'Granny Smith' apple

South African Journal of Botany 88 (2013) 125–131

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South African Journal of Botany journal homepage: www.elsevier.com/locate/sajb

Differential gene expression analysis of ‘Granny Smith’ apple (Malus domestica Borkh.) during fruit skin coloration☆ X.J. Zhang a, L.X. Wang a, Y.L. Liu a, X.X. Chen b, Y.Z. Yang a, Z.Y. Zhao a,c,⁎ a b c

College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, PR China College of Natural Resources and Environment, Northwest A&F University, Yangling, Shaanxi 712100, PR China Apple E&T Research Centre of Shaanxi Province, Yangling, Shaanxi 712100, PR China

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Article history: Received 25 March 2013 Received in revised form 26 June 2013 Accepted 7 July 2013 Available online 10 August 2013 Edited by AK Cowan Keywords: Apple Anthocyanin Quantitative real time-PCR ‘Granny Smith’ Gene expression

a b s t r a c t ‘Granny Smith’ apples growing under normal sunlight develop green skin, whereas the peel turns red due to anthocyanin accumulation after the removal of a bagging treatment. Two anthocyanins, Cyanidin 3-O-galactoside (cy3-gal) and Cyanidin 3-O-arabinoside (cy3-ara), were detected in the red ‘Granny Smith’ apple peels, and cy3-gal was determined to be chiefly responsible for the red color. The content of cy3-gal was more than 98% of the total anthocyanin in the red ‘Granny Smith’ peels. To better understand the molecular basis of anthocyanin biosynthesis in ‘Granny Smith’ apples, we performed a quantitative real-time PCR (qRT-PCR) analysis of anthocyanin biosynthetic genes (MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, and MdMYB1). Our results indicate that the expression of these genes (except MdCHS) was associated with increased anthocyanin accumulation in the skin of ‘Granny Smith’ apples. Four selected genes obtained from the ‘Granny Smith’ skin cDNA library, phytoene synthase (PSY), WD40 repeat protein, polygalacturonase (PG), and galactosidase (GAL), were also confirmed by qRT-PCR. We found that these genes were differently expressed during ‘Granny Smith’ apple skin coloration, suggesting that they are directly or indirectly involved in pigment accumulation. In conclusion, anthocyanin biosynthesis in ‘Granny Smith’ apples is the result of interactions between multiple enzymes in the anthocyanin biosynthesis pathway, and the coloring mechanism of ‘Granny Smith’ apples may be similar to that of red-skinned cultivars. © 2013 The Authors. Published by Elsevier B.V. on behalf of SAAB. All rights reserved.

1. Introduction Apple (Malus domestica Borkh.) is one of the most important fruit crops in temperate regions of the world. Apple color is an important factor when considering consumer appeal, with red-colored fruits being more popular (King and Cliff, 2002). Apple skin color is thought to be determined by the interaction of anthocyanin molecules, a class of flavonoids that is responsible for the red skin color (Lancaster and Dougall, 1992), with other compounds, such as chlorophyll and carotenoids. There are more anthocyanins in red-skinned apple cultivars than in non-red-skinned cultivars (Honda et al., 2002). In recent years, significant progress has been made in understanding the genetic regulation of anthocyanin biosynthesis in apples. The genes Abbreviations: CHS, chalcone synthase; F3H, flavanone 3-hydroxylase; DFR, Dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, flavonoid 3-Oglucosyltransferase; DAFB, days after full bloom; DABR, days after bag removal; qRT-PCR, quantitative real time-PCR; HPLC, high-performance liquid chromatography. ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: College of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, PR China. Tel./fax: +86 29 87082922. E-mail address: [email protected] (Z.Y. Zhao).

