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Comparative transcripome data for commercial maturity and physiological maturity of ‘Royal Gala’ apple fruit under room temperature storage condition
Scientia Horticulturae 225 (2017) 386–393
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Comparative transcripome data for commercial maturity and physiological maturity of ‘Royal Gala’ apple fruit under room temperature storage condition Yingwei Qi1, Qin Lei1, Yujie Zhang, Xiaoran Liu, Bin Zhou, Cuihua Liu, Xiaolin Ren
MARK
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College of Horticulture, Northwest A & F University, Yangling Shaanxi 712100, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Apple Transcriptome Ethylene Firmness Commercial maturity Physiological maturity
‘Royal Gala’ apple fruit (Malus domestica) is one of the popular apple fruit consumed world-wide. A great amount literature was accumulated for this classical apple variety about the molecular mechanisms of individual gene or gene family transcript changes during ripening, yet few of them on the large scale at transcriptomic levels. The present study was designed to use RNA-Seq to compare expression profiles between commercial ripening and physiological ripening stages of apple fruit in order to provide a deeper whole view for understanding the molecular control of ‘Royal Gala’ fruit ripening on transcript level. 13.78 G and 12.28 G clean reads were obtained for the commercial ripening and physiological ripening stages apple fruit, respectively acquired and were blasted against the apple genome. 78,553 genes were expressed in ripening apple fruit and among them 860 genes were differentially expressed between the two maturity stages, with 199 up-regulated and 661 downregulated. The enrichment of differentially expressed genes (DEGs) in gene ontology (GO) pathways revealed that genes in 8 GO terms were more activated at physiological maturity stage. Meanwhile, KEGG enrichment showed that secondary metabolism contained more up-regulated DEGs at physiological maturity stage than at commercial maturity stage. Furthermore, genes working on ripening-related processes were examined. For transcription factors regulating ethylene biosynthesis such as ERFs and HBs, the expression levels were significantly different between the two samples. Regarding fruit firmness decline, expression variation happened to genes encoding enzymes involving in cell wall metabolism, such as polygalacturonase PG1, xyloglucan endotransglucosylase XTHs and EXP3, as well as cell wall structure protein gene FLA. In conclusion, the RNA-Seq data presents the global expression changes of ripening related transcripts in ‘Royal Gala’ apple and novel identified genes and/or transcription factors may provide new clues to illuminate this complicated event.
1. Introduction Apple is an economically rosaceous fruit and academically important as a typical climacteric fruit. For apple, commercial maturity is a pre-climacteric stage, only basal ethylene or system 1 ethylene is produced. Comparably, physiological maturity is marked by an ethylene burst mainly contributed by system 2 ethylene. At physiological maturity apple fruit become ripe and near their optimum quality for fresh consumption, and it is the last stage of fruit ripening and fruit development, followed by fruit senescence (Alexander and Grierson, 2002). From commercial to physiological maturity, the ripening process is complex and coordinated during which physiological and biochemical changes happen. The most dramatic changes are the greatly increased production of the ripening hormone ethylene and respiratory
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Corresponding author. E-mail address: [email protected] (X. Ren). These two authors contribute equally to this work.
http://dx.doi.org/10.1016/j.scienta.2017.07.024 Received 23 February 2017; Received in revised form 5 July 2017; Accepted 19 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
carbon dioxide. Other major changes include the decline of fruit firmness, changes of ground color and over color, increase of sugar acid ratio. Intensive studies on ripening related genes expression characteristics during fruit ripening have been conducted, however, they were mainly restricted to small-scale quantitative PCR (ContrerasVergara et al., 2012; Giovannoni, 2004; Hubert and Mbeguie, 2012; Omidvar et al., 2013; Soe et al., 2013; Wei et al., 2010). Recently, researchers have made efforts by using transcriptomic technologies to reveal the extent and complexity of expression profiles for apple (Costa et al., 2010a). Considering that ‘Royal Gala’ apple is one of the widely grown and consumed apple varieties and also an early ripening cultivar with shorter ripening period, it could be a good model to study apple fruit ripening. Among all the factors promoting the physiological changes,
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2.3. Bioinformatic methods
gene transcriptional regulation especially transcriptomic co-adjustment is the key. Mining the transcriptomic profile differences between commercial maturity and physiological maturity will be helpful for understanding the mechanism of apple fruit ripening. Two main technologies were applied in collecting transcriptome data, microarray and RNA-Seq. Microarray did provide us chances to explore gene expression profiles related to fruit ripening and revealed genes crucial for a particular pathway on a large scale. According to Schaffer et al. (2007) 972 genes in cortex and 537 genes in skin that response to external ethylene treatment were mined with microarray by comparing transgenic line AO3, greatly down-regulated ACC OXIDASE and no detectable ethylene, with wild type ‘Royal Gala’ apple fruit. This study also disclosed 17 candidate genes on aroma biosynthesis pathways that are controlled by ethylene. Using microarray, 106 genes were found highly coordinated with fruit ripening in tree ripening ‘Royal Gala’ apple (Janssen et al., 2008). Gene expression profiles using homologous and heterologous (tomato) microarray of normal ripening “Mondial Gala” and on 1-methylcyclopropene (1-MCP) treated fruit revealed common orthologous ripening related genes (Costa et al., 2010a). Since microarray is based on hybridization of the complementary DNA strands with many probes, it is limited by the cross-hybridization and small amount of data (usually less than 10 Mb). RNA-Seq based on high-throughput next generation sequencing conquered the disadvantages on microarray (Wang et al., 2009b; Zhao et al., 2014) and it is becoming an alternative to microarray. RNA-Seq has been recruited to study fruit ripening like Chinese bayberry (Feng et al., 2012), Chinese pear (Huang et al., 2014; Xie et al., 2013), sweet orange (Yu et al., 2012), mango (Dautt-Castro et al., 2015) and apple (Tadiello et al., 2016). In this study, two RNA samples extracted from fruit in the two different maturity stages were sequenced on the Illumina HiSeq™2000/ MiSeq sequencing platform. The aim of the study is to gain a further and more reliable understanding of the molecular mechanisms of apple fruit ripening, especially before and during ethylene burst. Our analysis will provide molecular evidence for the changes of ethylene production and fruit firmness.
2.3.1. Mapping of clean reads Raw reads of fastq format were firstly processed through in-house perl scripts to get clean reads on which all the downstream analyses were based. At the same time, Q20, Q30 and GC content were calculated. The paired-end clean reads were then aligned to “Golden Delicious” apple genome (Velasco et al., 2010) using TopHat V2.0.9 (Trapnell et al., 2009) to generate mapping results.
2.3.2. Expression annotation and differentially expression analyses HTSeq v0.5.4p3 was used to count the reads numbers mapped to each gene. Reads Per Kilobase of exon model per Million mapped reads (RPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene (Mortazavi et al., 2008). With regard to differential expression analysis, since we had only one sample per condition for each sequenced library, the read counts were first adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was then performed using the DEGSeq R package (1.12.0). The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.005 and log2 (Fold change) of 1 were set as the threshold for significantly differential expression.
2.3.3. GO and KEGG enrichment analyses of differentially expressed genes (DEGs) Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other highthroughput experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways (Xie et al., 2011).
2. Materials and methods 2.1. Plant material ‘Royal Gala’ apple (Malus domestica) were harvested at commercial ripening stage in Qian County, Shaanxi province, China and immediately transported to the laboratory. Fruit free of mechanical damage and uniform size were selected and stored at room temperature (20 ± 1 °C). At days 0, 3, 6, 9, 12, 15, 18 respectively, randomly nine fruit were used to determine their physiological status and pericarp tissue of another fifteen fruit at same stage were sampled until the fruit reached the physiological maturation stage. All samples were frozen in liquid nitrogen before stored at −80 °C.
2.4. Time course quantitative real-time PCR validation Quantitative Real-Time PCR (qRT-PCR) was used to measure the expression of eighteen genes shown to be differentially regulated by RNA-Seq. Five fruit ripening stages were considered: 0 d after storage, and 9, 12, 15, 18 d after storage. For each stage, three biological replicates and each biological replicate composed by five apple fruit were used. RNA extraction, primer design, qRT-PCR experiment and data analysis were carried out as described by Zhao et al. (2015). The primer names and sequences are listed in Online Supplemental Table 1.
