element for fruit-specific and hormone responses, especially to auxin and ethylene, further supporting their participation in peach fruit ripening. Strong evidence makes it imperative to include this new candidate in PME-related studies. Further studies are needed to determine whether and how these cis elements function during fruit ripening.

Conclusion

In conclusion, the peach genome contains 71 PME and 30 PMEI genes that are unevenly distributed on all eight chromosomes. Comprehensive phylogenetic, gene structure, and chromosomal location analyses provided information on this gene family in peach. Analysis of the expression profiles of the PME and PMEI genes during peach fruit ripening in two types of peaches with different textures identified three PME and five PMEI genes with differential expression, and the upstream regulatory cis elements of these genes related to fruit ripening were investigated. One PME (Prupe.7G192800) and two PMEIs (Prupe.1G114500 and Prupe.2G279800) and their promoters were identified as potential targets for future studies on the biochemical metabolism and regulation of fruit ripening. The comprehensive data generated in this study will improve our understanding of the complex PME and PMEI gene families in peach. However, further studies are needed to reveal the detailed biochemical function and regulatory mechanism of PME and PMEI genes during peach fruit ripening.

Literature Cited

  • An, S.H., Choi, H.W., Hong, J.K. & Hwang, B.K. 2009 Regulation and function of the pepper pectin methylesterase inhibitor (CaPMEI1) gene promoter in defense and ethylene and methyl jasmonate signaling in plants Planta 230 1223 1237

    • Search Google Scholar
    • Export Citation
  • An, S.H., Sohn, K.H., Choi, H.W., Hwang, I.S., Lee, S.C. & Hwang, B.K. 2008 Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance Planta 228 61 78

    • Search Google Scholar
    • Export Citation
  • Balestrieri, C., Castaldo, D., Giovane, A., Quagliuolo, L. & Servillo, L. 1990 A glycoprotein inhibitor of pectin methylesterase in kiwifruit (Actinidia chinensis) Eur. J. Biochem. 193 183 187

    • Search Google Scholar
    • Export Citation
  • Brummell, D.A., Dal Cin, V., Crisosto, C.H. & Labavitch, J.M. 2004 Cell wall metabolism during maturation, ripening and senescence of peach fruit J. Expt. Bot. 55 2029 2039

    • Search Google Scholar
    • Export Citation
  • Brummell, D.A. & Harpster, M.H. 2001 Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants Plant Mol. Biol. 47 311 340

    • Search Google Scholar
    • Export Citation
  • Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V. & Henrissat, B. 2009 The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics Nucleic Acids Res. 37 D233 D238

    • Search Google Scholar
    • Export Citation
  • Deytieux-Belleau, C., Vallet, A., Donèche, B. & Geny, L. 2008 Pectin methylesterase and polygalacturonase in the developing grape skin Plant Physiol. Biochem. 46 638 646

    • Search Google Scholar
    • Export Citation
  • Di Matteo, A., Giovane, A., Raiola, A., Camardella, L., Bonivento, D., De Lorenzo, G., Bellincampi, D., Cervone, F. & Tsernoglou, D. 2005 Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein Plant Cell 17 849 858

    • Search Google Scholar
    • Export Citation
  • Eriksson, E.M., Bovy, A., Manning, K., Harrison, L., Andrews, J., De Silva, J., Tucker, G.A. & Seymour, G.B. 2004 Effect of the Colorless non-ripening mutation on cell wall biochemistry and gene expression during tomato fruit development and ripening Plant Physiol. 136 4184 4197

    • Search Google Scholar
    • Export Citation
  • Francis, K.E., Lam, S.Y. & Copenhaver, G.P. 2006 Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene Plant Physiol. 142 1004 1013

    • Search Google Scholar
    • Export Citation
  • Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D. & Bairoch, A. 2003 ExPASy: The proteomics server for in-depth protein knowledge and analysis Nucleic Acids Res. 31 3784 3788

    • Search Google Scholar
    • Export Citation
  • Giovane, A., Servillo, L., Balestrieri, C., Raiola, A., D’avino, R., Tamburrini, M., Ciardiello, M.A. & Camardella, L. 2004 Pectin methylesterase inhibitor Biochim. Biophys. Acta. Proteins Proteomics 1696 245 252

    • Search Google Scholar
    • Export Citation
  • Goodstein, D.M., Shu, S., Howson, R., Neupane, R., Hayes, R.D., Fazo, J., Mitros, T., Dirks, W., Hellsten, U., Putnam, N. & Rokhsar, D.S. 2012 Phytozome: A comparative platform for green plant genomics Nucleic Acids Res. 40 D1178 D1186

    • Search Google Scholar
    • Export Citation
  • Haji, T., Yaegaki, H. & Yamaguchi, M. 2001 Changes in ethylene production and flesh firmness of melting, nonmelting and stony hard peaches [Prunus persica] after harvest J. Jpn. Soc. Hort. Sci. 70 458 459

