پاورانگلیسی هلو

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abstract To understand better the regulatory mechanism of the carotenoid accumulation, the expression profile of relevant carotenoid genes and metabolites were compared between two peach cultivars with different colors during fruit development. Meanwhile, the change pattern of carotenoid content and expression of carotenoid metabolic genes in peaches after harvest in response to blue light were also investigated. As compared to the yellow fleshed-cultivar ‘Jinli’, lower carotenoid levels were observed in skin and pulp in white peach cultivar ‘Hujing’, which might be explained by differentially expression of PpCCD4 gene. With respect to ‘Jinli’, the carotenoid accumulation during fruit velopment in fruit skin was partially linked with the transcriptional egulation of PpFPPS, PpGGPS, PpLCYB and PpCHYB. However, in the lp, the accumulation might be also associated with the increased ranscriptions of PpPDS, along with the above four genes. Blue light treatment induced carotenoid accumulation in ‘Jinli’ peaches during storage. In addition, the treated-fruit displayed higher expression of all the eight genes analysed with a lesser extent on PpCCD4, which suggested that the much more increased carotenoid synthesis rate could result in the higher 

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t is well documented that the content and composition of carotenoid is developmentally regulated and affected by environmental stimuli (Cazzonelli & Pogson, 2010). Light has been reported to be an important environmental factor which can regulate carotenoid metabolism in plants (Wu et al., 2007; Zhang et al., 2012, 2015). In tomato irradiated with red light, the accumulation of lycopene, as well as an increase in total carotenoid content, was observed (Alba, Cordonnier-Pratt, & Pratt, 2000; Schofield & Paliyath, 2005). Blue light treatment induced carotenoid accumulation effectively in the juice sacs of Satsuma mandarin and Valencia orange (Zhang et al., 2012, 2015). The flesh color of yellow-fleshed peach fruit (Prunus persica L. Batsch) is produced by a group of carotenoids, which is an 

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Recently, it has been reported that the carotenoid cleavage dioxygenase (PpCCD4) was the major factor in determining carotenoid degradation in white peaches but no correlation was observed between carotenoid accumulation and the expression levels of carotenoid biosynthetic genes (Adami et al., 2013; Brandi et al., 2011). Despite recent efforts to understand the molecular biology of carotenogenesis takes place in peaches, several gaps remain in our understanding of the signals and mechanisms involved in carotenoid metabolism. To investigate further how carotenoid accumulation in peach fruit, in this study, firstly the concentration and composition of carotenoids and the expression of several carotenoid biosynthetic genes as well as PpCCD4 in fruit peel and pulp were 

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2. Materials and methods 2.1. Plant materials, sample collection, and blue light treatment Two peach cultivars (Prunus persica), ‘Hujing’ and ‘Jinli’, were obtained from the experimental farm of Fenghua Peach Fruit Research Institute (Ningbo, China). Trees were subjected to standard horticultural practices. Fruit ripening stages were defined according to Tonutti, Bonghi, Ruperti, Tornielli, and Ramina (1997) and Gabotti, Negrini, Morgutti, Nocito, and Cocucci (2015). At the desired times, fruit (ten fruit per each of the five plants of each cultivar considered per each ripening stage) of ‘Hujing’ and ‘Jinli’ were picked and quickly transferred to the laboratory, and fruit tissues at 98 (S1), 105 (S2), 120 (S3), 127 (S4), and 134 (S5) DAFB were sampled during the spring-summer season of 2015 (Fig. $2). For blue light treatment, fruit of each cultivar at commercial maturity stage were selected for uniform size and color, and then divided into two groups randomly. The blue light treatment 

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2.2. Extraction and HPLC analysis of carotenoids Extraction and purification of carotenoids in tissue samples (2 g) were performed according to a previously described method (Tuan et al., 2013; Wright & Kader, 1997). To determine the carotenoids content in each sample, the HPLC analyses were carried out as previously described by Taylor and Ramsay 520 2.3. Total RNA extraction and cDNA synthesis Total RNA was isolated using a Plant Total RNA Extraction Kit (Genotheramics, Suzhou, China) according to the manufacturer’s instructions. Extracted RNA was treated with RNase-free DNase (Omega, Norcross, GA) to remove any genomic DNA according to the instruction manual. An aliquot (2 

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2.4. Quantitative real-time PCR (q-PCR) analysis Q-PCR analysis was performed using the Mx3000P q-PCR System (Agilent Stratagene, Santa Clara, CA, USA) and the DyNAmoTM ColorFlash SYBR Green qPCR kit (Thermo Scientific, Pittsburgh, PA) following the manufacturer’s instructions. Amplifications were performed using a total volume of 12.5 IL reaction containing 0.5 IL of the synthesized cDNA, 0.25 IL of 101M each forward and reverse primers, 6.5 IL _of the SYBR Green PCR Master Mix and 5 IL of RNase-free water. The data were analysed and normalized to PpTEF2 to minimize variation in cDNA template levels. All gene expression analyses were performed with three independent biological replicates, and primer sequences used for real time PCR are listed in Supplementary Table $1.2.5. Data processing and statistical analysis The contents of carotenoids and transcript abundance of carotenoidmetabolic genes of two peach cultivars were displayed with GraphPad Prism (v.5.01). All values are 

