Transcriptome changes during fruit development and ripening of sweet orange (Citrus sinensis)
© Yu et al; licensee BioMed Central Ltd. 2012
Received: 24 September 2011
Accepted: 10 January 2012
Published: 10 January 2012
The transcriptome of the fruit pulp of the sweet orange variety Anliu (WT) and that of its red fleshed mutant Hong Anliu (MT) were compared to understand the dynamics and differential expression of genes expressed during fruit development and ripening.
The transcriptomes of WT and MT were sampled at four developmental stages using an Illumina sequencing platform. A total of 19,440 and 18,829 genes were detected in MT and WT, respectively. Hierarchical clustering analysis revealed 24 expression patterns for the set of all genes detected, of which 20 were in common between MT and WT. Over 89% of the genes showed differential expression during fruit development and ripening in the WT. Functional categorization of the differentially expressed genes revealed that cell wall biosynthesis, carbohydrate and citric acid metabolism, carotenoid metabolism, and the response to stress were the most differentially regulated processes occurring during fruit development and ripening.
A description of the transcriptomic changes occurring during fruit development and ripening was obtained in sweet orange, along with a dynamic view of the gene expression differences between the wild type and a red fleshed mutant.
The typical course of fruit development involves expansion, sweetening and increasing pigmentation . From the consumers' point of view, the appearance, texture and taste of the fruit are all of high importance. These properties involve attaining a suitable composition of sugars, organic acids, amino acids and carotenoids. The underlying mechanisms of fruit development and ripening have been extensively studied in tomato , but are not well explored in non-climacteric fruits. Citrus is a widely grown fruit crops, which exhibits non-climacteric ripening behaviour. Its fruit contains a juicy pulp made of vesicles within segments . The growth and development of the citrus fruit can be divided into three stages: cell division, an expansion phase involving cell enlargement and water accumulation, and the ripening stage . In the latter stage, carotenoids and other soluble solids are accumulated, chlorophyll is lost, the cell wall is extensively modified, the organic acid content is reduced, and the concentration of a number of volatiles increases.
Citrus fruit provides a convenient vehicle to study gene regulation during non-climacteric fruit development and ripening. In grape (Vitis vinifera), another non-climacteric fruit, several comprehensive mRNA expression profiling studies have been presented to describe fruit development and ripening [5–8], while in citrus, an EST sequencing project showed that 20% of the sequences were metallothionein . Application of a citrus cDNA microarray has suggested that unique genetic regulatory networks arise during fruit development , while a more global transcriptome analysis was able to identify ethylene-responsive genes in the mandarin fruit . More recently, a comprehensive study of the clementine fruit transcriptome has proposed a mechanism for citrate utilization . However, these transcriptomic studies of fruit development in citrus have mainly been based on microarray analysis, the next generation sequencing technology provides new opportunities for more accurate and powerful deep transcriptome analysis of fruit development.
The ripening of citrus fruit is accompanied by carbohydrate build-up, acid reduction, and carotenoid accumulation. Citrus fruits accumulate most of their sucrose in the juice cells . Glucose is decomposed via glycolysis and the pentose phosphate pathway (OPP). As the major acid present in citrus fruit, citric acid contributes > 90% of the total organic acid content. Citric acid synthesis is thought to take place in the mitochondria via the tricarboxylic acid (TCA) cycle. Although the genes encoding some of the key enzymes (specially, citrate synthase (CS), aconitase , and NADP-isocitrate dehydrogenase (NADP-IDH) ) have been isolated, their activity cannot completely account for variation in the level of citric acid in citrus fruit . Carotenoids are also important components of the citrus fruit, and their composition and content in sweet orange fruits have been extensively studied [17, 18]. Cross-talk between carotenoid, sugar and organic acid metabolism has been documented [19, 20]. The presence of sucrose may promote colour break , while its deficiency delays lycopene accumulation in tomato . The down-regulation of CS and NAD-dependent IDH results in a decrease in the levels of both organic acids and carotenoids .