    • Search Google Scholar
    • Export Citation
  • Haji, T., Yaegaki, H. & Yamaguchi, M. 2005 Inheritance and expression of fruit texture melting, non-melting and stony hard in peach Sci. Hort. 105 241 248

    • Search Google Scholar
    • Export Citation
  • Hayama, H., Shimada, T., Fujii, H., Ito, A. & Kashimura, Y. 2006 Ethylene-regulation of fruit softening and softening-related genes in peach J. Expt. Bot. 57 4071 4077

    • Search Google Scholar
    • Export Citation
  • Higo, K., Ugawa, Y., Iwamoto, M. & Korenaga, T. 1999 Plant cis-acting regulatory DNA elements (PLACE) database: 1999 Nucleic Acids Res. 27 297 300

  • Hothorn, M., Wolf, S., Aloy, P., Greiner, S. & Scheffzek, K. 2004 Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins Plant Cell 16 3437 3447

    • Search Google Scholar
    • Export Citation
  • Hu, B., Jin, J., Guo, A.Y., Zhang, H., Luo, J. & Gao, G. 2015 GSDS 2.0: An upgraded gene feature visualization server Bioinformatics 31 1296 1297

  • Jolie, R.P., Duvetter, T., Van Loey, A.M. & Hendrickx, M.E. 2010 Pectin methylesterase and its proteinaceous inhibitor: A review Carbohydr. Res. 345 2583 2595

    • Search Google Scholar
    • Export Citation
  • Koch, J.L. & Nevins, D.J. 1989 Tomato fruit cell wall I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins Plant Physiol. 91 816 822

    • Search Google Scholar
    • Export Citation
  • Lacoux, J., Gutierrez, L., Dantin, F., Beaudoin, B., Roger, D. & Laine, E. 2003 Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation Protoplasma 222 205 209

    • Search Google Scholar
    • Export Citation
  • Lamesch, P., Berardini, T.Z., Li, D., Swarbreck, D., Wilks, C., Sasidharan, R., Muller, R., Dreher, K., Alexander, D.L., Garcia-Hernandez, M., Karthikeyan, A.S., Lee, C.H., Nelson, W.D., Ploetz, L., Singh, S., Wensel, A. & Huala, E. 2012 The Arabidopsis Information Resource (TAIR): Improved gene annotation and new tools Nucleic Acids Res. 40 D1202 D1210

    • Search Google Scholar
    • Export Citation
  • Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouzé, P. & Rombauts, S. 2002 PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences Nucleic Acids Res. 30 325 327

    • Search Google Scholar
    • Export Citation
  • Lionetti, V., Cervone, F. & Bellincampi, D. 2012 Methyl esterification of pectin plays a role during plant–pathogen interactions and affects plant resistance to diseases J. Plant Physiol. 169 1623 1630

    • Search Google Scholar
    • Export Citation
  • Lionetti, V., Raiola, A., Camardella, L., Giovane, A., Obel, N., Pauly, M., Favaron, F., Cervone, F. & Bellincampi, D. 2007 Overexpression of pectin methylesterase inhibitors in arabidopsis restricts fungal infection by Botrytis cinerea Plant Physiol. 143 1871 1880

    • Search Google Scholar
    • Export Citation
  • Lionetti, V., Raiola, A., Mattei, B. & Bellincampi, D. 2015 The grapevine VvPMEI1 gene encodes a novel functional pectin methylesterase inhibitor associated to grape berry development PLoS One 10 e0133810

    • Search Google Scholar
    • Export Citation
  • Livak, K.J. & Schmittgen, T.D. 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method Methods 25 402 408

    • Search Google Scholar
    • Export Citation
  • Micheli, F. 2001 Pectin methylesterases: Cell wall enzymes with important roles in plant physiology Trends Plant Sci. 6 414 419

  • Micheli, F., Sundberg, B., Goldberg, R. & Richard, L. 2000 Radial distribution pattern of pectin methylesterases across the cambial region of hybrid aspen at activity and dormancy Plant Physiol. 124 191 199

    • Search Google Scholar
    • Export Citation
  • Montgomery, J., Goldman, S., Deikman, J., Margossian, L. & Fischer, R.L. 1993 Identification of an ethylene-responsive region in the promoter of a fruit ripening gene Proc. Natl. Acad. Sci. USA 90 5939 5943

    • Search Google Scholar
    • Export Citation
  • Murayama, H., Arikawa, M., Sasaki, Y., Dal Cin, V., Mitsuhashi, W. & Toyomasu, T. 2009 Effect of ethylene treatment on expression of polyuronide-modifying genes and solubilization of polyuronides during ripening in two peach cultivars having different softening characteristics Postharvest Biol. Technol. 52 196 201

    • Search Google Scholar
    • Export Citation
  • Pan, L., Zeng, W., Niu, L., Lu, Z., Wang, X., Liu, H., Cui, G., Zhu, Y., Chu, J., Li, W., Fang, W., Cai, Z., Li, G. & Wang, Z. 2015 PpYUC11, a strong candidate gene for the stony hard phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis during fruit ripening J. Expt. Bot. 66 7031 7044