encoding anthocyanin biosynthesis enzymes have been isolated and studied (Honda et al., 2002; Kim et al., 2003). The transcriptional levels of these genes are positively correlated with anthocyanin accumulation (Honda et al., 2002; Ben-Yehudah et al., 2005), and the MYB-bHLHWD40 regulatory protein complex controls their expression (Koes et al., 2005; Allan et al., 2008). In apples, MYB transcription factors (MdMYB1, MdMYBA, and MdMYB10) and bHLH transcription factors (MdbHLH3 and MdbHLH33) have been isolated and characterized (Takos et al., 2006; Ban et al., 2007a; Espley et al., 2007). In addition to genetic factors, external factors, such as light, temperature, mineral nutrition, and orchard management practices, also affect apple anthocyanin biosynthesis, with light being the most important and essential factor (Saure, 1990), and the transcription of MdMYB1 and several anthocyanin biosynthetic genes is regulated by light (Takos et al., 2006; Ban et al., 2007a; Ju et al., 1997; Ubi et al., 2006). Recent studies have proven that bagging treatment is an effective practice to study the effect of light on fruit anthocyanin synthesis (Ju, 1998; Zhang et al., 2011; Wei et al., 2011). It is also a useful technique for studying anthocyanin biosynthesis and gene expression in apples (Ju, 1998; Takos et al., 2006; Ban et al., 2007a). ‘Granny Smith’ apples display a green background with a pink blush in exposed areas (Reay, 1999). We found that, during the fruit ripening phase, bagged fruits that are re-exposed to sunlight accumulate

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X.J. Zhang et al. / South African Journal of Botany 88 (2013) 125–131

significant levels of anthocyanin in the skin (Fig. 1). Previous research commonly used red-skinned apple cultivars as experimental materials to elucidate the molecular regulation of anthocyanin biosynthesis (Takos et al., 2006; Xu et al., 2012). However, informations regarding the genetic regulation of anthocyanin biosynthesis for non-redskinned cultivars are rare. The present study was aimed to understand the molecular mechanisms of color variability in red ‘Granny Smith’ apple skin, by comparison with the non-bagged fruits in terms of anthocyanin content and transcription of anthocyanin biosynthetic genes. In addition, in order to identify potential color-related genes of ‘Granny Smith’, a light-specific subtractive cDNA library was constructed using SSH technology (Diatchenko et al., 1996), which has been widely used to identify differentially expressed genes (Ban et al., 2007b; Han et al., 2011; Zhang et al., 2011; Zhou et al., 2012). Moreover, this cDNA library would provide a foundation for future cloning and functional analysis of genes related to ‘Granny Smith’ coloration.

into the pGEM-T-Easy Vector (Promega, USA) and then were transformed into Escherichia coli strain TOP10 (Tiangen Biotech (Beijing) Co., LTD) and screened on the LB solid medium plate containing IPTG + X-gal + Amp at 37 °C overnight. Subsequently, recombination rate was tested by blue– white screening.

2. Materials and methods

2.4. Anthocyanin extraction and measurement

2.1. Plant materials

For the high-performance liquid chromatography (HPLC) analysis, the anthocyanins were extracted from 1 g of finely ground plant material in 1 mL 1% (v/v) HCl-methanol for 24 h at 4 °C on a rotating wheel in the dark. The samples were clarified by centrifugation at 13,000 ×g for 15 min at 4 °C, and then 1.5 mL of supernatant was transferred to an autosampler vial. The analysis was conducted using an HPLC PDA (Waters, USA), which was equipped with a model 1525 binary solvent delivery system (Waters, USA), an on-line degasser and a 2707 autosampler (Waters, USA). The data acquisition and processing were performed with Breeze software. For all the samples, the anthocyanin separations were performed using a 250 × 4.6 mm i.d., 5 μm C18 column (Diamonsil, China). Solvent A was 10% (v/v) formic acid, and solvent B was methanol. The gradient of solvent B was as follows: 0 min, 17%; 1 min, 17%; 10 min, 35%; 20 min, 37%; and 25 min, 100%. The gradient was run at a flow rate of 1 mL · min−1 at a column temperature of 40 °C, and a 5 μL sample was injected. Absorbance was measured at 530 nm. The standards were Cyanidin 3-O-galactoside, Cyanidin 3-Oglucoside (Sigma, USA), Cyanidin 3-O-arabinoside, and Cyanidin 3-Orutinoside (Polyphenols Laboratories, Hanaveien). Anthocyanins were expressed as mg/100 g fresh wight (FW).