The fruit of the bud mutant Hong Anliu is characterized by a high sucrose and low citric acid level and its tendency to accumulate lycopene is responsible for the red pigmentation of its pulp . The mutant appears to be highly isogenic with its progenitor wild type variety Anliu, as revealed by a genotypic analysis based on microsatellites and AFLPs. The mutant has therefore provided a platform to study cross-talk between primary (sugar and organic acid synthesis) and secondary (carotenoid synthesis) metabolism enzymes, which are fundamental in the determination of citrus fruit quality. Suppressive subtraction hybridization combined with cDNA microarray analysis has been applied to determine that the differentially expressed genes were mainly enriched in the stage of 170 DAF (days after flowering) in the mutant fruit . At this stage, a total of 582 genes were found to be differentially expressed between the wild type Anliu (hereafter WT) and Hong Anliu (hereafter MT) as revealed by RNA-seq analysis . However, how genes are dynamically and differentially expressed during fruit development and ripening has not yet been determined. Here, the developmental changes of fruit transcriptome of sweet orange were investigated.
Plant material and RNA preparation
WT and MT plants were both cultivated in the same orchard at the Institute of Citrus Research (Guilin, Guangxi Province, China), with the same climatic conditions. Fruit samples were harvested at 120, 150, 190, and 220 DAF (days after flowering) from three different trees in 2009. At each developmental stage, ten representative fruits were sampled from each tree. The pulp was separated from the peel, and the pulp was sliced. The sliced WT pulps samples were combined with one another (similarly for the MT samples), snap-frozen in liquid nitrogen and kept at -80°C until required [24, 25]. One aliquot was used to extract RNA isolation, as described previously . The remainder of the powder was used for the determination of sugar and organic acid composition and concentration, and the content of H2O2.
RNA-seq and functional assignment
The WT and MT fruit pulp harvested at 120, 150, 190, and 220 DAF was subjected to RNA-seq using an Illumina Genome Analyzer at Beijing Genomics Institute (Shenzhen) in 2009. The abundance of each tag was normalized to one transcript per million (TPM) for between sample comparison purposes. The raw data was filtered to remove low quality sequences including ambiguous nucleotides, adaptor sequences, and below 3 TPM, as described previously . The sequencing data can be accessed at the website: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=dxqjxoygumyauzm&acc=GSE22505. To link the expressed signatures to known genes from orange, the TIGR unigene dataset (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=orange) was used as a reference database. The Z-score method using the p-value as a statistical significance index  was applied to identify differentially expressed genes. A cluster analysis was performed according to Eisen et al. ; the log2 of TPM for each gene was used for the hierarchical clustering analysis. Gene Ontology (GO) categorization was carried out as described previously . The ultra-geometric test was applied to perform GO enrichment analysis. In the significance analysis of the enrichment of a GO item, the p-value represents the probability of satisfying the hypothesis that the designated genes involved in the GO item has not been enriched (statistical significance at P = 0.05).
Real-time quantitative RT-PCR
The differential expression of a selection of the genes identified as being differentially expressed was validated by applying real-time quantitative RT-PCR (qRT-PCR). The sequences of the primer pairs (designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA, USA)) are listed in additional file 1. All qRT-PCRs were performed using an ABI 7500 Real Time System (Applied Biosystems) using the actin gene as the reference . Primers for both the target gene and the reference were diluted in SYBR GREEN PCR Master Mix (Applied Biosystems) and 20 μL of the reaction mix were added to each well. Reactions were performed via an initial incubation at 50°C for 2 min and at 95°C for 10 min, and then cycled at 95°C for 15 s, and 60°C for 60 s for 40 cycles. The resulting data were handled by the instrument on-board software Sequence Detector Version 1.3.1 (Applied Biosystems).
Analysis of sugar, organic acid and H2O2
Soluble sugar and organic acid composition and concentrations were determined by gas chromatography (GC) using 3 g of the powdered pulp as described previously  with minor modifications. The powder was suspended in chilled 80% methanol and then held in a 75°C water bath for 30 min. After a 2 h ultrasonic extraction and centrifugation at 4000g for 10 min, the supernatant was collected and 1 mL internal standard (2.5% w/v phenyl-β-D-glucopyranoside, 2.5% w/v methyl-α-D-glucopyranoside) was added. The solution was made up to 50 mL with 80% methanol, and a 2 mL aliquot was centrifuged at 12000g for 15 min. A 0.5 mL aliquot of this final supernatant was vacuum-dried and then re-dissolved in 800 μL 2% w/v hydroxylamine hydrochloride in pyridine at 75°C for 1 h. Then 400 μL hexamethyldisilazane and 200 μL trimethylchlorosilane were added and the sample was held at 75°C for 2 h. A 0.5 μL aliquot was used for GC analysis in an Agilent 6890N device (Santa Clara, CA, USA) equipped with a flame ionization detector. A capillary column (HP-5, 5% phenyl-methylpolysiloxane, 30 m × 25 μm i.d. × 0.1 μm) was employed, with nitrogen as the carrier gas at a flow rate of 45 mL/min, and flow-rates of hydrogen and air set to 40 mL/min and 450 mL/min, respectively. Sugars and organic acids were identified through a comparison of retention times using standard compounds from Sigma (St. Louis, MO, USA). The concentration of H2O2 was measured using a hydrogen peroxide detection kit supplied by Nanjing Jiancheng Institute of Biological Technology (Nanjing, China). A 0.8 g sample of powdered pulp was suspended in 7.2 ml saline (0.90% w/v of NaCl) and centrifuged for 10 min at 10, 000g. The intensity of yellow complex formed by the reaction of molybdate and H2O2, as measured spectrophotometrically at 405 nm, was used to assess the concentration of H2O2. Three replicates were conducted for each sample.