    • Search Google Scholar
    • Export Citation
  • Peaucelle, A., Braybrook, S.A., Le Guillou, L., Bron, E., Kuhlemeier, C. & Höfte, H. 2011 Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis Curr. Biol. 21 1720 1726

    • Search Google Scholar
    • Export Citation
  • Peaucelle, A., Louvet, R., Johansen, J.N., Höfte, H., Laufs, P., Pelloux, J. & Mouille, G. 2008 Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins Curr. Biol. 18 1943 1948

    • Search Google Scholar
    • Export Citation
  • Pelletier, S., Van Orden, J., Wolf, S., Vissenberg, K., Delacourt, J., Ndong, Y.A., Pelloux, J., Bischoff, V., Urbain, A., Mouille, G., Lemonnier, G., Renou, J. & Höfte, H. 2010 A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls New Phytol. 188 726 739

    • Search Google Scholar
    • Export Citation
  • Pelloux, J., Rusterucci, C. & Mellerowicz, E.J. 2007 New insights into pectin methylesterase structure and function Trends Plant Sci. 12 267 277

  • Pinzón-Latorre, D. & Deyholos, M.K. 2013 Characterization and transcript profiling of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) gene families in flax (Linum usitatissimum) BMC Genomics 14 742 766

    • Search Google Scholar
    • Export Citation
  • Prasanna, V., Prabha, T.N. & Tharanathan, R.N. 2007 Fruit ripening phenomena-an overview Crit. Rev. Food Sci. Nutr. 47 1 19

  • Punta, M., Coggill, P.C., Eberhardt, R.Y., Mistry, J., Tate, J., Boursnell, C., Pang, N., Forslund, K., Ceric, G., Clements, J., Heger, A., Holm, L., Sonnhammer, E.L.L., Eddy, S.R., Bateman, A. & Finn, R.D. 2011 The Pfam protein families database Nucleic Acids Res. 40 D290 D301

    • Search Google Scholar
    • Export Citation
  • Raiola, A., Camardella, L., Giovane, A., Mattei, B., De Lorenzo, G., Cervone, F. & Bellincampi, D. 2004 Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors1 FEBS Lett. 557 199 203

    • Search Google Scholar
    • Export Citation
  • Reca, I.B., Lionetti, V., Camardella, L., D’Avino, R., Giardina, T., Cervone, F. & Bellincampi, D. 2012 A functional pectin methylesterase inhibitor protein (SolyPMEI) is expressed during tomato fruit ripening and interacts with PME-1 Plant Mol. Biol. 79 429 442

    • Search Google Scholar
    • Export Citation
  • Richard, L., Qin, L.X. & Goldberg, R. 1996 Clustered genes within the genome of Arabidopsis thaliana encoding pectin methylesterase-like enzymes Gene 170 207 211

    • Search Google Scholar
    • Export Citation
  • Röckel, N., Wolf, S., Kost, B., Rausch, T. & Greiner, S. 2008 Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins Plant J. 53 133 143

    • Search Google Scholar
    • Export Citation
  • Saldanha, A.J. 2004 Java Treeview-extensible visualization of microarray data Bioinformatics 20 3246 3248

  • Siedlecka, A., Wiklund, S., Péronne, M.A., Micheli, F., Leśniewska, J., Sethson, I., Edlund, U., Richard, L., Sundberg, B. & Mellerowicz, E.J. 2008 Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus Plant Physiol. 146 554 565

    • Search Google Scholar
    • Export Citation
  • Srivastava, S., Gupta, S.M., Sane, A.P. & Nath, P. 2012 Isolation and characterization of ripening related pectin methylesterase inhibitor gene from banana fruit Physiol. Mol. Biol. Plants 18 191 195

    • Search Google Scholar
    • Export Citation
  • Tamura, K., Dudley, J., Nei, M. & Kumar, S. 2007 MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0 Mol. Biol. Evol. 24 1596 1599

    • Search Google Scholar
    • Export Citation
  • Tatsuki, M., Nakajima, N., Fujii, H., Shimada, T., Nakano, M., Hayashi, K.I., Hayama, H., Yoshioka, H. & Nakamura, Y. 2013 Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch) J. Expt. Bot. 64 1049 1059

    • Search Google Scholar
    • Export Citation
  • Trapnell, C., Williams, B.A., Pertea, G., Mortazavi, A., Kwan, G., Van Baren, M.J., Salzberg, S.L., Wold, B.J. & Pachter, L. 2010 Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation Nat. Biotechnol. 28 511 515

    • Search Google Scholar
    • Export Citation
  • Van Berloo, R. 1999 Computer note. GGT: Software for the display of graphical genotypes J. Hered. 90 328 329

