Identification of candidate genes involved in the sugar metabolism and accumulation during pear fruit post-harvest ripening of ‘Red Clapp’s Favorite’ (Pyrus communis L.) by transcriptome analysis
© The Author(s) 2017
Received: 19 April 2017
Accepted: 11 September 2017
Published: 21 September 2017
Pear (Pyrus spp.) is a popular fruit that is commercially cultivated in most temperate regions. In fruits, sugar metabolism and accumulation are important factors for fruit organoleptic quality. Post-harvest ripening is a special feature of ‘Red Clapp’s Favorite’.
In this study, transcriptome sequencing based on the Illumina platform generated 23.8 - 35.8 million unigenes of nine cDNA libraries constructed using RNAs from the ‘Red Clapp’s Favorite’ pear variety with different treatments, in which 2629 new genes were discovered, and 2121 of them were annotated. A total of 2146 DEGs, 3650 DEGs, 1830 DEGs from each comparison were assembled. Moreover, the gene expression patterns of 8 unigenes related to sugar metabolism revealed by qPCR. The main constituents of soluble sugars were fructose and glucose after pear fruit post-harvest ripening, and five unigenes involved in sugar metabolism were discovered.
Our study not only provides a large-scale assessment of transcriptome resources of ‘Red Clapp’s Favorite’ but also lays the foundation for further research into genes correlated with sugar metabolism.
KeywordsPear (Pyrus communis L.) Quantitative real-time PCR (qPCR) RNA-Seq Sugar determination
Pear (Pyrus spp.), one of the most important and oldest temperate fruit tree species (belonging to the subfamily Pomoideae in the family Rosaceae), has been grown in temperate regions since antiquity in both Europe and China . A large number of pear cultivars are functional diploids (2n = 34). The primary edible pear species are the Japanese pear (Pyrus pyrifolia Nakai), the European pear (P. communis L.) and Chinese pears (P. bretschneideri Rehd. and P. ussuriensis Maxim.), which are grown for commercial fruit production. The Japanese pear and Chinese pears are grown in East Asia, while the European pear is cultivated in Europe and other temperate regions of the Southern Hemisphere .
Improving fruit quality has become an important direction of fruit tree cultivation. In fruits, sugar metabolism and accumulation are important factors for fruit organoleptic quality. Sugar, the primary product of photosynthesis and a substrate of respiration, is required for carbon skeleton construction and energy supply in plants . Sugars are known to play key roles in both plant metabolic and defense responses as signaling molecules [4–8]. Moreover, soluble sugars (sucrose, glucose and fructose) are important components of fruit taste, directly influencing consumer preferences for fresh fruit . Glucose and fructose take part in cell division, and sucrose is actively involved in differentiation and maturation . It has been reported that the levels and ratios of these sugars differ in various tree species and rely on the major catalytic enzymes in sugar metabolism . Soluble acid invertase (INV) converts sucrose into fructose and glucose . In pear and many other woody Rosaceae plants, photosynthetic products, primarily in the form of sorbitol, are produced by leaves and transported to the fruit and other organs, which leads to the invertase-catalyzed hydrolysis of sorbitol to glucose and fructose . It is well known that environmental factors (such as temperature and light) have a certain impact on sugar metabolism in post-harvest fruit . For example, Wang  showed that peach fruit stored at 5 °C produced lower levels of sucrose and higher levels of glucose and fructose than fruit stored at 0 °C. It is well documented that the accumulation of soluble sugars could be improved by modifying the enzymatic activity of sucrose metabolism of post-harvest lemon fruit after exposure to UV-B . Transcriptome sequencing has become a powerful tool to profile transcriptomes due to its reproducibility, sensitivity, high throughput, low cost and accuracy . Transcriptome sequencing is an effective technique for the acquisition of sequences for new genes and provides opportunities to study specific cellular pathways and gene expression patterns [16–18]. In this work, expression data regarding differentially expressed genes were analyzed, and the respective putative functions of the sequences were identified through the described screening process. Our study aimed to provide important information for further functional studies of novel genes of ‘Red Clapp’s Favorite’ related to sugar metabolism and accumulation using RNA-Seq technology.
Plant materials and treatments
The plant materials of ‘Red Clapp’s Favorite’ (Pyrus communis L.) used in this study were obtained from Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences in Henan Province, China. Group 1 (T04, T07 and T10) of the pear pulps was collected at maturity. Group 2 (T05, T08 and T11) of the pear pulps was subjected to low temperature (5 °C) for 10 days after picking. Group 3 (T06, T09 and T12) was treated at normal temperature (25 °C) for 3 days after treatment at low temperature (5 °C, 10 days), the fruit went soft. All of the treatments were performed using three replicates with three fruits for each replicate. All collected samples were immediately frozen in liquid nitrogen and stored at −80 °C until RNA extraction.
RNA extraction and cDNA library construction
The extraction of RNA, construction of cDNA libraries and the transcriptome sequencing assay were performed by Biomarker Biotechnology Corporation (Beijing, China). RNA degradation and contamination were checked on 1% agarose gels. RNA purity and concentration were measured using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Sequencing libraries were generated using the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) according to the manufacturer’s recommendations. The library quality was monitored on the Agilent Bioanalyzer 2100 system. The first-strand cDNA synthesis for qPCR was obtained by the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa, Japan).
Data analysis and functional annotation
In this formula, cDNA Fragments represents the number of fragments that aligned to a specific transcript. Mapped Fragments (Millions) represents the total number of fragments that aligned to all transcripts. Transcript Length (kb) represents the length of the transcript.