The fruits used in this experiment were obtained from the orchard of the Northwest A&F University apple experimental station in the Baishui, Shaanxi Province in 2010. For the bagging treatment, each fruit was bagged with two-layer paper bags (Hong Tai, China) on May 25th, approximately 30 days after full bloom (DAFB). The bags were 13 × 16 cm, and the inner layer was made of red paper with a wax coating, while the outside layer was taupe. The bags were removed at 165 DAFB. The fruits in the bagged group were collected at 0, 2, 4, 6, 8, 10, and 15 days after the bag removal (DABR). The control group fruits were grown under normal sunlight conditions, and they were collected at 165, 167, 169, 171, 173, 175, and 180 DAFB. At each point in time, 10 individual fruits were collected from different locations in the tree canopy. The peels, including 1 mm of cortical tissue, were carefully peeled off with a knife, frozen immediately in liquid nitrogen and stored at −80 °C until use. 2.2. Apple skin SSH library construction Total RNA from the skin tissues of ‘Granny Smith’ apple was isolated using a hot borate method (Yao et al., 2005). Isolation of mRNA was performed by Oligotex® mRNA Mini Kit (QIAGEN GmbH, Germany), and cDNA was synthesized by the SMART cDNA synthesis kit (Clontech, Palo Alto, CA, USA). The SSH library of the ‘Granny Smith’ fruit peel was constructed using the PCR-SelectTM Subtractive Hybridization kit (Clontech, Palo Alto, CA, USA) according to the manufacturer's instructions, with the cDNA of the peel exposed to sunlight for 0 day as the driver and the mixed peel cDNA (2, 4, 6, 8 and 10 DABR) as the tester. The second PCR products, after subtraction, were recycled and linked

2.3. Sequencing and BLAST analysis White colonies were isolated on selective media, Luria-Bertani ampicillin (LB-Amp) solid medium, and these clones were grown overnight in 100 μL LB-Amp medium at 37 °C, respectively. Two μL of bacterium culture for each colony was used to amplify the inserts with SP6 and T7 primers. The cDNA fragments were sequenced by Invitrogen Trading Co. (Shanghai). All the inserted sequences were queried for similarity in the NCBI database using the BLASTX sequence comparison software at the website (http://www.ncbi.nlm.nih.gov/BLAST).

2.5. Analysis of gene expression profile by qRT-PCR Isolation of total RNA from the pericarp of ‘Granny Smith’ apples and first-strand cDNA synthesis were performed as described above. The transcript levels of MdCHS, MdF3H, MdDFR, MdANS, MdUFGT, MdMYB1, and four selected genes were analyzed by qRT-PCR using SYBR green master mix (Takara, Japan) and an IQ5 real-time PCR cycler (Bio-Rad Laboratories, USA) in a reaction volume of 25 μL. All the gene-specific primers were designed using the Primer Premier 5.0 program (Supplementary

Fig. 1. The ‘Granny Smith’ apple. a. Control fruit (175 DAFB); b. bagged fruit (10 DABR).


Table 1). According to the manufacturer's instructions, the reaction mixtures were heated to 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s, 60 °C for 30 s and 72 °C for 30 s, followed by 72 °C for 7 min. A melting curve was generated for each sample at the end of each run to ensure the purity of the amplified products. The raw data were analyzed with Bio-Rad IQ5 software.

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domain class transcription factor, WD-repeat protein, C3HL domain class transcription factor, Ttg1-like protein, and MYB domain class transcription factor. The functional descriptions of the other three gene categories are listed in Supplementary Table 2. The remaining 43 ESTs had no match in GenBank, suggesting that they represent unique ‘Granny Smith’ genes or are from nonconserved regions of known genes. Putative functions of all ESTs were annotated based on the highest scoring matches (e-values lower than 10e− 4) by BLAST in NCBI.

2.6. Statistical analysis Statistical analysis was conducted with the SPSS 17.0. ANOVA was used for analysis of anthocyanin content and gene expression levels. The significance of the differences between means was estimated by Duncan test (P ≤ 0.05). Experimental data were the average of three replications ± s.e.

3.2. Changes of anthocyanin content during fruit coloration The anthocyanin composition and concentration of ‘Granny Smith’ apple peels were assessed by HPLC. The control group fruits have green skin during fruit maturation, and anthocyanins were barely detected. Two anthocyanins, cy3-gal and cy3-ara, were detected in the red ‘Granny Smith’ apple peels in our study (Fig. 2). Cy3-gal is chiefly responsible for the red color, ranging from 0 to 30.92 mg/100 g FW during ‘Granny Smith’ coloration (Fig. 3b). Cy3-ara was present in smaller amounts, ranging from 0 to 0.43 mg/100 g FW (Fig. 3a). The content of cy3-gal was greater than 98% of the total anthocyanin content of the red ‘Granny Smith’ peels. There was very little anthocyanin biosynthesis in the skin tissues of bagged fruits at 0 DABR, but it increased rapidly at 4 DABR, then accumulated progressively.