  • Verde, I., Abbott, A.G., Scalabrin, S., Jung, S., Shu, S., Marroni, F., Zhebentyayeva, T., Dettori, M.T., Grimwood, J., Cattonaro, F., Zuccolo, A., Rossini, L., Jenkins, J., Vendramin, E., Meisel, L.A., Decroocq, V., Sosinski, B., Prochnik, S., Mitros, T., Policriti, A., Cipriani, G., Dondini, L., Ficklin, S., Goodstein, D.M., Xuan, P., DelFabbro, C., Aramini, V., Copetti, D., Gonzalez, S., Horner, D.S., Falchi, R., Lucas, S., Mica, E., Maldonado, J., Lazzari, B., Bielenberg, D., Pirona, R., Miculan, M., Barakat, A., Testolin, R., Stella, A., Tartarini, S., Tonutti, P., Arus, P., Orellana, A., Wells, C., Main, D., Vizzotto, G., Silva, H., Salamini, F., Schmutz, J., Morgante, M. & Rokhsar, D.S. 2013 The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution Nat. Genet. 45 487 494

    • Search Google Scholar
    • Export Citation
  • Wang, M., Yuan, D., Gao, W., Li, Y., Tan, J. & Zhang, X. 2013 A comparative genome analysis of PME and PMEI families reveals the evolution of pectin metabolism in plant cell walls PLoS One 8 e72082

    • Search Google Scholar
    • Export Citation
  • Wolf, S., Rausch, T. & Greiner, S. 2009 The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus Plant J. 58 361 375

    • Search Google Scholar
    • Export Citation
  • Yamagata, H., Yonesu, K., Hirata, A. & Aizono, Y. 2002 TGTCACA motif is a novel cis-regulatory enhancer element involved in fruit-specific expression of the cucumisin gene J. Biol. Chem. 277 11582 11590

    • Search Google Scholar
    • Export Citation
  • Yu, Y., Liang, Y., Lv, M., Wu, J., Lu, G. & Cao, J. 2014 Genome-wide identification and characterization of polygalacturonase genes in Cucumis sativus and Citrullus lanatus Plant Physiol. Biochem. 74 263 275

    • Search Google Scholar
    • Export Citation
  • Zeng, W., Pan, L., Liu, H., Niu, L., Lu, Z., Cui, G. & Wang, Z. 2015 Characterization of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes during nectarine fruit development and ripening Tree Genet. Genomes 11 1 10

    • Search Google Scholar
    • Export Citation
  • Zhang, G.Y., Feng, J., Wu, J. & Wang, X.W. 2010 BoPMEI1, a pollen-specific pectin methylesterase inhibitor, has an essential role in pollen tube growth Planta 231 1323 1334

    • Search Google Scholar
    • Export Citation
Supplemental Table 1.

Fragments per kilobase of exon per million fragments mapped (FPKM) values for pectin methylesterase (PME) genes during ‘Zhongyoutao 13’ (‘CN13’) and ‘Zhongyoutao 16’ (‘CN16’) peach fruit ripening.

Supplemental Table 1.
Supplemental Table 2.

Fragments per kilobase of exon per million fragments mapped (FPKM) values for pectin methylesterase inhibitor (PMEI) genes during ‘Zhongyoutao 13’ (‘CN13’) and ‘Zhongyoutao 16’ (‘CN16’) peach fruit ripening.

Supplemental Table 2.
Supplemental Table 3.

The putative cis-acting regulatory elements of plant hormones response presented in the 5′-upstream region of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes related to fruit development and ripening of peach.

Supplemental Table 3.
Supplemental Table 4.

The types and number of putative cis-acting regulatory elements of plant hormones response presented in the 5′-upstream region of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes related to fruit ripening of peach.

Supplemental Table 4.

If the inline PDF is not rendering correctly, you can download the PDF file here.

Contributor Notes

The research was financially supported by the National Natural Science Foundation of China (No. 31501732), the Agricultural Science and Technology Innovation Program (ASTIP) (CAAS-ASTIP-2017-ZFRI), the Major Science and Technology Project in Henan Province (No. 151100110900), the Project of Basic and Advanced Technology Research of Henan Province of China (Grant No. 152300410137), and Central Public-interest Scientific Institution Basal Research Fund (No. 1610192016203).

These authors contributed equally to this work.

Corresponding author. E-mail: wangzhiqiang@caas.cn.

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    Genomic distribution of pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes on chromosomes of peach. PMEI genes were shown by triangle, others were PME genes. The chromosome numbers are demonstrated at the top of each chromosome.

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    Phylogenetic analysis of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) protein sequences from peach and arabidopsis. (A) Phylogenetic relationships among 71 peach PMEs and 66 arabidopsis PMEs determined based on amino acid sequences. (B) Phylogenetic relationships among 30 peach PMEIs and 71 arabidopsis PMEIs determined based on amino acid sequences. Black circle = arabidopsis PMEs and PMEIs, square frame = peach PMEs and PMEIs.

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    Gene structure of pectin methylesterase (PME) (A) and pectin methylesterase inhibitor (PMEI) (B) genes in peach. Exons and introns are indicated by filled boxes and single lines, respectively. Untranslated regions (UTRs) are indicated by thick lines at both ends of the diagrams. CDS = coding sequence.