Identification of differentially expressed genes (DEGs)
Differentially expressed genes (DEGs) between two groups were identified using the DESeq R package. DESeq provides statistical routines for determining differential expression in digital gene expression data using a negative binomial distribution model. The resulting P values were adjusted using Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value <0.05 found by DESeq were considered differentially expressed.
Real-time quantitative PCR analysis and sugar content determination
The primers for selected DEGs and Tubulin
Forward primer (5′-3′)
Reverse primer (5′-3′)
A 100 mg sample of each pear was weighed and extracted for LC-ESI-MS/MS of sugar content determination. Methanol, acetonitrile and ethanol were purchased from Merck Company (Germany). Standards were purchased from Sigma-Aldrich, were dissolved in methanol and preserved at −20 °C for LC-MS analysis.
Summary statistics of Illumina sequencing for ‘Red Clapp’s Favorite’
% ≥ Q30
Mapped reads ratio
Statistical table for the annotation of new genes
New gene number
Number of DEGs fall into four patterns
Number of DEGs
Analysis of DEGs
Unigenes for sugar metabolism analysis and qPCR validation
Analysis of sugar content and related genes
Five of the abovementioned unigenes that were related to sugar metabolism were selected. The relative expression of two unigenes (PCP030959 and PCP008001) increased rapidly with the increase in fructose content (Fig. 6b). The unigene PCP013141 was significantly increased with the increase in glucose content (Fig. 6c). The unigene PCP005278 showed a small increase with the increase in glucose content (Fig. 6c).
RNA-Seq is a feasible and economical way to detect genes of interest in a short time, and its popularity among researchers continues to increase [20–22]. In modern times, transcript information, gene structure and functional annotation, alternative splicing, and DEGs can be obtained by RNA-Seq technology [23, 24]. This technology has been widely applied to model and non-model species, such as Arabidopsis (Arabidopsis thaliana) , rice (Oryza sativa) [26, 27], cotton (Gossypium hirsutum) , licorice (Glycyrrhiza uralensis Fisch) , Scutellaria baicalensis Georgi , and Chinese wolfberry (Lycium chinense Mill.) . In this study, a total of 23.8 - 35.8 million clean reads were obtained from 9 cDNA libraries using this technology (Table 2) and were assembled into a total of 2146 DEGs, 3650 DEGs, and 1830 DEGs, respectively, from each comparison (Fig. 1).
Great taste is a prerequisite for consumer satisfaction . Pear eating quality is influenced by climatic conditions, post-storage ripening and harvest time . Physiological maturity of ‘Red Clapp’s Favorite’ is the stage of development when the fruit ripens adequately after harvest . Hence, fruit ripening processes are very important because they influence the changes that appear during fruit storage, transport and shelf life and changes in aroma and color . Sugar metabolism is an important part of the pear ripening process. In this research, we used RNA-Seq technology to study DEGs in the ripening process. These DEGs showed functional diversity. In the GO functional analysis, DEGs were involved in extracellular region, extracellular region part, extracellular matrix, extracellular matrix part, virion and virion part of the cellular component category (Fig. 3a–c). The DEGs were involved in molecular function categories such as nucleic acid binding transcription factor activity, protein binding transcription factor activity, protein binding transcription factor activity, nutrient reservoir activity, transporter activity, nutrient reservoir activity and guanyl-nucleotide exchange factor activity (Fig. 3a–c). The DEGs were also involved in the biological process category, including signaling, growth, rhythmic process and locomotion (Fig. 3a–c). All of these results show that sugar metabolism is a complex physiological and biochemical process.
We analyzed the eight differentially expressed unigenes related to sugar metabolism with qPCR, and the results showed similar expression trends to those of RNA-Seq (Fig. 5a–d). Only the multiple of up-regulated or down regulated was different, which validated that the RNA-Seq analysis was generally more accurate and robust. Soluble sugars, including fructose, sucrose, glucose and sorbitol, are an important factor in determining fruit quality and flavor. Fructose showed the highest sweetness, followed by sucrose; the sweetness of glucose and sorbitol was the lowest. Chen et al. showed that fructose was the dominant sugar in eight pear varieties, followed by glucose and sucrose . In this study, fructose was the main sugar after pear post-harvest ripening, followed by glucose, sucrose and sorbitol. This result might indicate that fructose is the key factor in the pear eating quality of ‘Red Clapp’ (Fig. 6a). Interestingly, we discovered five unigenes that might be involved in sugar metabolism (Fig. 6b, c). In future studies, we plan to clone the specific genes related to sugar metabolism and verify their functions.
In this study, transcriptome sequencing was performed on the Illumina platform, generating 23.8 - 35.8 million unigenes of nine cDNA libraries constructed using RNAs from the ‘Red Clapp’s Favorite’ pear variety with different treatments. A number of DEGs and novel genes were obtained from each group and were assembled. Moreover, the gene expression patterns of 8 unigenes related to sugar metabolism revealed by qPCR confirmed the RNA-Seq data. The main constituents of soluble sugars were fructose and glucose after pear fruit post-harvest ripening, and five unigenes involved in sugar metabolism were discovered. This study lays the foundation for further research into genes correlated with sugar metabolism.
This work was supported by CAAS-ASTIP, the Earmarked Fund for China Agriculture Research System (CARS-29), and the Central Public-Interest Scientific Institution Basal Research Fund (CPSI-BRF).
This work was funded by CAAS-ASTIP, CARS-29, and CPSI-BRF.
Availability of data and materials
We have provided detailed information about the materials and methods in our manuscript, so we will not provide data or supporting materials in this section.
Conceived and designed the experiments: XL. Performed the experiments: LW, YC, SW, and HX. Analyzed the data: YS and JY. Wrote the paper: LW and XL. Revised and approved the final version of the paper: XL.
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