3. Results 3.1. Construction of the subtracted cDNA library A subtracted cDNA library was constructed to identify the molecular determinants of ‘Granny Smith’ apple skin color. A total of 1000 randomly chosen white transformants were selected, and more than 90% of the colonies produced a single band. The insert sizes ranged from 200 to 800 bp. One hundred and sixteen high-quality ESTs were obtained after removing the low-quality and repetitive sequences. Of these genes, 73 had high homology to genes of known function (Supplementary Table 2). The annotation of the genes obtained from the ‘Granny Smith’ apple skin SSH library was predicted by BLAST, and the genes were sorted into functional categories based on BLAST analysis. The largest category was metabolism-related genes, with a total of 36 ESTs. This group included genes with homology to phytoene synthase, cysteine protease inhibitor, isoflavone reductase, pyruvate decarboxylase, sarcosine oxidase, etc. The second largest category was stress/defense related genes, totaling 13 ESTs. Five transcription factors were also present in this cDNA library, including BZIP

3.3. qRT-PCR for anthocyanin biosynthetic genes qRT-PCR was used to measure the relative expression of anthocyanin related genes in ‘Granny Smith’ apples. In the bagged group, the transcript levels of all six genes were very low at 0 DABR, and they increased rapidly in the skin tissues exposed to sunlight (Fig. 4). MdF3H transcripts rapidly increased, reaching a maximum level at 2 DABR, then decreasing to a lower level at 10 DABR, and then increased again. MdCHS and MdDFR transcripts increased, reaching a maximum level of expression at 4 DABR, then gradually decreased. The transcripts of the downstream genes (MdANS and MdUFGT) in the anthocyanin pathway

1 0.060

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0.045 0.030 0.015

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0.045 0.030 0.015 0.000 4.40

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Time (Min) Fig. 2. HPLC separation of anthocyanins in peels of ‘Granny Smith’ apple. a. Bagged fruit (10 DABR); b. Control fruit (175 DAFB). Peak 1: Cyanidin 3-O-galactoside; Peak 2: Cyanidin 3-Oarabinoside.

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Fig. 3. Contents of anthocyanins in red ‘Granny Smith’ apple peel after bag removal. a. Cyanidin 3-O-arabinoside; b. Cyanidin 3-O-galactoside. Data are means ± s.e. of three replications. Different letters above the bars indicate significant (P b 0.05) differences according to the Duncan test.

and the regulation the MdMYB1 gene reached maximum expression levels at 6, 2, and 2 DABR, respectively (Fig. 4). In the control group, the expressions levels of all six genes (expect MdCHS) were significantly lower than those in the bagged group. Among these genes, MdCHS and MdUFGT transcripts peaked at 169 DAFB, whereas the others showed no major fluctuations during fruit coloration. 3.4. qRT-PCR for selected genes Four selected genes were detected using gene-specific primers. All of these genes were differentially expressed in ‘Granny Smith’ apple skin during fruit coloration (Fig. 5). In the bagged group, transcript levels of WD40 gene were up-regulated and reached the peak at 4 DABR, then gradually decreased, similar to those in the control group. Expression of PSY gene was up-regulated from 0 DABR, and reached a peak at 10 DABR in bagged fruits, however, it was down-regulated in the control group. The GAL gene transcript level rapidly increased, reaching a maximum level of expression at 4 DABR, then decreased in the bagged group. However, in the control group, GAL levels appeared to fluctuate, as the levels were high at 169 DAFB, decreased dramatically at 171 DAFB, and then increased steadily up to 180 DAFB. The transcript levels of the PG gene increased steadily in the control groups up to 169 DAFB, then rapidly decreased. In the bagged groups, PG expression followed a similar pattern to that of the GAL gene in the control groups (Fig. 5). 4. Discussion To study the bagging effect on anthocyanin accumulation in ‘Granny Smith’ apple skin, the anthocyanin content and the expression level of its regulatory genes were compared between bagged and non-bagged fruit. Previous research has shown that the HPLC chromatograms of red-skinned apple extracts revealed three anthocyanins (cy3-gal, cy3glu, and cy3-ara), and cy3-gal is the most abundant anthocyanin