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    Hierarchical cluster analysis of the expression levels of pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes in melting flesh (MF) and stony hard (SH) peaches during fruit ripening. (A) 11 PME genes and 15 PMEI genes in ‘Goldhoney 3’ and ‘Yumyeong’. (B) 11 PME genes and 15 PMEI genes in ‘Zhongyoutao 13’ (‘CN13’) and ‘Zhongyoutao 16’ (‘CN16’). The log2 ratios and scale bars are shown in the figure, which were obtained using Java TreeView (Saldanha, 2004).

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    Expression of eight pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes during peach fruit ripening. (A) Expression of eight PME and PMEI genes in ‘Goldhoney 3’ (‘GH3’) and ‘Yumyeong’ (‘YM’). (B) Expression of eight PME and PMEI genes in ‘Zhongyoutao 13’ (‘CN13’) and ‘Zhongyoutao 16’ (‘CN16’). Expression levels were normalized to input RNA, and the value of ‘CN13’ and ‘GH3’ at stage S3 was set to 1. Data are means ± sd of at least three individual experiments.

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    Effects of ethylene (C2H4) and 1-naphthaleneacetic acid (NAA) treatment on postharvest ‘Zhongyoutao 16’ (‘CN16’) fruit. Values are means ± sd of three biological replicates. CK = control.

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    Cis elements in the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes promoter predicted by database analysis. Cylinder represents fruit specific element including TGTCACA-motif and TCCAAAA-motif; parallelogram represents gibberellic acid (GA) response element including TATC-box, gibberellin-responsive element (GARE-motif) and P-box. Triangle represents ethylene response element including ethylene responsive element (ERE), GCC-box and C-repeat/drought-responsive element (CRT/DRE). Rectangle represents auxin response element including TGA-element, TGA-box, S000270; sun represents abscisic acid (ABA) response element including abscisic acid-responsive element (ABRE). Rhombus represents salicylic acid (SA) response element including TCA-element; smiling face represents methyl jasmonate (MeJA) response element including TGACG-motif.

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    Effects of 1-naphthaleneacetic acid (NAA) and ethylene (Eth) treatment on expression of eight pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) genes in ‘Zhongyoutao 16’ (‘CN16’) peach fruit. Expression levels were normalized to the amount of input RNA, and values at 0 h were set to 1. Data are means ± sd of at least three individual experiments. CK = control.

  • An, S.H., Choi, H.W., Hong, J.K. & Hwang, B.K. 2009 Regulation and function of the pepper pectin methylesterase inhibitor (CaPMEI1) gene promoter in defense and ethylene and methyl jasmonate signaling in plants Planta 230 1223 1237

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  • An, S.H., Sohn, K.H., Choi, H.W., Hwang, I.S., Lee, S.C. & Hwang, B.K. 2008 Pepper pectin methylesterase inhibitor protein CaPMEI1 is required for antifungal activity, basal disease resistance and abiotic stress tolerance Planta 228 61 78

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  • Balestrieri, C., Castaldo, D., Giovane, A., Quagliuolo, L. & Servillo, L. 1990 A glycoprotein inhibitor of pectin methylesterase in kiwifruit (Actinidia chinensis) Eur. J. Biochem. 193 183 187

    • Search Google Scholar
    • Export Citation
  • Brummell, D.A., Dal Cin, V., Crisosto, C.H. & Labavitch, J.M. 2004 Cell wall metabolism during maturation, ripening and senescence of peach fruit J. Expt. Bot. 55 2029 2039

    • Search Google Scholar
    • Export Citation
  • Brummell, D.A. & Harpster, M.H. 2001 Cell wall metabolism in fruit softening and quality and its manipulation in transgenic plants Plant Mol. Biol. 47 311 340

    • Search Google Scholar
    • Export Citation
  • Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V. & Henrissat, B. 2009 The Carbohydrate-Active EnZymes database (CAZy): An expert resource for glycogenomics Nucleic Acids Res. 37 D233 D238

    • Search Google Scholar
    • Export Citation
  • Deytieux-Belleau, C., Vallet, A., Donèche, B. & Geny, L. 2008 Pectin methylesterase and polygalacturonase in the developing grape skin Plant Physiol. Biochem. 46 638 646

    • Search Google Scholar
    • Export Citation
  • Di Matteo, A., Giovane, A., Raiola, A., Camardella, L., Bonivento, D., De Lorenzo, G., Bellincampi, D., Cervone, F. & Tsernoglou, D. 2005 Structural basis for the interaction between pectin methylesterase and a specific inhibitor protein Plant Cell 17 849 858

    • Search Google Scholar
    • Export Citation
  • Eriksson, E.M., Bovy, A., Manning, K., Harrison, L., Andrews, J., De Silva, J., Tucker, G.A. & Seymour, G.B. 2004 Effect of the Colorless non-ripening mutation on cell wall biochemistry and gene expression during tomato fruit development and ripening Plant Physiol. 136 4184 4197