2.2. RNA extraction, cDNA library preparation and sequencing 2.5. Ethylene and fruit firmness analysis Frozen tissue was grounded in liquid nitrogen and total RNA was extracted according to previously reported CTAB method by Gasic et al. (2012) with minor modification. RNA quality was verified initially by Nanodrop ND-1000 and agarose gel electrophoresis, its completeness and concentration was further measured using Agilent 2100 Bioanalyzer (Agilent, USA) and Qubit® 2.0 Flurometer (Life Technologies, CA, USA). A total amount of 3 micrograms of qualified RNA per sample was applied for cDNA library preparation using NEB Next® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The libraries prepared were then sequenced on an Illumina Hiseq™ 2000 platform and 100 bp paired-end reads were generated.
Internal ethylene concentration was measured by withdrawing an 1 mL gas sample from the core of each fruit using a syringe and injecting the gas sample into a gas chromatograph (Shimadzu GC-14A, Kyoto, Japan). Nitrogen was used as the carrier gas at a flow rate of 1 mL s−1 and pressure of 0.1 Mpa. The injector and detector port temperatures were 60 and 110 °C, respectively. An external standard of ethylene (1.0 mL L−1) was used for calibration and quantification. Fruit firmness was determined on opposite sides of fruit equatorial axis after peel removal by using GUSS Fruit Texture Analyzer fitted with an 11 mm flat plate probe (Model GS-15; Strand, South Africa) and the results expressed in newton (N). 387
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Table 1 Overview of sequencing yields, quality and alignments.
Fig. 1. Ethylene production and firmness changes during ‘Royal Gala’ apple fruit ripening at room temperature storage. The total number of tests n = 9.
Accession
0d
15 d
Raw reads Clear reads Clear based Q20 (%) Error rate (%) Total mapped reads (percentage of clear reads) uniquely mapped reads (percentage of clear reads) multiple mapped reads (percentage of clear reads) Ratioof exon, intergenic and intron reads mapped to genome region
143160288 137810896 13.78Gb 96.43 0.04 86.06% 69.91%
137405112 132881896 13.28Gb 96.915 0.035 75.45% 62.20%
16.15%
13.25%
85.8: 12.3: 1.9
86.1: 12.0: 2.0
almost 1:1. When aligned with genome data, exon, intergenic and intron reads mapped to genome region for 0 d were 85.8%, 12.3%, 1.9% and 15 d were 86.1%, 12.0%, 2.0%, respectively. The detection of intergenic and intron reads may be caused by contamination of immature mRNA or incomplete genome annotation. Around 70% of mapped reads are Non-splice reads. Compared against known apple genes by Cufflinks and Cuffcompare, it was found that the assembled 78,553 genes include 15,012 novel genes that were expressed in ripening apple fruit. The discovery of novel genes in our RNA-Seq data could be explained by low coverage of apple reference genome (Bai et al., 2014). Moreover, all 78,553 genes were positioned into apple genome and 59,287 (75.47%) were functionally described in the known “Golden Delicious” apple database. The expression levels of genes in commercial and physiological maturity stages were calculated by RPKM (Reads Per Kilo bases per Million reads) method, with RPKM value lower than 1 (36492 and 35184 of 78,553 genes in two samples respectively) were filtered. Based on expression level, genes were classified into five groups (Table 2).
3. Results and discussion 3.1. Ethylene and fruit firmness changes during apple fruit ripening In order to sample the right apple fruit for RNA-Seq, internal ethylene production and fruit firmness were measured every three days to track the fruit physiological changes during the postharvest storage at room temperature. As reported in many studies, apple fruit ripening during postharvest stage is accompanied by a great amount of ethylene release and firmness decline (Gwanpua et al., 2014; Harb et al., 2012; Ireland et al., 2014). In the present study, internal ethylene production is comparatively lower at the very beginning (Fig. 1), indicating the fruit were pre-climacteric. Ethylene production reached peak at 15 days, and then decreased at 18 days of storage, meaning ‘Royal Gala’ apple came to their physiological maturity at 15 days of storage. Fruit firmness was continuously declining during storage, from 85.45 ± 1.63 to 44.83 ± 1.18 N. Change patterns of ethylene production and fruit firmness decline are consistent with former studies on ‘Mclntosh’ apple fruit (Harb et al., 2012) and those apple fruit of ‘Fuji’ and ‘Golden Delicious’ stored at room temperature (Wei et al., 2010). It was noticed that ‘Royal Gala’ apple fruit firmness dramatically decreased in the first three days. Similar result was observed by Costa et al. (2010b) where about 30N of firmness was lost in five days in ‘Mondial Gala’ under room temperature. Moreover, ‘Golden Delicious’ and ‘Fuji’ fruit firmness also dropped quickly in the first seven days after harvest as showed in the study carried out by Wei et al. (2010).
3.3. Analysis of differentially expressed genes Totally 860 among 78,553 genes were identified to be expressed differentially during apple fruit ripening with ∣ log2(RPKM15d/RPKM0d) ∣ > 1 and FDR < 0.005. The list is shown in Supplemental file 2. Among the 860 genes, 565 genes were matched to known gene products, 30 genes were matched to unknown genes, and 265 were novel genes against known plant gene sequence in the GenBank database. At physiological maturity stage, 199 genes were up-regulated and 661 genes were down-regulated comparing with commercial maturity stage indicating that somehow physiological activities were more active in the initiation of ripening than the physiological ripening stage, which come to the same conclusion with the results in Bayberry (Feng et al., 2012) and pear (Xie et al., 2013). GO analysis showed that 643 of all 860 DEGs could be annotated in significantly enriched 113 GO terms in three categories, biological process (59 GO terms), cellular component (9 GO terms) and molecular function (45 GO terms) category. Single-organism metabolic process (GO:0044710, 183/643), extracellular region (GO:0005576 35/643) and oxidoreductase activity (GO:0016705, 15/643) were the most abundant GO terms within these three categories, respectively. Within
3.2. Genome-wide transcriptome analysis by RNA-Seq Based on the physiological changes acquired during fruit ripening during room temperature storage, Cortex of ‘Royal Gala’ apple in commercial maturity stage (0 d) and physiological maturity stage (15 d) were sampled for RNA sequencing. RNA sequencings were performed on an Illumina HiSeq™2000 platform. A total of 14.28 G and 13.55 G raw reads were generated for these two maturity stage samples considering that a single paired-end read is 100 bp long. After filtered, about 13.78 G and 13.28 G clean reads were produced, with Q20 percentage is over 96% and error rate percentage is lower than 0.04%. Pearson correlation (R2) between two samples is 0.912 (Table 1). Read density and quality of our RNA-Seq data is good enough for gene expression analysis. The sequence reads were aligned against the “Golden Delicious” apple genome (Velasco et al., 2010) using TopHat v2.0.9 software (Trapnell et al., 2009). Total mapped reads, uniquely mapped reads and multiple mapped reads of the clean reads were 86.06%, 69.91%, 16.15% and 75.45%, 62.20%, 13.25% for sample 0 d and 15 d, respectively. Reads map to“ + ” to reads map to “-” in both samples are
Table 2 Percentage of genes with different expression levels in 0 d and 15 d samples.