present in red cultivars (Kondo et al., 2002; Liu et al., 2013). In the present work, only two anthocyanins, cy3-gal and cy3-ara, were detected in red ‘Granny Smith’ apple peels (Fig. 2), whereas anthocyanins were barely detected in non-bagged fruit. These results indicate that the bagged fruits had a much greater potential to accumulate anthocyanin than the control group. Correspondingly, the transcription of the six biosynthetic genes significantly increased in the bagged fruit. Moreover, the differences in four late biosynthetic genes were more obvious than the upstream gene, MdCHS, which had a similar expression level in both the bagged group and the control group (Fig. 4). Among these genes, MdUFGT is the most important gene for anthocyanin biosynthesis in apples (Ju et al., 1997). Our results showed that the transcript levels of MdUFGT in red skin, bagged ‘Granny Smith’ fruits were higher than those of the control fruits, and our previous study revealed that only UFGT activity significantly correlated with anthocyanin accumulation in ‘Granny Smith’ apples (Liu et al., 2013), suggesting that UFGT may be an important factor in anthocyanin biosynthesis in ‘Granny Smith’ apples. It is well known that anthocyanin biosynthetic genes were regulated by MYB-bHLH-WD40 protein complex, which was influenced by external environment factors (Koes et al., 2005; Allan et al., 2008), and light is known to be the most important external factor regulating anthocyanin synthesis (Lancaster and Dougall, 1992; Saure, 1990). In our research, the transcription of MdMYB1 and biosynthetic genes was low in darkgrown fruits but increased dramatically after bag removal (Fig. 4). Moreover, anthocyanin content and the transcript levels of MdMYB1 and four late genes in the anthocyanin pathway were dramatically elevated in the re-exposed fruits compared to the control group. Here, the transcription of MdMYB1 was positively correlated with the transcript levels of biosynthetic genes. Similar results were reported by Takos et al. (2006) and Xu et al. (2012). Taken together, our results indicate that anthocyanin accumulation in red peels of ‘Granny Smith’ apples is the result of interactions between multiple key enzymes in the anthocyanin pathway, similar to red-skinned cultivars. We also examined the genotype of ‘Granny Smith’ apples using two alleles markers (Cheng et al., 1996; Takos et al., 2006), and our results showed that ‘Granny Smith’ apple has the same genotype as some red-skinned cultivars (date not shown). This finding supported that green-skinned cultivars may contain the ‘red’ gene (Ju et al., 1997), and the expression of anthocyanin biosynthetic genes in greenskinned apples might be blocked in some way (Kim et al., 2003), which can be reversed by bagging treatment. Moreover, other researchers have suggested that there may be a delayed ripening response that prevents pigment synthesis and accumulation in green-skinned apples (Cheng et al., 1996). All of these indicated that anthocyanin accumulation in ‘Granny Smith’ apple is not an accidental phenomenon, and the coloring mechanism of ‘Granny Smith’ apples may be either similar or identical to that of red-skinned cultivars. To better understand the effect of bagging treatment on ‘Granny Smith’ apples anthocyanin biosynthesis, a subtracted cDNA library was constructed to identify the molecular determinants of ‘Granny Smith’ apple skin color. The transcription of four selected genes (PSY, WD40, PG, and GAL), which were involved in apple coloration or softening, was determined by qRT-PCR. The results showed that these selected genes were differentially expressed in ‘Granny Smith’ skin during fruit coloration (Fig. 5), suggesting that they are directly or indirectly involved in pigment accumulation. Carotenoid color ranges from yellow to red to orange and plays an important role in many fruits coloration (Bartley and Scolnik, 1995). PSY is a key regulatory gene in the carotenoid biosynthesis pathway; accordingly, this gene has been a potential target of genetic manipulation in plants to enhance carotenoid levels (Busch et al., 2002; Kato et al., 2004). PSY genes have been isolated and analyzed from numerous higher plants (Busch et al., 2002; Kato et al., 2004; Qin et al., 2011), but little is known about their functions and potentials to improve carotenoid levels in rosaceous plants, such as apple, pear, and peach. In this