    • Search Google Scholar
    • Export Citation
  • Francis, K.E., Lam, S.Y. & Copenhaver, G.P. 2006 Separation of Arabidopsis pollen tetrads is regulated by QUARTET1, a pectin methylesterase gene Plant Physiol. 142 1004 1013

    • Search Google Scholar
    • Export Citation
  • Gasteiger, E., Gattiker, A., Hoogland, C., Ivanyi, I., Appel, R.D. & Bairoch, A. 2003 ExPASy: The proteomics server for in-depth protein knowledge and analysis Nucleic Acids Res. 31 3784 3788

    • Search Google Scholar
    • Export Citation
  • Giovane, A., Servillo, L., Balestrieri, C., Raiola, A., D’avino, R., Tamburrini, M., Ciardiello, M.A. & Camardella, L. 2004 Pectin methylesterase inhibitor Biochim. Biophys. Acta. Proteins Proteomics 1696 245 252

    • Search Google Scholar
    • Export Citation
  • Goodstein, D.M., Shu, S., Howson, R., Neupane, R., Hayes, R.D., Fazo, J., Mitros, T., Dirks, W., Hellsten, U., Putnam, N. & Rokhsar, D.S. 2012 Phytozome: A comparative platform for green plant genomics Nucleic Acids Res. 40 D1178 D1186

    • Search Google Scholar
    • Export Citation
  • Haji, T., Yaegaki, H. & Yamaguchi, M. 2001 Changes in ethylene production and flesh firmness of melting, nonmelting and stony hard peaches [Prunus persica] after harvest J. Jpn. Soc. Hort. Sci. 70 458 459

    • Search Google Scholar
    • Export Citation
  • Haji, T., Yaegaki, H. & Yamaguchi, M. 2005 Inheritance and expression of fruit texture melting, non-melting and stony hard in peach Sci. Hort. 105 241 248

    • Search Google Scholar
    • Export Citation
  • Hayama, H., Shimada, T., Fujii, H., Ito, A. & Kashimura, Y. 2006 Ethylene-regulation of fruit softening and softening-related genes in peach J. Expt. Bot. 57 4071 4077

    • Search Google Scholar
    • Export Citation
  • Higo, K., Ugawa, Y., Iwamoto, M. & Korenaga, T. 1999 Plant cis-acting regulatory DNA elements (PLACE) database: 1999 Nucleic Acids Res. 27 297 300

  • Hothorn, M., Wolf, S., Aloy, P., Greiner, S. & Scheffzek, K. 2004 Structural insights into the target specificity of plant invertase and pectin methylesterase inhibitory proteins Plant Cell 16 3437 3447

    • Search Google Scholar
    • Export Citation
  • Hu, B., Jin, J., Guo, A.Y., Zhang, H., Luo, J. & Gao, G. 2015 GSDS 2.0: An upgraded gene feature visualization server Bioinformatics 31 1296 1297

  • Jolie, R.P., Duvetter, T., Van Loey, A.M. & Hendrickx, M.E. 2010 Pectin methylesterase and its proteinaceous inhibitor: A review Carbohydr. Res. 345 2583 2595

    • Search Google Scholar
    • Export Citation
  • Koch, J.L. & Nevins, D.J. 1989 Tomato fruit cell wall I. Use of purified tomato polygalacturonase and pectinmethylesterase to identify developmental changes in pectins Plant Physiol. 91 816 822

    • Search Google Scholar
    • Export Citation
  • Lacoux, J., Gutierrez, L., Dantin, F., Beaudoin, B., Roger, D. & Laine, E. 2003 Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation Protoplasma 222 205 209

    • Search Google Scholar
    • Export Citation
  • Lamesch, P., Berardini, T.Z., Li, D., Swarbreck, D., Wilks, C., Sasidharan, R., Muller, R., Dreher, K., Alexander, D.L., Garcia-Hernandez, M., Karthikeyan, A.S., Lee, C.H., Nelson, W.D., Ploetz, L., Singh, S., Wensel, A. & Huala, E. 2012 The Arabidopsis Information Resource (TAIR): Improved gene annotation and new tools Nucleic Acids Res. 40 D1202 D1210

    • Search Google Scholar
    • Export Citation
  • Lescot, M., Déhais, P., Thijs, G., Marchal, K., Moreau, Y., Van de Peer, Y., Rouzé, P. & Rombauts, S. 2002 PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences Nucleic Acids Res. 30 325 327

    • Search Google Scholar
    • Export Citation
  • Lionetti, V., Cervone, F. & Bellincampi, D. 2012 Methyl esterification of pectin plays a role during plant–pathogen interactions and affects plant resistance to diseases J. Plant Physiol. 169 1623 1630

    • Search Google Scholar
    • Export Citation
  • Lionetti, V., Raiola, A., Camardella, L., Giovane, A., Obel, N., Pauly, M., Favaron, F., Cervone, F. & Bellincampi, D. 2007 Overexpression of pectin methylesterase inhibitors in arabidopsis restricts fungal infection by Botrytis cinerea Plant Physiol. 143 1871 1880