388
RPKM Interval
0d
15 d
0–1 1–3 3–15 15–60 > 60
42061(53.54%) 10290(13.10%) 16179(20.60%) 7340(9.34%) 2683(3.42%)
43369(55.21%) 9439(12.02%) 15695(19.98%) 7444(9.48%) 2606(3.32%)
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that all of the qRT-PCR results were consistent with the RNA-Seq, which strongly confirmed the RNA-Seq results were reliable (Fig. 3). 3.5. Genes related to ethylene biosynthesis and perceptiveness when ‘Royal Gala’ apple ripen Ethylene is the main hormone regulator of climacteric fruit ripening, of which apple was a typical example. In this study, internal ethylene of ‘Royal Gala’ apple was increased from very low levels at 0 d to its peak value at 15 d after harvest. Several enzymes worked on ethylene biosynthesis pathway, among which 1-aminocyclopropane-1carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxydase (ACO) are well-known as the key enzymes. Moreover, since ethylene burst was mainly contributed by system 2 ethylene, we were oriented towards revealing transcripts worked on system 2 ethylene production. Comparing all the DEGs involved in ethylene production in these two ripening stages. It was found that physiological stage producing both system 1 and 2 ethylene, while only system 1 ethylene was produced in commercial day. Our analysis recovered two ACS transcripts: Novel12596 (best hit: U89156), Log2 fold change = 6.37, FDR = 1.84E-96 and Novel12597 (best hit:AB010102), Log2 fold change = 7.02, FDR = 6.21E-18, and three ACO transcripts: MDP0000195885 (best hit: JQ675681), Log2 fold change = 1.28, FDR = 0 (ACO1), MDP0000200737 (best hit: XM_008372823), Log2 fold change = 1.06, FDR = 1.83E-18 (ACO2) and MDP0000200896 (best hit: XM_008339460), Log2 fold change = 2.14, FDR = 3.63E11(ACO7) were significantly up-regulated when fruit ripen. Closely analysis of two up-regulated MdACS transcripts showed that they hit either MdACS1-1 (U89156) or MdACS1-2 (AB010102). MdACS1 and MdACS3 are the most studied ACS transcripts in apple. MdACS1, unlike MdACS3a whose expression preceded MdACS1 and it was responsible for transiting from system 1 to system 2 ethylene biosynthesis, was reported only expressed in ripening fruit and its expression was increased with ripening, so it was considered responsible for system 2 ethylene biosynthesis (Sunako et al., 1999; Wang et al., 2009a). RNASeq data confirmed the previous results. MdACO1, like MdACS1 was intensively reported to have ripening related expression pattern during apple fruit ripening (Atkinson et al., 1998; Costa et al., 2005; Dal Cin et al., 2007; Tsantili et al., 2007). Extensive expressed sequence tag (EST) data on apple also revealed that MdACO1 showed a ripeningrelated expression pattern associated with climacteric (Park et al., 2006). According to Dandekar et al. (2004) down regulated MdACO showed less ethylene production during ripening. We identified one MdACO transcripts: MDP0000195885 (best hit: JQ675681, MaACO1-c allele) that shared very high sequence similarity with the reported MdACO1. The other one MDP0000200737 (best hit: XM_008372823) was MdACO2 which was the closest allele of MdACO1 in the phylogeny of the ACO genes (Clouse and Carraro, 2014). The third one MDP0000200896 (MdACO7) shared a bit less similarity with MdACO1. Former study using microarray on tree ripening apple found the expression of one ethylene receptors ETR2/EIN4 (EE663937) had changed significantly during ripening (Janssen et al., 2008). However, none of the ethylene receptor expression was recognized to be significantly changed by our RNA-Seq results which could be explained either by the sample difference or by experimental technique difference. Transcription factors such as ERFs, MYBs and HBs play crucial roles on regulating ethylene bio-function. Most ERFs were reported to be ripening down-regulated and involved in ripening by interacting with the promoters of ACS and ACO, and SIERF6 repressed line in tomato enhanced ethylene levels (Lee et al., 2012; Xiao et al., 2013; Yin et al., 2010). We identified three down-regulated ERFs (MDP0000175375, Log2 Fold change = −4.14, FDR = 0.00010483; MDP0000235028, Log2 Fold change = −2.97, FDR = 1.66E-05; MDP0000689946, Log2 Fold change = −2.71, FDR = 2.81E-05), and two up-regulated ERFs (MDP0000155743, Log2 Fold change = 1.64, FDR = 6.97E-20; Novel
Fig. 2. The GO term distribution of DEGs by RNA-Seq between day 0 (0D) and day 15 (15D). The top 30 enriched GO terms in the comparison groups between day 0 (0D) and day 15 (15D). Asterisks (*) indicate significant (P-value < 0.05) enrichment GO terms. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component, and molecular function.
them, 11 GO terms were activated at physiological maturity, including 9 in biological process and 2 in molecular function. Most of biological processes were related to fruit ripening, ethylene biosynthesis and secondary metabolism. The two molecular function GO terms are 1aminocyclopropane-1-carboxylate oxidase activity and oxidoreductase activity. These results perfectly matched the physiological status of physiological maturation during which ethylene burst and ripening happened. The most enriched 30 GO terms which were thought to be indicator of significantly altered biological program between the two maturity stages is shown in Fig. 2. These activated GO terms were mainly responsive of ethylene and aroma biosynthesis, which would be abundantly produced when apple fruit ripen. By retrieving KEGG pathways from KEGG database based on sequence similarity, all the DEGs can be mapped into 140 biochemical pathways (Supplemental file 3). The pathways with highest representations were metabolic pathways (Ko01100, 45), Biosynthesis of secondary metabolites (Ko01110, 29), Microbial metabolism in diverse environments (ko01120, 10), Biosynthesis of amino acids (ko01230, 8), Carbon metabolism (ko01200, 8). 3.4. Time course analysis and validation of selected DEGs by quantitative PCR To validate RNA-Seq data and to check how expression of these genes change at different storage time (0, 6, 12, 15, 18 d), quantitative real-time PCR (qRT-PCR) was used to examine the relative expression pattern of eighteen transcripts. 6 of 18 genes (XTH8, XTH1, XTH4, OMT7, AFS and AST) were selected from transcripts in the result and the rest 12 genes were randomly picked from the whole DEGs. These genes are from an array of different processes including redox activity and metabolism making the validation more rational. Housekeeping gene GAPDH (Genbank number: CN929227) was used to normalize qRT-PCR results after a preliminary experiment comparing the stability of different housekeeping genes in this experiment. Our data showed 389
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Fig. 3. qRT-PCR revealing genes expression patterns and validation of the selected differentially expressed transcripts from RNA-Seq quantitative results during gala apple fruit postharvest storage under 20 °C. Error bars represent means ± SE (n = 3 with 5 apple fruit each).
3.6. Genes related to fruit firmness when ‘Royal Gala’ apple ripen
04681, Log2 Fold change = 2.65, FDR = 1.68E-06). This was reasonable since different ERFs might act as transcriptional activators or repressors on ethylene biosynthesis as reported previously (Fujimoto et al., 2000; Ohme-Takagi et al., 2000). Transcription factor HD-Zip homeo box protein LeHB1 was observed to play an important role in a positive manner in tomato ripening by interacting with the promoter of LeACO1 (Lin et al., 2008). Our transcriptomic comparison found two of MdHB (MDP0000564897, Log2 Fold change = 1.01, FDR = 6.84E-11, best hit = XM_008340338 (HBA); MDP0000615948, Log2 Fold change = 1.23, FDR = 1.28E-05, best hit = XM_008383196 (HBB)) that were highly expressed during ‘Royal Gala’ apple fruit ripening and were significantly up-regulated at physiological maturity, indicating that MdHBs in our RNA-Seq might also regulate apple fruit ripening as they did in tomato. In tomato, GRAS domain transcription factor (Solyc07g052960) was considered as ripening-associated and ethylene related transcription factor since it was specifically expressed at ripening fruit and it was down-regulated from pink coloring to red ripe tomato (Fujisawa et al., 2012). Similar result was observed in our RNASeq (MDP0000264347, Log2 Fold change = −2.93, FDR = 7.33E-11). Despite lack of direct evidence of GRAS regulating ethylene pathway, it was targeted by RIN and its expression pattern suggested that it together with the two MdHBs that screened in this study, was worth further studying.