The fruit transcriptome sampled at four developmental stages
Summary of the RNA-seq data collected from MT and WT at each of four selected fruit developmental stages
Total Sequence Collected
Low Quality Tags
Transcriptome changes during fruit development and ripening
Differentially expressed genes during fruit development and ripening
List of 10 genes involved in major processes associated with fruit development and ripening based on Gene Ontology categorization
150vs120 Fold change
190vs150 Fold change
220vs190 Fold change
Epsilon lycopene cyclase
Reversibly glycosylated protein
Pectinesterase PPE8B precursor
Sucrose-phosphate synthase 1
Sucrose-phosphate synthase 1
Sucrose-phosphate synthase 1
Superoxide dismutase [Cu-Zn]
Cu/Zn superoxide dismutase
Cu/Zn-superoxide dismutase copper chaperone precursor
Superoxide dismutase [Cu-Zn]
Superoxide dismutase [Mn], mitochondrial precursor
Cu/Zn-superoxide dismutase copper chaperone precursor
Heat shock protein 70
Serine/threonine-protein phosphatase PP1
2-oxoacid dehydrogenase family protein
22 kDa polypeptide
Group 5 late embryogenesis abundant protein
Developmental difference between fruit transcriptome of MT and WT
When the expression profiles of the genes differentially expressed between MT and WT were subjected to a cluster analysis (additional file 9), over one half (492/883) turned out to be up-regulated in MT at all the developmental stages except 150 DAF. Some examples of this large cluster included genes encoding Cu/Zn superoxide dismutase (TC12069), ascorbate peroxidase (EY685405, TC22775 and TC14669), MYB1 (EY649968) and plastidic glucose 6-phosphate/phosphate translocator (EY722703). Only five genes were detected as differentially expressed at all four stages; one encoded a cysteine protease (Cp5) (TC5370), which had been reported exhibiting the acid-activatable cysteine protease forms , three had no assigned functions, and one shared no homology with any entry in. The five genes could be classified into two groups (two genes in one group, three genes in the other), with their expression patterns of being opposite to each other but with both groups having a turning point at 150 DAF (additional file 10).
Gene ontology enrichment analysis for the genes differentially expressed between WT and MT during fruit development and ripening
Enriched GO terms
Genes in group
capsanthin/capsorubin synthase activity
transferase activity, transferring glycosyl groups
EY691346, TC18663, TC10188, TC8764, TC320, TC9277, TC11658, EY679857, TC24546, TC11943, TC8402, CK939135, TC15309, TC22218, EY661193
protein N-terminus binding
TC9238, EY702245, TC17456
cell wall organization
TC4981, CK933828, DN618740, EY725863, TC18738, TC3660, TC12562, EY746957, TC14614
protein import into nucleus, docking
EY701416, EY733548, DY305879
carotenoid metabolic process
EY722043, TC15628, TC5834, TC9367
Verification of differentially expressed genes during fruit development and ripening
Changes in fruit soluble sugars, organic acids, carotenoids and H2O2 content
Transcriptome dynamics during fruit development and ripening
Fruit ripening is a highly coordinated, genetically programmed and irreversible process which involves a series of physiological, biochemical, and organoleptic changes allowing for the development of an edible ripe fruit . Fruit ripening in citrus is accompanied by carbohydrate build-up, acid reduction, carotenoid accumulation and chlorophyll degradation . The bud mutant MT produces fruit with high sucrose and lycopene, but low citric acid content . Here, transcriptome changes over the course of fruit development and ripening in MT and WT were monitored and annotated. We must point out that we collected samples from three different trees and pooled for RNA-seq analysis for each developmental stage. We did not include biological replicates considering that we used a pair of genotypes (WT and MT) and the data from WT and MT could corroborate to each other to some extent. We sequenced each pool once technically since the next generation sequencing data are highly replicable with relatively little technical variation . We also used real-time qRT-PCR to verify the transcription profile revealed by RNA-seq data (Figure 4), with an overall correlation coefficient of 0.8379 which indicated the RNA-seq data was reliable. The RNA-seq approach detected a similar number of genes (18,829 genes in WT and 19,440 genes in MT) in both genotypes, with no apparent difference between the global number of genes expressed in the two genotypes at any of the four stages of fruit development and ripening sampled (additional file 4). The WT and MT patterns of gene expression were also largely alike (20 of the 24 expression patterns were similar). Thus, we believed that overall picture of the transcriptome captured by RNA-seq is robust.