    • Search Google Scholar
    • Export Citation
  • Lionetti, V., Raiola, A., Mattei, B. & Bellincampi, D. 2015 The grapevine VvPMEI1 gene encodes a novel functional pectin methylesterase inhibitor associated to grape berry development PLoS One 10 e0133810

    • Search Google Scholar
    • Export Citation
  • Livak, K.J. & Schmittgen, T.D. 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method Methods 25 402 408

    • Search Google Scholar
    • Export Citation
  • Micheli, F. 2001 Pectin methylesterases: Cell wall enzymes with important roles in plant physiology Trends Plant Sci. 6 414 419

  • Micheli, F., Sundberg, B., Goldberg, R. & Richard, L. 2000 Radial distribution pattern of pectin methylesterases across the cambial region of hybrid aspen at activity and dormancy Plant Physiol. 124 191 199

    • Search Google Scholar
    • Export Citation
  • Montgomery, J., Goldman, S., Deikman, J., Margossian, L. & Fischer, R.L. 1993 Identification of an ethylene-responsive region in the promoter of a fruit ripening gene Proc. Natl. Acad. Sci. USA 90 5939 5943

    • Search Google Scholar
    • Export Citation
  • Murayama, H., Arikawa, M., Sasaki, Y., Dal Cin, V., Mitsuhashi, W. & Toyomasu, T. 2009 Effect of ethylene treatment on expression of polyuronide-modifying genes and solubilization of polyuronides during ripening in two peach cultivars having different softening characteristics Postharvest Biol. Technol. 52 196 201

    • Search Google Scholar
    • Export Citation
  • Pan, L., Zeng, W., Niu, L., Lu, Z., Wang, X., Liu, H., Cui, G., Zhu, Y., Chu, J., Li, W., Fang, W., Cai, Z., Li, G. & Wang, Z. 2015 PpYUC11, a strong candidate gene for the stony hard phenotype in peach (Prunus persica L. Batsch), participates in IAA biosynthesis during fruit ripening J. Expt. Bot. 66 7031 7044

    • Search Google Scholar
    • Export Citation
  • Peaucelle, A., Braybrook, S.A., Le Guillou, L., Bron, E., Kuhlemeier, C. & Höfte, H. 2011 Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis Curr. Biol. 21 1720 1726

    • Search Google Scholar
    • Export Citation
  • Peaucelle, A., Louvet, R., Johansen, J.N., Höfte, H., Laufs, P., Pelloux, J. & Mouille, G. 2008 Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins Curr. Biol. 18 1943 1948

    • Search Google Scholar
    • Export Citation
  • Pelletier, S., Van Orden, J., Wolf, S., Vissenberg, K., Delacourt, J., Ndong, Y.A., Pelloux, J., Bischoff, V., Urbain, A., Mouille, G., Lemonnier, G., Renou, J. & Höfte, H. 2010 A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls New Phytol. 188 726 739

    • Search Google Scholar
    • Export Citation
  • Pelloux, J., Rusterucci, C. & Mellerowicz, E.J. 2007 New insights into pectin methylesterase structure and function Trends Plant Sci. 12 267 277

  • Pinzón-Latorre, D. & Deyholos, M.K. 2013 Characterization and transcript profiling of the pectin methylesterase (PME) and pectin methylesterase inhibitor (PMEI) gene families in flax (Linum usitatissimum) BMC Genomics 14 742 766

    • Search Google Scholar
    • Export Citation
  • Prasanna, V., Prabha, T.N. & Tharanathan, R.N. 2007 Fruit ripening phenomena-an overview Crit. Rev. Food Sci. Nutr. 47 1 19

  • Punta, M., Coggill, P.C., Eberhardt, R.Y., Mistry, J., Tate, J., Boursnell, C., Pang, N., Forslund, K., Ceric, G., Clements, J., Heger, A., Holm, L., Sonnhammer, E.L.L., Eddy, S.R., Bateman, A. & Finn, R.D. 2011 The Pfam protein families database Nucleic Acids Res. 40 D290 D301

    • Search Google Scholar
    • Export Citation
  • Raiola, A., Camardella, L., Giovane, A., Mattei, B., De Lorenzo, G., Cervone, F. & Bellincampi, D. 2004 Two Arabidopsis thaliana genes encode functional pectin methylesterase inhibitors1 FEBS Lett. 557 199 203

    • Search Google Scholar
    • Export Citation
  • Reca, I.B., Lionetti, V., Camardella, L., D’Avino, R., Giardina, T., Cervone, F. & Bellincampi, D. 2012 A functional pectin methylesterase inhibitor protein (SolyPMEI) is expressed during tomato fruit ripening and interacts with PME-1 Plant Mol. Biol. 79 429 442

    • Search Google Scholar
    • Export Citation
  • Richard, L., Qin, L.X. & Goldberg, R. 1996 Clustered genes within the genome of Arabidopsis thaliana encoding pectin methylesterase-like enzymes Gene 170 207 211