‘Royal Gala’ apple is a typical mealy fruit and its firmness decreases consistently during ripening. Fruit firmness changing characteristic is thought to be mainly involved in loss of turgor pressure, starch degradation and cell wall modification. Among these factors, cell wall which is responsible for cell shape and mechanical support was the main contributor of fruit firmness and its modification determined fruit firmness change (Chaib et al., 2007; Goulao and Oliveira, 2008). Several cell wall modification enzymes involved in fruit cell wall modification, such as polygalacturonase (PG), pectin methylasterase (PME), xyloglucan endotransglycosylase/hysrolase (XTH), expansin (EXP), βgalactosidase (β-Gal) and pectate lyase (PL). All these genes encoding cell wall metabolism enzymes were analyzed, and the expression of PG1, XTH7, purple acid phosphatases (PAPs) and EXPA3 was significantly different between fruit of the two maturity stages. The cell wall hydrolase (polygalacturonase1) PG1 acts on the middle lamella to solubilize intercellular adhesion and it is a main factor responsible for fruit texture changing referred by QTL (Costa et al., 2010b; Dunlevy et al., 2013; Longhi et al., 2012). Suppression of PG1 led to firmer fruit, higher CDTA-soluble pectin percentage and higher intercellular adhesion (Atkinson et al., 2012). The expression of PG1 was reported to be not only exclusively in fruit (Tomato Genome, 2012) but also increased during fruit ripening (Costa et al., 2010b) 390
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dramatically down-regulated (MDP0000904458, log2 Fold change = −5.56, FDR = 2.21E-38) during ripening. FLAs, a subclass of arabinogalactan proteins (AGPs) which were found in the cell wall as structural complex molecules (Keegstra, 2010), had been proposed to function in cell wall cross-link by binding with pectin (Ellis et al., 2010). We hypothesized that the down-regulated FLA caused decrease of FLA synthesis and might even cause the loosing of cell wall adhesion. Although the function of FLA during fruit ripening had not been investigated, its cell wall location, interaction with pectin and dramatic expression change in ripening hint its potential role on fruit firmness change during ripening. Phosphorylation also has a role in regulating enzymatic activity in the cell wall. Here we identified two cell wall-bound PAPs transcripts (MDP0000636860, Log2 Fold change = 2.51, FDR = 3.37E-18; MDP0000824136, Log2 Fold change = 2.52, FDR = 1.62E-27), which were significantly up-regulated when ‘Royal Gala’ apple was ripen. PAPs were reported to dephosphorylate proteins like α-xylosidase and β-glucosidase resulting in a decrease in their activities, causing more cell wall oligosaccharides accumulated (Kaida et al., 2010). In olive, PAP expression was reported to be highly increased in abscission zone of ripen fruit, which might be associated with its role of limiting phosphorylation (Parra et al., 2013). These changes indicated that dynamic changes and rearrangements of the cell wall that occur during ‘Royal Gala’ fruit ripening.
indicating its specific role on late stages of ripening (Brummell et al., 2004). RNA-Seq data showed that PG1 expression was in a high level during whole ripening process and was up-regulated at physiological maturity stage (MDP0000326734, Log2 Fold change = 1.01,FDR = 1.04E-135). PME is also thought to play important role in fruit softening, since PME is the main pectin de-esterification enzyme and its production is the substrate of PG. PME expression levels and patterns may vary among apple cultivars, therefore may play different roles. In ‘Fuji’ apple, expression level of PME was constant up to day 42, and only up-regulated in the late stage of storage at room temperature; differently, PME expression in ‘Golden Delicious’ apple fruit increased rapidly and significantly during the early postharvest time, and after going to the peak on day 28 it was decreased (Wei et al., 2010). In our study, data showed that PME expression was up-regulated in the physiological maturity of ‘Gala’ apple fruit although its expression level was relatively low, and there was no significant difference between the two samples. Lower expression level of PME gene (MdPME1) was also observed by Goulao et al. (2008) as these researchers found that its expression was hardly detected by RT-PCR even with 40 cycles, while for other genes 35 cycles were sufficient enough. Another example of rearrangement of cell wall substrate was achieved by function of XTHs proteins. XTH was gene family encoding xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), the activities of which cut or cut and rejoined xyloglucan chains which were responsive for cell wall extension, since the structural change and rearrangement of cell wall was mutual during ripening (Goulao and Oliveira, 2008). We found several XTH transcripts that were differentially expressed and regulated during ripening, two were significantly down-regulated (MDP0000269483, log2 Fold change = −2.57, FDR = 1.50E-07, best hit = MdXTH4; MDP0000181544, log2 Fold change = −6.31, FDR = 0.00077076, best hit = MdXTH8) and one was significantly up-regulated (MDP0000398765, log2 Fold change = 2.90, FDR = 4.34E-06, best hit = MdXTH1). These results confirmed former reports that different XTH transcripts expressed in ripening fruit (Rose et al., 2002). Apple XTH genes were historically clustered into three groups according to their sequence similarity (Atkinson et al., 2009; Munoz-Bertomeu et al., 2013). The transcripts expressed in ripening fruit were grouped into group 3 in tomato (Munoz-Bertomeu et al., 2013; Saladie et al., 2006), grouped into 1 and 3 in “Mondial Gala” apple (Goulao et al., 2008), 2 and 3 “Golden Delicious” (Munoz-Bertomeu et al., 2013) and 2 and 3 in “Granny Smith” (Atkinson et al., 2009); grouped in 2 and 3 in kiwifruit (Atkinson et al., 2009) and grouped into group A and B in persimmon (Zhu et al., 2013). In the present study, MdXTH1 and MdXTH8 were belonged to group 1 and the down-regulated MdXTH4 and MdXTH7 were belonged to group 2 (data not shown). This indicated that the phylogenetic tree could not represent the expression or function classification (Eklof and Brumer, 2010) and for different cultivars, different transcripts were functioning for regulating fruit ripening. Expansins responsive for depolymerization of hemicelluloses (Brummell et al., 1999) are composed by a big family (Zhang et al., 2014) were observed to have different expression patterns during apple fruit ripening in different cultivars (Goulao et al., 2008). The expression of MdExp3 (referring to MdExp2 in other reference) in “Golden Delicious” was almost parallel with ethylene production (Wakasa et al., 2003), while MdEXPA3 in “Mondial Gala” apple like LeExp1 in tomato was reported with highest expression level at harvest and reduced to low level thereafter (Goulao et al., 2008). Analysis of DEGs showed that one transcript, MdEXPA3, (MDP0000139058, log2 Fold change = −2.62, FDR = 3.74E-152) was down-regulated when apple fruit ripening, confirmed the former results that MdEXPA3 showed higher expression level in fruit at harvest, and after that its expression was significantly reduced (Goulao et al., 2008). Besides cell wall metabolism enzymes, cell wall structural proteins changes also played crucial role on fruit firmness. One transcript fasciclin-like AGP (FLA) that encoded cell wall structural proteins was
4. Conclusions RNA-Seq was carried out to investigate complex changing process between commercial and physiological maturation of ‘Royal Gala’ apple fruit at the transcriptome level. GO annotations showed that 643 of all 860 DEGs belonging to a wide range of functional categories, especially on ethylene biosynthesis and secondary metabolism, were significantly enriched. Various gene families and pathways involved in this ripening process were identified. Among them, novel genes such as HBs, ERFs and ACC that are thought to be related to apple fruit ethylene biosynthesis were significantly up-regulated. These enriched DEGs will be selected in our future gene functions research. Overall, the observed results provide a broad overview into the molecular mechanisms underlying the postharvest ripening process of one of the most important fresh eating fruit. Conflicts of interest Authors of this article declare no conflict of interest. Acknowledgement This work was supported by Modern Agricultural Industry Technology System of China for Apple (Grant No.Z225020701). The funder had no role in research design, data collection and analysis, decision to publish and/or preparation of manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2017.07.024. References Alexander, L., Grierson, D., 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. J. Exp. Bot. 53, 2039–2055. Atkinson, R.G., Bolitho, K.M., Wright, M.A., Iturriagagoitia-Bueno, T., Reid, S.J., Ross, G.S., 1998. Apple ACC-oxidase and polygalacturonase: ripening-specific gene expression and promoter analysis in transgenic tomato. Plant Mol. Biol. 38, 449–460. Atkinson, R.G., Johnston, S.L., Yauk, Y.-K., Sharma, N.N., Schröder, R., 2009. Analysis of xyloglucan endotransglucosylase/hydrolase (XTH) gene families in kiwifruit and apple. Postharvest Biol. Technol. 51, 149–157.