An overwhelming proportion of the genes identified (84.8% in MT and 89.7% in WT) varied in their level of expression over the course of fruit development and ripening, reflecting the occurrence of a massive genetic re-programming. A large number of these genes were expressed in a stage-specific manner, which implicates their involvement in physiological processes which take place only at a specific developmental stage(s). A major group of the differentially expressed genes was involved in cell wall modification, which is not surprising since the major textural changes associated with the softening of fruit are due to enzyme-mediated alterations in the structure and composition of the cell wall , especially the cell walls of juice sacs in citrus. Changes in the activity of several cell wall-related genes were known to result in the abnormal development of juice sac granulation [35, 36], while modifications in cell wall structure or in the components of the membranes of the segments and juice sacs during fruit development and ripening clearly influenced the formation of the fruit pulp melting characteristic . One of the cell wall-related genes revealed here encoded a pectinesterase, an enzyme which modifies the assembly and disassembly of pectin, a common component of the primary cell wall. In tomato, the gene for pectinesterase was highly expressed prior to ripening, and was down-regulated by ethylene as ripening begins . Here, the expression of the gene encoding the pectinesterase PPE8B precursor decreased as the WT fruit matured (Figure 4A), while in the blood orange, the expression of a pectinesterase gene has been shown to increase during fruit development and ripening . It might be due to the different members identified in the two studies. The hemicellulose xyloglucan is a common component of the cell wall, and is hydrolysed and transglycosylated by xyloglucan endotransglycosylase in growing tissues and ripening fruits . Here, a gene encoding this enzyme was up-regulated during fruit development and ripening (Figure 4A), indicating its probable role in cell wall degradation during ripening. Both the early enlargement of the citrus fruit driven by cell expansion and the later ripening process require the presence of expansins to loosen the cell walls , and several genes encoding expansins were detected in the present study and their expression was higher at the later stages of fruit development and ripening.
The accumulation of carbohydrates represents one of the most obvious changes which occur during citrus fruit development and ripening. The perceived changes in expression of genes involved in carbohydrate metabolism here were consistent with the findings of other transcriptomic analyses in citrus . The type of sugar deposited to a high level in the cell vacuole in citrus is predominantly sucrose, unlike in grape, where it is glucose and fructose . Reflecting this difference, the expression profile of the gene encoding sucrose synthase in citrus  was rather different from that in the grape . Nevertheless, in both species, sugar is important for the regulation of colour development, and perhaps also for other ripening processes . It was notable that the gene encoding a sucrose phosphate synthase was induced during fruit development and ripening, perhaps because the activity of this enzyme may be required for the re-synthesis of sucrose to allow its further transport to the vacuole . Of some interest also was the behaviour of a gene encoding valencene synthase, an enzyme the activity of which was known to be induced by ethylene, and which was part of the natural ripening process in citrus . Valencene is an important component of the aroma of the ripe sweet orange fruit. Both in the present experiments (Figure 4A) and elsewhere , valencene synthase transcript accumulated in the ripening fruit.
Molecular processes involved in the formation of the fruit traits of red flesh sweet orange
Suppressive subtraction hybridization, in combination with cDNA microarray analysis, has identified a set of 267 genes which were differentially expressed between MT and WT . RNA-seq technology is more capable of identifying a nearly complete inventory of transcripts and by this method a total of 582 genes were found to be differentially expressed between WT and MT at the stage of 170 DAF . In the current study, we tested on four developmental stages of WT and MT and extended our understanding of the global and dynamic changes during fruit development and ripening in MT and WT. Almost all of the members of the 267 gene set revealed by SSH strategy were also identified in the present study, although some discrepant expression patterns were apparent between this new data set and previously assembled microarray-based set. For example, the abundance of the transcript encoding cysteine protease, which appeared to differ between MT and WT in both studies, was documented by the microarray analysis as being lower at all developmental stages, whereas it appeared to be higher at 150 DAF in MT in the present study (additional file 10).