    • Search Google Scholar
    • Export Citation
  • Röckel, N., Wolf, S., Kost, B., Rausch, T. & Greiner, S. 2008 Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins Plant J. 53 133 143

    • Search Google Scholar
    • Export Citation
  • Saldanha, A.J. 2004 Java Treeview-extensible visualization of microarray data Bioinformatics 20 3246 3248

  • Siedlecka, A., Wiklund, S., Péronne, M.A., Micheli, F., Leśniewska, J., Sethson, I., Edlund, U., Richard, L., Sundberg, B. & Mellerowicz, E.J. 2008 Pectin methyl esterase inhibits intrusive and symplastic cell growth in developing wood cells of Populus Plant Physiol. 146 554 565

    • Search Google Scholar
    • Export Citation
  • Srivastava, S., Gupta, S.M., Sane, A.P. & Nath, P. 2012 Isolation and characterization of ripening related pectin methylesterase inhibitor gene from banana fruit Physiol. Mol. Biol. Plants 18 191 195

    • Search Google Scholar
    • Export Citation
  • Tamura, K., Dudley, J., Nei, M. & Kumar, S. 2007 MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0 Mol. Biol. Evol. 24 1596 1599

    • Search Google Scholar
    • Export Citation
  • Tatsuki, M., Nakajima, N., Fujii, H., Shimada, T., Nakano, M., Hayashi, K.I., Hayama, H., Yoshioka, H. & Nakamura, Y. 2013 Increased levels of IAA are required for system 2 ethylene synthesis causing fruit softening in peach (Prunus persica L. Batsch) J. Expt. Bot. 64 1049 1059

    • Search Google Scholar
    • Export Citation
  • Trapnell, C., Williams, B.A., Pertea, G., Mortazavi, A., Kwan, G., Van Baren, M.J., Salzberg, S.L., Wold, B.J. & Pachter, L. 2010 Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation Nat. Biotechnol. 28 511 515

    • Search Google Scholar
    • Export Citation
  • Van Berloo, R. 1999 Computer note. GGT: Software for the display of graphical genotypes J. Hered. 90 328 329

  • Verde, I., Abbott, A.G., Scalabrin, S., Jung, S., Shu, S., Marroni, F., Zhebentyayeva, T., Dettori, M.T., Grimwood, J., Cattonaro, F., Zuccolo, A., Rossini, L., Jenkins, J., Vendramin, E., Meisel, L.A., Decroocq, V., Sosinski, B., Prochnik, S., Mitros, T., Policriti, A., Cipriani, G., Dondini, L., Ficklin, S., Goodstein, D.M., Xuan, P., DelFabbro, C., Aramini, V., Copetti, D., Gonzalez, S., Horner, D.S., Falchi, R., Lucas, S., Mica, E., Maldonado, J., Lazzari, B., Bielenberg, D., Pirona, R., Miculan, M., Barakat, A., Testolin, R., Stella, A., Tartarini, S., Tonutti, P., Arus, P., Orellana, A., Wells, C., Main, D., Vizzotto, G., Silva, H., Salamini, F., Schmutz, J., Morgante, M. & Rokhsar, D.S. 2013 The high-quality draft genome of peach (Prunus persica) identifies unique patterns of genetic diversity, domestication and genome evolution Nat. Genet. 45 487 494

    • Search Google Scholar
    • Export Citation
  • Wang, M., Yuan, D., Gao, W., Li, Y., Tan, J. & Zhang, X. 2013 A comparative genome analysis of PME and PMEI families reveals the evolution of pectin metabolism in plant cell walls PLoS One 8 e72082

    • Search Google Scholar
    • Export Citation
  • Wolf, S., Rausch, T. & Greiner, S. 2009 The N-terminal pro region mediates retention of unprocessed type-I PME in the Golgi apparatus Plant J. 58 361 375

    • Search Google Scholar
    • Export Citation
  • Yamagata, H., Yonesu, K., Hirata, A. & Aizono, Y. 2002 TGTCACA motif is a novel cis-regulatory enhancer element involved in fruit-specific expression of the cucumisin gene J. Biol. Chem. 277 11582 11590

    • Search Google Scholar
    • Export Citation
  • Yu, Y., Liang, Y., Lv, M., Wu, J., Lu, G. & Cao, J. 2014 Genome-wide identification and characterization of polygalacturonase genes in Cucumis sativus and Citrullus lanatus Plant Physiol. Biochem. 74 263 275

    • Search Google Scholar
    • Export Citation
  • Zeng, W., Pan, L., Liu, H., Niu, L., Lu, Z., Cui, G. & Wang, Z. 2015 Characterization of 1-aminocyclopropane-1-carboxylic acid synthase (ACS) genes during nectarine fruit development and ripening Tree Genet. Genomes 11 1 10

    • Search Google Scholar
    • Export Citation
  • Zhang, G.Y., Feng, J., Wu, J. & Wang, X.W. 2010 BoPMEI1, a pollen-specific pectin methylesterase inhibitor, has an essential role in pollen tube growth Planta 231 1323 1334

    • Search Google Scholar
    • Export Citation
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