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Comparative transcripome data for commercial maturity and physiological maturity of ‘Royal Gala’ apple fruit under room temperature storage condition Yingwei Qi1, Qin Lei1, Yujie Zhang, Xiaoran Liu, Bin Zhou, Cuihua Liu, Xiaolin Ren
MARK
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College of Horticulture, Northwest A & F University, Yangling Shaanxi 712100, China
A R T I C L E I N F O
A B S T R A C T
Keywords: Apple Transcriptome Ethylene Firmness Commercial maturity Physiological maturity
‘Royal Gala’ apple fruit (Malus domestica) is one of the popular apple fruit consumed world-wide. A great amount literature was accumulated for this classical apple variety about the molecular mechanisms of individual gene or gene family transcript changes during ripening, yet few of them on the large scale at transcriptomic levels. The present study was designed to use RNA-Seq to compare expression profiles between commercial ripening and physiological ripening stages of apple fruit in order to provide a deeper whole view for understanding the molecular control of ‘Royal Gala’ fruit ripening on transcript level. 13.78 G and 12.28 G clean reads were obtained for the commercial ripening and physiological ripening stages apple fruit, respectively acquired and were blasted against the apple genome. 78,553 genes were expressed in ripening apple fruit and among them 860 genes were differentially expressed between the two maturity stages, with 199 up-regulated and 661 downregulated. The enrichment of differentially expressed genes (DEGs) in gene ontology (GO) pathways revealed that genes in 8 GO terms were more activated at physiological maturity stage. Meanwhile, KEGG enrichment showed that secondary metabolism contained more up-regulated DEGs at physiological maturity stage than at commercial maturity stage. Furthermore, genes working on ripening-related processes were examined. For transcription factors regulating ethylene biosynthesis such as ERFs and HBs, the expression levels were significantly different between the two samples. Regarding fruit firmness decline, expression variation happened to genes encoding enzymes involving in cell wall metabolism, such as polygalacturonase PG1, xyloglucan endotransglucosylase XTHs and EXP3, as well as cell wall structure protein gene FLA. In conclusion, the RNA-Seq data presents the global expression changes of ripening related transcripts in ‘Royal Gala’ apple and novel identified genes and/or transcription factors may provide new clues to illuminate this complicated event.
1. Introduction Apple is an economically rosaceous fruit and academically important as a typical climacteric fruit. For apple, commercial maturity is a pre-climacteric stage, only basal ethylene or system 1 ethylene is produced. Comparably, physiological maturity is marked by an ethylene burst mainly contributed by system 2 ethylene. At physiological maturity apple fruit become ripe and near their optimum quality for fresh consumption, and it is the last stage of fruit ripening and fruit development, followed by fruit senescence (Alexander and Grierson, 2002). From commercial to physiological maturity, the ripening process is complex and coordinated during which physiological and biochemical changes happen. The most dramatic changes are the greatly increased production of the ripening hormone ethylene and respiratory
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1
Corresponding author. E-mail address: [email protected] (X. Ren). These two authors contribute equally to this work.
http://dx.doi.org/10.1016/j.scienta.2017.07.024 Received 23 February 2017; Received in revised form 5 July 2017; Accepted 19 July 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.
carbon dioxide. Other major changes include the decline of fruit firmness, changes of ground color and over color, increase of sugar acid ratio. Intensive studies on ripening related genes expression characteristics during fruit ripening have been conducted, however, they were mainly restricted to small-scale quantitative PCR (ContrerasVergara et al., 2012; Giovannoni, 2004; Hubert and Mbeguie, 2012; Omidvar et al., 2013; Soe et al., 2013; Wei et al., 2010). Recently, researchers have made efforts by using transcriptomic technologies to reveal the extent and complexity of expression profiles for apple (Costa et al., 2010a). Considering that ‘Royal Gala’ apple is one of the widely grown and consumed apple varieties and also an early ripening cultivar with shorter ripening period, it could be a good model to study apple fruit ripening. Among all the factors promoting the physiological changes,
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2.3. Bioinformatic methods
gene transcriptional regulation especially transcriptomic co-adjustment is the key. Mining the transcriptomic profile differences between commercial maturity and physiological maturity will be helpful for understanding the mechanism of apple fruit ripening. Two main technologies were applied in collecting transcriptome data, microarray and RNA-Seq. Microarray did provide us chances to explore gene expression profiles related to fruit ripening and revealed genes crucial for a particular pathway on a large scale. According to Schaffer et al. (2007) 972 genes in cortex and 537 genes in skin that response to external ethylene treatment were mined with microarray by comparing transgenic line AO3, greatly down-regulated ACC OXIDASE and no detectable ethylene, with wild type ‘Royal Gala’ apple fruit. This study also disclosed 17 candidate genes on aroma biosynthesis pathways that are controlled by ethylene. Using microarray, 106 genes were found highly coordinated with fruit ripening in tree ripening ‘Royal Gala’ apple (Janssen et al., 2008). Gene expression profiles using homologous and heterologous (tomato) microarray of normal ripening “Mondial Gala” and on 1-methylcyclopropene (1-MCP) treated fruit revealed common orthologous ripening related genes (Costa et al., 2010a). Since microarray is based on hybridization of the complementary DNA strands with many probes, it is limited by the cross-hybridization and small amount of data (usually less than 10 Mb). RNA-Seq based on high-throughput next generation sequencing conquered the disadvantages on microarray (Wang et al., 2009b; Zhao et al., 2014) and it is becoming an alternative to microarray. RNA-Seq has been recruited to study fruit ripening like Chinese bayberry (Feng et al., 2012), Chinese pear (Huang et al., 2014; Xie et al., 2013), sweet orange (Yu et al., 2012), mango (Dautt-Castro et al., 2015) and apple (Tadiello et al., 2016). In this study, two RNA samples extracted from fruit in the two different maturity stages were sequenced on the Illumina HiSeq™2000/ MiSeq sequencing platform. The aim of the study is to gain a further and more reliable understanding of the molecular mechanisms of apple fruit ripening, especially before and during ethylene burst. Our analysis will provide molecular evidence for the changes of ethylene production and fruit firmness.
2.3.1. Mapping of clean reads Raw reads of fastq format were firstly processed through in-house perl scripts to get clean reads on which all the downstream analyses were based. At the same time, Q20, Q30 and GC content were calculated. The paired-end clean reads were then aligned to “Golden Delicious” apple genome (Velasco et al., 2010) using TopHat V2.0.9 (Trapnell et al., 2009) to generate mapping results.
2.3.2. Expression annotation and differentially expression analyses HTSeq v0.5.4p3 was used to count the reads numbers mapped to each gene. Reads Per Kilobase of exon model per Million mapped reads (RPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene (Mortazavi et al., 2008). With regard to differential expression analysis, since we had only one sample per condition for each sequenced library, the read counts were first adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was then performed using the DEGSeq R package (1.12.0). The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.005 and log2 (Fold change) of 1 were set as the threshold for significantly differential expression.
2.3.3. GO and KEGG enrichment analyses of differentially expressed genes (DEGs) Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the GOseq R package, in which gene length bias was corrected. GO terms with corrected P-value less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other highthroughput experimental technologies (http://www.genome.jp/kegg/). We used KOBAS software to test the statistical enrichment of differential expression genes in KEGG pathways (Xie et al., 2011).
2. Materials and methods 2.1. Plant material ‘Royal Gala’ apple (Malus domestica) were harvested at commercial ripening stage in Qian County, Shaanxi province, China and immediately transported to the laboratory. Fruit free of mechanical damage and uniform size were selected and stored at room temperature (20 ± 1 °C). At days 0, 3, 6, 9, 12, 15, 18 respectively, randomly nine fruit were used to determine their physiological status and pericarp tissue of another fifteen fruit at same stage were sampled until the fruit reached the physiological maturation stage. All samples were frozen in liquid nitrogen before stored at −80 °C.
2.4. Time course quantitative real-time PCR validation Quantitative Real-Time PCR (qRT-PCR) was used to measure the expression of eighteen genes shown to be differentially regulated by RNA-Seq. Five fruit ripening stages were considered: 0 d after storage, and 9, 12, 15, 18 d after storage. For each stage, three biological replicates and each biological replicate composed by five apple fruit were used. RNA extraction, primer design, qRT-PCR experiment and data analysis were carried out as described by Zhao et al. (2015). The primer names and sequences are listed in Online Supplemental Table 1.