The major biological processes occurring in the mitochondria (TCA cycle, coupling electron transfer, and oxidative phosphorylation) were remarkably altered in MT. The intermediates of the TCA cycle can be channelled into the syntheses of fats, terpenoids, porphyrins, nucleotides, and amino acids. In MT, the level of citric acid, the major organic acid present in citrus fruit, was consistently around 25% that present in WT fruit (Figure 5). However, no major difference was detected in the expression of the genes encoding CS and aconitase-iron regulated protein (Figure 4A), two predominant enzymes involved in the TCA cycle. Five differentially expressed genes, all associated with mitochondria-related processes (coupling electron transfer and oxidative phosphorylation) were down-regulated in MT compared with WT (Figure 4B). These included the genes encoding a NADH-ubiquinone oxidoreductase 75kDa subunit (TC7962), cytochrome C oxidase (TC18256), and cytochrome oxidase subunit 3 (TC156), suggesting that MT mitochrondria were capable of less efficient electron transport than that WT ones. If, as a result, flux through the TCA cycle is decreased, the accumulation of citric acid is likely to be compromised. In addition, PDS and ZDS catalyzing desaturation of phytoene to lycopene involve net electron transfer . In tomato, a NAD(P)H dehydrogenase complex which participating in electron transfer was involved in carotenoid biosynthetic pathway , suggesting the possible exist of cross-talk between electron transfer and carotenoid accumulation in sweet orange.
In the plant cell, the mitochondrial electron transport chain is a major site of reactive oxygen species (ROS) production . Here, the concentration of one of the primary ROS molecules (H2O2) was higher in MT than that in WT at 120 DAF (Figure 5D). The delicate balance between antioxidant defence and ROS production can be disrupted by either compromised antioxidant defence or the inhibition of electron flow . Here, the primary anti-oxidant enzymes (SOD, APX and GR) were more active in MT than in WT pulp, suggesting that the level of oxidative stress may be greater in MT than in WT . The expression of a large number of stress-related genes was also substantially different in MT and WT (additional file 8). Functioning as an important ROS scavenging pathway, the ascorbate-glutathione cycle requires the cofactor NADPH, which is provided by the OPP pathway . This pathway is also a major source of NADPH for many biosynthetic processes, including carotenoid biosynthesis , in non-photosynthetic organs such as the fruit. The gene encoding glucose 6-phosphate dehydrogenase, which is considered as the first and rate-limiting enzyme of the OPP pathway in all cells , was up-regulated in MT. A statistical analysis of the qRT-PCR result confirmed that the level of transcription of this gene was significantly higher in MT than in WT, especially at 120 DAF (Figure 4C). Carotenoid biosynthesis, another effective anti-oxidative process , was also higher in MT than in WT. Lycopene is the most potent antioxidant among the carotenoids . The expression level of several carotenoid biosynthesis genes, encoding namely PSY, ZDS, lycopene β-cyclase (LCYb) and CCS, was greatly changed in MT. PSY is generally accepted to be a rate-limiting enzyme in carotenoid biosynthesis pathway. The CCS product is an enzyme which is mechanistically similar to LCYb, and the low transcript level of CCS may well be responsible for the accumulation of lycopene in red grapefruits (Citrus paradisi) . The qRT-PCR analysis confirmed that both upstream genes (PSY and ZDS) were up-regulated and both downstream ones (LCYb and CCS) down-regulated in MT (Figure 4D), consistent with the mechanism regulating lycopene accumulation in tomato .
The present study has provided a dynamic view of the transcriptome during fruit development and ripening of a sweet orange red-fleshed mutant and its progenitor wild type. Cell wall biosynthesis, carbohydrate metabolism, the TCA cycle, and carotenoid biosynthesis were all differentially regulated during fruit development and ripening. These differentially regulated processes may well be important for the formation of the pleiotropic fruit trait of Hong Anliu sweet orange.
This work was supported by the National Basic research program of China (973 program; No. 2011CB100601), and the National Natural Science Foundation of China (No. 30830078 and 31071779).
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