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Fig. 4. Expression profiling of ‘Granny Smith’ anthocyanin biosynthesis genes during fruit color change. The genes amplified included: MdCHS (AB074485), MdF3H (AB074486), MdDFR (AF117268), MdANS (AB074487), MdUFGT (AF117267) and MdMYB1 (DQ886414). Each column height indicates the relative mRNA abundance, relative to 165 DAFB which was set to 1. All qRT-PCR reactions were normalized using the Ct value corresponding to MdActin (AB638619). a. Control group; b. Bagged group. Data are means ± s.e. of three replications. Different letters above the bars indicate significant (P b 0.05) differences according to the Duncan test.

study, PSY gene was up-regulated in bagged fruits (Fig. 5) during fruit coloration, suggesting that the PSY gene plays an important role in the regulation of apple skin color. Another color-related gene, WD40 repeat protein, combines with MYB and bHLH to regulate anthocyanin biosynthesis (Morita et al., 2006; Gonzalez et al., 2008). In latter reports, WD40 repeat protein was shown to be involved in anthocyanin biosynthesis regulation (Stommel et al., 2009; Matus et al., 2010; Zohar et al., 2011). The MdTTG1 gene encoding an apple WD40 repeat protein has been reported that it could regulate anthocyanin accumulation in transgenic Arabidopsis (Brueggemann et al., 2010). In our study, the transcription of WD40 gene was low in the dark-grown fruits, but increased dramatically after bag removal and was up-regulated in bagged fruits skins during fruit coloration (Fig. 5). The data suggest that this gene is responsive to light and involved in anthocyanin biosynthesis in apples. Fruit coloration and softening are two important indexes to evaluate fruit maturity, and fruit softening is associated with changes in the composition of the cell wall (Fischer and Bennett, 1991;

Barnavon et al., 2001). Recent work suggests that the changes in the cell wall result from the action of cell wall-modifying enzymes, such as polygalacturonase (PG) and galactosidase (GAL) (Tateishi et al., 2007; Liu et al., 2011; Zheng et al., 2012). In our study, the expression of PG and GAL during fruit ripening was increased during ‘Granny Smith’ fruit coloration, similar to previous studies (Wu et al., 1993; Ross, 1994; Mann et al., 2008). Moreover, the expression levels of PG and GAL were higher in fruit maturity prophase, and they were also higher in the bagged fruits than the control group (Fig. 5). 5. Conclusions ‘Granny Smith’ skin rapidly becomes red when the fruits are reexposed to sunlight after bag removal. Two anthocyanins, cy3-gal and cy3-ara, were detected in the red ‘Granny Smith’ apple peels, and cy3gal was chiefly responsible for red color. Expression analysis of anthocyanin biosynthetic genes (MdCHS, MdF3H, MdDFR, MdANS, and MdUFGT)

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Fig. 5. Expression profiling of four selected genes from the SSH library of ‘Granny Smith’. The genes amplified included: PSY (JK750344); GAL (JK973900); PG (JK750375) and WD40 (JK974070). Each column height indicates the relative mRNA abundance, relative to 165 DAFB which was set to 1. All qRT-PCR reactions were normalized using the Ct value corresponding to MdActin (AB638619). a. Control group; b. Bagged group. Data are means ± s.e. of three replications. Different letters above the bars indicate significant (P b 0.05) differences according to the Duncan test.

showed that all of the selected genes (except MdCHS) in the anthocyanin pathway were markedly elevated in the re-exposed fruits, and their expression was highly correlated with anthocyanin accumulation. MdMYB1 was found to regulate these structural genes in anthocyanin biosynthesis pathway. To gain a better understanding of the molecular regulation of anthocyanin biosynthesis in ‘Granny Smith’, we constructed a subtractive cDNA library and obtained 73 unigenes that are mainly involved in metabolism and disease/defense. The expression changes of four selected genes (PSY, WD40, PG, and GAL) were confirmed using qRT-PCR, and the results showed that these genes are directly or indirectly involved in ‘Granny Smith’ coloration during fruit maturation. Taken together, anthocyanin accumulation in ‘Granny Smith’ apples is the result of interactions between multiple key enzymes in the anthocyanin pathway, and the coloring mechanism of ‘Granny Smith’ may be similar or identical to that of red-skinned cultivars. Acknowledgments This research was supported by the earmarked fund for Modern Agro-industry Technology Research System, China (CARS-28). We are all extremely grateful to Dr. Zahoor Ahmad Bhat, who devoted much time and effort to revise the manuscript for us. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.sajb.2013.07.009.

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