2.2. RNA extraction, cDNA library preparation and sequencing 2.5. Ethylene and fruit firmness analysis Frozen tissue was grounded in liquid nitrogen and total RNA was extracted according to previously reported CTAB method by Gasic et al. (2012) with minor modification. RNA quality was verified initially by Nanodrop ND-1000 and agarose gel electrophoresis, its completeness and concentration was further measured using Agilent 2100 Bioanalyzer (Agilent, USA) and Qubit® 2.0 Flurometer (Life Technologies, CA, USA). A total amount of 3 micrograms of qualified RNA per sample was applied for cDNA library preparation using NEB Next® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. The libraries prepared were then sequenced on an Illumina Hiseq™ 2000 platform and 100 bp paired-end reads were generated.
Internal ethylene concentration was measured by withdrawing an 1 mL gas sample from the core of each fruit using a syringe and injecting the gas sample into a gas chromatograph (Shimadzu GC-14A, Kyoto, Japan). Nitrogen was used as the carrier gas at a flow rate of 1 mL s−1 and pressure of 0.1 Mpa. The injector and detector port temperatures were 60 and 110 °C, respectively. An external standard of ethylene (1.0 mL L−1) was used for calibration and quantification. Fruit firmness was determined on opposite sides of fruit equatorial axis after peel removal by using GUSS Fruit Texture Analyzer fitted with an 11 mm flat plate probe (Model GS-15; Strand, South Africa) and the results expressed in newton (N). 387
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Table 1 Overview of sequencing yields, quality and alignments.
Fig. 1. Ethylene production and firmness changes during ‘Royal Gala’ apple fruit ripening at room temperature storage. The total number of tests n = 9.
Accession
0d
15 d
Raw reads Clear reads Clear based Q20 (%) Error rate (%) Total mapped reads (percentage of clear reads) uniquely mapped reads (percentage of clear reads) multiple mapped reads (percentage of clear reads) Ratioof exon, intergenic and intron reads mapped to genome region
143160288 137810896 13.78Gb 96.43 0.04 86.06% 69.91%
137405112 132881896 13.28Gb 96.915 0.035 75.45% 62.20%
16.15%
13.25%
85.8: 12.3: 1.9
86.1: 12.0: 2.0
almost 1:1. When aligned with genome data, exon, intergenic and intron reads mapped to genome region for 0 d were 85.8%, 12.3%, 1.9% and 15 d were 86.1%, 12.0%, 2.0%, respectively. The detection of intergenic and intron reads may be caused by contamination of immature mRNA or incomplete genome annotation. Around 70% of mapped reads are Non-splice reads. Compared against known apple genes by Cufflinks and Cuffcompare, it was found that the assembled 78,553 genes include 15,012 novel genes that were expressed in ripening apple fruit. The discovery of novel genes in our RNA-Seq data could be explained by low coverage of apple reference genome (Bai et al., 2014). Moreover, all 78,553 genes were positioned into apple genome and 59,287 (75.47%) were functionally described in the known “Golden Delicious” apple database. The expression levels of genes in commercial and physiological maturity stages were calculated by RPKM (Reads Per Kilo bases per Million reads) method, with RPKM value lower than 1 (36492 and 35184 of 78,553 genes in two samples respectively) were filtered. Based on expression level, genes were classified into five groups (Table 2).
3. Results and discussion 3.1. Ethylene and fruit firmness changes during apple fruit ripening In order to sample the right apple fruit for RNA-Seq, internal ethylene production and fruit firmness were measured every three days to track the fruit physiological changes during the postharvest storage at room temperature. As reported in many studies, apple fruit ripening during postharvest stage is accompanied by a great amount of ethylene release and firmness decline (Gwanpua et al., 2014; Harb et al., 2012; Ireland et al., 2014). In the present study, internal ethylene production is comparatively lower at the very beginning (Fig. 1), indicating the fruit were pre-climacteric. Ethylene production reached peak at 15 days, and then decreased at 18 days of storage, meaning ‘Royal Gala’ apple came to their physiological maturity at 15 days of storage. Fruit firmness was continuously declining during storage, from 85.45 ± 1.63 to 44.83 ± 1.18 N. Change patterns of ethylene production and fruit firmness decline are consistent with former studies on ‘Mclntosh’ apple fruit (Harb et al., 2012) and those apple fruit of ‘Fuji’ and ‘Golden Delicious’ stored at room temperature (Wei et al., 2010). It was noticed that ‘Royal Gala’ apple fruit firmness dramatically decreased in the first three days. Similar result was observed by Costa et al. (2010b) where about 30N of firmness was lost in five days in ‘Mondial Gala’ under room temperature. Moreover, ‘Golden Delicious’ and ‘Fuji’ fruit firmness also dropped quickly in the first seven days after harvest as showed in the study carried out by Wei et al. (2010).
3.3. Analysis of differentially expressed genes Totally 860 among 78,553 genes were identified to be expressed differentially during apple fruit ripening with ∣ log2(RPKM15d/RPKM0d) ∣ > 1 and FDR < 0.005. The list is shown in Supplemental file 2. Among the 860 genes, 565 genes were matched to known gene products, 30 genes were matched to unknown genes, and 265 were novel genes against known plant gene sequence in the GenBank database. At physiological maturity stage, 199 genes were up-regulated and 661 genes were down-regulated comparing with commercial maturity stage indicating that somehow physiological activities were more active in the initiation of ripening than the physiological ripening stage, which come to the same conclusion with the results in Bayberry (Feng et al., 2012) and pear (Xie et al., 2013). GO analysis showed that 643 of all 860 DEGs could be annotated in significantly enriched 113 GO terms in three categories, biological process (59 GO terms), cellular component (9 GO terms) and molecular function (45 GO terms) category. Single-organism metabolic process (GO:0044710, 183/643), extracellular region (GO:0005576 35/643) and oxidoreductase activity (GO:0016705, 15/643) were the most abundant GO terms within these three categories, respectively. Within
3.2. Genome-wide transcriptome analysis by RNA-Seq Based on the physiological changes acquired during fruit ripening during room temperature storage, Cortex of ‘Royal Gala’ apple in commercial maturity stage (0 d) and physiological maturity stage (15 d) were sampled for RNA sequencing. RNA sequencings were performed on an Illumina HiSeq™2000 platform. A total of 14.28 G and 13.55 G raw reads were generated for these two maturity stage samples considering that a single paired-end read is 100 bp long. After filtered, about 13.78 G and 13.28 G clean reads were produced, with Q20 percentage is over 96% and error rate percentage is lower than 0.04%. Pearson correlation (R2) between two samples is 0.912 (Table 1). Read density and quality of our RNA-Seq data is good enough for gene expression analysis. The sequence reads were aligned against the “Golden Delicious” apple genome (Velasco et al., 2010) using TopHat v2.0.9 software (Trapnell et al., 2009). Total mapped reads, uniquely mapped reads and multiple mapped reads of the clean reads were 86.06%, 69.91%, 16.15% and 75.45%, 62.20%, 13.25% for sample 0 d and 15 d, respectively. Reads map to“ + ” to reads map to “-” in both samples are
Table 2 Percentage of genes with different expression levels in 0 d and 15 d samples.
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RPKM Interval
0d
15 d
0–1 1–3 3–15 15–60 > 60
42061(53.54%) 10290(13.10%) 16179(20.60%) 7340(9.34%) 2683(3.42%)
43369(55.21%) 9439(12.02%) 15695(19.98%) 7444(9.48%) 2606(3.32%)
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that all of the qRT-PCR results were consistent with the RNA-Seq, which strongly confirmed the RNA-Seq results were reliable (Fig. 3). 3.5. Genes related to ethylene biosynthesis and perceptiveness when ‘Royal Gala’ apple ripen Ethylene is the main hormone regulator of climacteric fruit ripening, of which apple was a typical example. In this study, internal ethylene of ‘Royal Gala’ apple was increased from very low levels at 0 d to its peak value at 15 d after harvest. Several enzymes worked on ethylene biosynthesis pathway, among which 1-aminocyclopropane-1carboxylate synthase (ACS) and 1-aminocyclopropane-1-carboxylate oxydase (ACO) are well-known as the key enzymes. Moreover, since ethylene burst was mainly contributed by system 2 ethylene, we were oriented towards revealing transcripts worked on system 2 ethylene production. Comparing all the DEGs involved in ethylene production in these two ripening stages. It was found that physiological stage producing both system 1 and 2 ethylene, while only system 1 ethylene was produced in commercial day. Our analysis recovered two ACS transcripts: Novel12596 (best hit: U89156), Log2 fold change = 6.37, FDR = 1.84E-96 and Novel12597 (best hit:AB010102), Log2 fold change = 7.02, FDR = 6.21E-18, and three ACO transcripts: MDP0000195885 (best hit: JQ675681), Log2 fold change = 1.28, FDR = 0 (ACO1), MDP0000200737 (best hit: XM_008372823), Log2 fold change = 1.06, FDR = 1.83E-18 (ACO2) and MDP0000200896 (best hit: XM_008339460), Log2 fold change = 2.14, FDR = 3.63E11(ACO7) were significantly up-regulated when fruit ripen. Closely analysis of two up-regulated MdACS transcripts showed that they hit either MdACS1-1 (U89156) or MdACS1-2 (AB010102). MdACS1 and MdACS3 are the most studied ACS transcripts in apple. MdACS1, unlike MdACS3a whose expression preceded MdACS1 and it was responsible for transiting from system 1 to system 2 ethylene biosynthesis, was reported only expressed in ripening fruit and its expression was increased with ripening, so it was considered responsible for system 2 ethylene biosynthesis (Sunako et al., 1999; Wang et al., 2009a). RNASeq data confirmed the previous results. MdACO1, like MdACS1 was intensively reported to have ripening related expression pattern during apple fruit ripening (Atkinson et al., 1998; Costa et al., 2005; Dal Cin et al., 2007; Tsantili et al., 2007). Extensive expressed sequence tag (EST) data on apple also revealed that MdACO1 showed a ripeningrelated expression pattern associated with climacteric (Park et al., 2006). According to Dandekar et al. (2004) down regulated MdACO showed less ethylene production during ripening. We identified one MdACO transcripts: MDP0000195885 (best hit: JQ675681, MaACO1-c allele) that shared very high sequence similarity with the reported MdACO1. The other one MDP0000200737 (best hit: XM_008372823) was MdACO2 which was the closest allele of MdACO1 in the phylogeny of the ACO genes (Clouse and Carraro, 2014). The third one MDP0000200896 (MdACO7) shared a bit less similarity with MdACO1. Former study using microarray on tree ripening apple found the expression of one ethylene receptors ETR2/EIN4 (EE663937) had changed significantly during ripening (Janssen et al., 2008). However, none of the ethylene receptor expression was recognized to be significantly changed by our RNA-Seq results which could be explained either by the sample difference or by experimental technique difference. Transcription factors such as ERFs, MYBs and HBs play crucial roles on regulating ethylene bio-function. Most ERFs were reported to be ripening down-regulated and involved in ripening by interacting with the promoters of ACS and ACO, and SIERF6 repressed line in tomato enhanced ethylene levels (Lee et al., 2012; Xiao et al., 2013; Yin et al., 2010). We identified three down-regulated ERFs (MDP0000175375, Log2 Fold change = −4.14, FDR = 0.00010483; MDP0000235028, Log2 Fold change = −2.97, FDR = 1.66E-05; MDP0000689946, Log2 Fold change = −2.71, FDR = 2.81E-05), and two up-regulated ERFs (MDP0000155743, Log2 Fold change = 1.64, FDR = 6.97E-20; Novel
Fig. 2. The GO term distribution of DEGs by RNA-Seq between day 0 (0D) and day 15 (15D). The top 30 enriched GO terms in the comparison groups between day 0 (0D) and day 15 (15D). Asterisks (*) indicate significant (P-value < 0.05) enrichment GO terms. Most consensus sequences were grouped into three major functional categories, namely biological process, cellular component, and molecular function.
them, 11 GO terms were activated at physiological maturity, including 9 in biological process and 2 in molecular function. Most of biological processes were related to fruit ripening, ethylene biosynthesis and secondary metabolism. The two molecular function GO terms are 1aminocyclopropane-1-carboxylate oxidase activity and oxidoreductase activity. These results perfectly matched the physiological status of physiological maturation during which ethylene burst and ripening happened. The most enriched 30 GO terms which were thought to be indicator of significantly altered biological program between the two maturity stages is shown in Fig. 2. These activated GO terms were mainly responsive of ethylene and aroma biosynthesis, which would be abundantly produced when apple fruit ripen. By retrieving KEGG pathways from KEGG database based on sequence similarity, all the DEGs can be mapped into 140 biochemical pathways (Supplemental file 3). The pathways with highest representations were metabolic pathways (Ko01100, 45), Biosynthesis of secondary metabolites (Ko01110, 29), Microbial metabolism in diverse environments (ko01120, 10), Biosynthesis of amino acids (ko01230, 8), Carbon metabolism (ko01200, 8). 3.4. Time course analysis and validation of selected DEGs by quantitative PCR To validate RNA-Seq data and to check how expression of these genes change at different storage time (0, 6, 12, 15, 18 d), quantitative real-time PCR (qRT-PCR) was used to examine the relative expression pattern of eighteen transcripts. 6 of 18 genes (XTH8, XTH1, XTH4, OMT7, AFS and AST) were selected from transcripts in the result and the rest 12 genes were randomly picked from the whole DEGs. These genes are from an array of different processes including redox activity and metabolism making the validation more rational. Housekeeping gene GAPDH (Genbank number: CN929227) was used to normalize qRT-PCR results after a preliminary experiment comparing the stability of different housekeeping genes in this experiment. Our data showed 389
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Fig. 3. qRT-PCR revealing genes expression patterns and validation of the selected differentially expressed transcripts from RNA-Seq quantitative results during gala apple fruit postharvest storage under 20 °C. Error bars represent means ± SE (n = 3 with 5 apple fruit each).
3.6. Genes related to fruit firmness when ‘Royal Gala’ apple ripen
04681, Log2 Fold change = 2.65, FDR = 1.68E-06). This was reasonable since different ERFs might act as transcriptional activators or repressors on ethylene biosynthesis as reported previously (Fujimoto et al., 2000; Ohme-Takagi et al., 2000). Transcription factor HD-Zip homeo box protein LeHB1 was observed to play an important role in a positive manner in tomato ripening by interacting with the promoter of LeACO1 (Lin et al., 2008). Our transcriptomic comparison found two of MdHB (MDP0000564897, Log2 Fold change = 1.01, FDR = 6.84E-11, best hit = XM_008340338 (HBA); MDP0000615948, Log2 Fold change = 1.23, FDR = 1.28E-05, best hit = XM_008383196 (HBB)) that were highly expressed during ‘Royal Gala’ apple fruit ripening and were significantly up-regulated at physiological maturity, indicating that MdHBs in our RNA-Seq might also regulate apple fruit ripening as they did in tomato. In tomato, GRAS domain transcription factor (Solyc07g052960) was considered as ripening-associated and ethylene related transcription factor since it was specifically expressed at ripening fruit and it was down-regulated from pink coloring to red ripe tomato (Fujisawa et al., 2012). Similar result was observed in our RNASeq (MDP0000264347, Log2 Fold change = −2.93, FDR = 7.33E-11). Despite lack of direct evidence of GRAS regulating ethylene pathway, it was targeted by RIN and its expression pattern suggested that it together with the two MdHBs that screened in this study, was worth further studying.
‘Royal Gala’ apple is a typical mealy fruit and its firmness decreases consistently during ripening. Fruit firmness changing characteristic is thought to be mainly involved in loss of turgor pressure, starch degradation and cell wall modification. Among these factors, cell wall which is responsible for cell shape and mechanical support was the main contributor of fruit firmness and its modification determined fruit firmness change (Chaib et al., 2007; Goulao and Oliveira, 2008). Several cell wall modification enzymes involved in fruit cell wall modification, such as polygalacturonase (PG), pectin methylasterase (PME), xyloglucan endotransglycosylase/hysrolase (XTH), expansin (EXP), βgalactosidase (β-Gal) and pectate lyase (PL). All these genes encoding cell wall metabolism enzymes were analyzed, and the expression of PG1, XTH7, purple acid phosphatases (PAPs) and EXPA3 was significantly different between fruit of the two maturity stages. The cell wall hydrolase (polygalacturonase1) PG1 acts on the middle lamella to solubilize intercellular adhesion and it is a main factor responsible for fruit texture changing referred by QTL (Costa et al., 2010b; Dunlevy et al., 2013; Longhi et al., 2012). Suppression of PG1 led to firmer fruit, higher CDTA-soluble pectin percentage and higher intercellular adhesion (Atkinson et al., 2012). The expression of PG1 was reported to be not only exclusively in fruit (Tomato Genome, 2012) but also increased during fruit ripening (Costa et al., 2010b) 390
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dramatically down-regulated (MDP0000904458, log2 Fold change = −5.56, FDR = 2.21E-38) during ripening. FLAs, a subclass of arabinogalactan proteins (AGPs) which were found in the cell wall as structural complex molecules (Keegstra, 2010), had been proposed to function in cell wall cross-link by binding with pectin (Ellis et al., 2010). We hypothesized that the down-regulated FLA caused decrease of FLA synthesis and might even cause the loosing of cell wall adhesion. Although the function of FLA during fruit ripening had not been investigated, its cell wall location, interaction with pectin and dramatic expression change in ripening hint its potential role on fruit firmness change during ripening. Phosphorylation also has a role in regulating enzymatic activity in the cell wall. Here we identified two cell wall-bound PAPs transcripts (MDP0000636860, Log2 Fold change = 2.51, FDR = 3.37E-18; MDP0000824136, Log2 Fold change = 2.52, FDR = 1.62E-27), which were significantly up-regulated when ‘Royal Gala’ apple was ripen. PAPs were reported to dephosphorylate proteins like α-xylosidase and β-glucosidase resulting in a decrease in their activities, causing more cell wall oligosaccharides accumulated (Kaida et al., 2010). In olive, PAP expression was reported to be highly increased in abscission zone of ripen fruit, which might be associated with its role of limiting phosphorylation (Parra et al., 2013). These changes indicated that dynamic changes and rearrangements of the cell wall that occur during ‘Royal Gala’ fruit ripening.
indicating its specific role on late stages of ripening (Brummell et al., 2004). RNA-Seq data showed that PG1 expression was in a high level during whole ripening process and was up-regulated at physiological maturity stage (MDP0000326734, Log2 Fold change = 1.01,FDR = 1.04E-135). PME is also thought to play important role in fruit softening, since PME is the main pectin de-esterification enzyme and its production is the substrate of PG. PME expression levels and patterns may vary among apple cultivars, therefore may play different roles. In ‘Fuji’ apple, expression level of PME was constant up to day 42, and only up-regulated in the late stage of storage at room temperature; differently, PME expression in ‘Golden Delicious’ apple fruit increased rapidly and significantly during the early postharvest time, and after going to the peak on day 28 it was decreased (Wei et al., 2010). In our study, data showed that PME expression was up-regulated in the physiological maturity of ‘Gala’ apple fruit although its expression level was relatively low, and there was no significant difference between the two samples. Lower expression level of PME gene (MdPME1) was also observed by Goulao et al. (2008) as these researchers found that its expression was hardly detected by RT-PCR even with 40 cycles, while for other genes 35 cycles were sufficient enough. Another example of rearrangement of cell wall substrate was achieved by function of XTHs proteins. XTH was gene family encoding xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET), the activities of which cut or cut and rejoined xyloglucan chains which were responsive for cell wall extension, since the structural change and rearrangement of cell wall was mutual during ripening (Goulao and Oliveira, 2008). We found several XTH transcripts that were differentially expressed and regulated during ripening, two were significantly down-regulated (MDP0000269483, log2 Fold change = −2.57, FDR = 1.50E-07, best hit = MdXTH4; MDP0000181544, log2 Fold change = −6.31, FDR = 0.00077076, best hit = MdXTH8) and one was significantly up-regulated (MDP0000398765, log2 Fold change = 2.90, FDR = 4.34E-06, best hit = MdXTH1). These results confirmed former reports that different XTH transcripts expressed in ripening fruit (Rose et al., 2002). Apple XTH genes were historically clustered into three groups according to their sequence similarity (Atkinson et al., 2009; Munoz-Bertomeu et al., 2013). The transcripts expressed in ripening fruit were grouped into group 3 in tomato (Munoz-Bertomeu et al., 2013; Saladie et al., 2006), grouped into 1 and 3 in “Mondial Gala” apple (Goulao et al., 2008), 2 and 3 “Golden Delicious” (Munoz-Bertomeu et al., 2013) and 2 and 3 in “Granny Smith” (Atkinson et al., 2009); grouped in 2 and 3 in kiwifruit (Atkinson et al., 2009) and grouped into group A and B in persimmon (Zhu et al., 2013). In the present study, MdXTH1 and MdXTH8 were belonged to group 1 and the down-regulated MdXTH4 and MdXTH7 were belonged to group 2 (data not shown). This indicated that the phylogenetic tree could not represent the expression or function classification (Eklof and Brumer, 2010) and for different cultivars, different transcripts were functioning for regulating fruit ripening. Expansins responsive for depolymerization of hemicelluloses (Brummell et al., 1999) are composed by a big family (Zhang et al., 2014) were observed to have different expression patterns during apple fruit ripening in different cultivars (Goulao et al., 2008). The expression of MdExp3 (referring to MdExp2 in other reference) in “Golden Delicious” was almost parallel with ethylene production (Wakasa et al., 2003), while MdEXPA3 in “Mondial Gala” apple like LeExp1 in tomato was reported with highest expression level at harvest and reduced to low level thereafter (Goulao et al., 2008). Analysis of DEGs showed that one transcript, MdEXPA3, (MDP0000139058, log2 Fold change = −2.62, FDR = 3.74E-152) was down-regulated when apple fruit ripening, confirmed the former results that MdEXPA3 showed higher expression level in fruit at harvest, and after that its expression was significantly reduced (Goulao et al., 2008). Besides cell wall metabolism enzymes, cell wall structural proteins changes also played crucial role on fruit firmness. One transcript fasciclin-like AGP (FLA) that encoded cell wall structural proteins was
4. Conclusions RNA-Seq was carried out to investigate complex changing process between commercial and physiological maturation of ‘Royal Gala’ apple fruit at the transcriptome level. GO annotations showed that 643 of all 860 DEGs belonging to a wide range of functional categories, especially on ethylene biosynthesis and secondary metabolism, were significantly enriched. Various gene families and pathways involved in this ripening process were identified. Among them, novel genes such as HBs, ERFs and ACC that are thought to be related to apple fruit ethylene biosynthesis were significantly up-regulated. These enriched DEGs will be selected in our future gene functions research. Overall, the observed results provide a broad overview into the molecular mechanisms underlying the postharvest ripening process of one of the most important fresh eating fruit. Conflicts of interest Authors of this article declare no conflict of interest. Acknowledgement This work was supported by Modern Agricultural Industry Technology System of China for Apple (Grant No.Z225020701). The funder had no role in research design, data collection and analysis, decision to publish and/or preparation of manuscript. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.scienta.2017.07.024. References Alexander, L., Grierson, D., 2002. Ethylene biosynthesis and action in tomato: a model for climacteric fruit ripening. J. Exp. Bot. 53, 2039–2055. Atkinson, R.G., Bolitho, K.M., Wright, M.A., Iturriagagoitia-Bueno, T., Reid, S.J., Ross, G.S., 1998. Apple ACC-oxidase and polygalacturonase: ripening-specific gene expression and promoter analysis in transgenic tomato. Plant Mol. Biol. 38, 449–460. Atkinson, R.G., Johnston, S.L., Yauk, Y.-K., Sharma, N.N., Schröder, R., 2009. Analysis of xyloglucan endotransglucosylase/hydrolase (XTH) gene families in kiwifruit and apple. Postharvest Biol. Technol. 51, 149–157.
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