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ال لك 3.1. Carotenoid content and composition analysis in fruit peel of two peach cultivars during fruit development The total carotenoid content in the peel of the yellow fleshedpeaches (Jinli) and white cultivar (Hujing) at five developmental stages was analysed. At early ripening stage S1, peel of ‘Jinli’ and ‘Hujing’ had similar total carotenoid levels and accumulated only a few carotenoid compounds at about 0.04 lg g1 fresh weight (FW) in both cultivars. However, a significant increase in the carotenoid content of ‘Jinli’ was observed from the S2 stage, while carotenoid content in ‘Hujing’ remained low. At the S5 stage, the total carotenoid content in peach peels rose to 1.3 lg g1 FW in ‘Jinli’ but only 0.10 lg g1 FW ‘Hujing’ (Fig. 1). HPLC measurements showed that lutein, zeaxanthin, b-carotene and b-cryptoxanthin were predominant in both two cultivars during ripening. All of these carotenoids progressively increased during fruit development in ‘Jinli’ but nearly remained constant in ‘Hujing’ during the whole process of fruit development (Fig. 1) 

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increasing transcript level during fruit development in peels of ‘Jinli’ fruit, which was consistent with the accumulation of the total carotenoids. More FFPS transcripts were detected in peels of ‘Jinli’ as compared to ‘Hujing’. However, ‘Hujing’ showed more transcripts of PpPSY, PpPDS, PpZDS and PpCCD4 during fruit development with a few exceptions (Fig. 2). From stage S1 to S2, the expression of PpGGPS was lower in the peel of ‘Jinli’ than in ‘Hujing’, but the gene expression level was approximately twice as great in ‘Jinli’ as in ‘Hujing’ thereafter. The expression levels of PpLCYB and PpCHYB increased with fruit development in the peel of ‘Hujing’ and peaked at stage 

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3.3. Carotenoid content and composition analysis in pulp of two peach cultivars during fruit development The changes and profiles in pulp of ‘Jinli’ fruit were similar to those in peels. The total carotenoid accumulated with ripening principally due to the increased lutein, zeaxanthin, b-carotene and _ b-cryptoxanthin during ripening. In the pulp of ‘Jinli’, the total carotenoid content was usually higher than in the peel. However, almost no carotenoids were detected in the pulp of the white cultivar ‘Hujing’ (Fig. 3). 3.4. Expression pattern of carotenoid metabolic genes in pulp of two peach cultivars during fruit development In pulp of ‘Jinli’, expression of most carotenogenic genes increased gradually from stage S1 to reach a maximum at stage S4. Abundance of PpFPPS and PpCHYB was higher in pulp of ‘Jinli’ than in ‘Hujing’ tissue during fruit development but higher transcripts of PpPSY, PpZDS and PpCCD4 were observed in ‘Hujing’ in this process with an exception of PpPSY expression at stage S2. In 

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3.5. Effect of blue light treatment on carotenoid content and Composition In the present study, the effect of blue light treatment on the accumulation of the main carotenoids in peach fruit of ‘Jinli’ and ‘Hujing’ after harvest was also investigated. During the whole 50۲296, the total carotenoid content and b-cryptoxanthin level in ‘Jinli’ increased with storage time. In respect to zeaxanthin and b-carotene in ‘Jinli’, their levels were increased firstly and peaked at day 15 and day 10, respectively, followed by a decline 9۹0۹ ‏ا‎ Lutein content in ‘Jinli’ decreased during the first 5 days of storage 

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and increased during the remaining days. The contents of all these four carotenoids could be induced by blue light treatment and as a result, the total carotenoid content was higher in the light treated-‘Jinli’ fruit than in control fruit (Fig. 5). No significant changes in levels of total carotenoid as well as each carotenoid such as lutein, zeaxanthin and b-cryptoxanthin were observed in ‘Hujing’ fruit during storage with a slight decrease in b-carotene content. Blue light did not have any influence on carotenoid changes and profiles in this cultivar (Fig. 5). 3.6. Effect of blue light treatment on gene expression related to carotenoid metabolism Most of genes including PpPSY, PpPDS, PpZDS and PpCHYB expressed higher in ‘Jinli’ than in ‘Hujing’ during storage. However, the higher carotenoid degradative PoCCD4 gene was detected 

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expression of PpFPPS and inhibited the decline of PpGGPS expression. Consequently, higher transcripts of these two genes were detected in the blue light treated-peaches regardless of the cultivars. The other six genes such as PpPSY, PpPDS, PpZDS, PpLCYB, PpCHYB and PpCCD4 in both cultivars showed a similar change pattern during storage. The expression in peaches increased duringthe first 5 or 10 days and decreased thereafter with exceptions ofPpZDS and PpLCYB in ‘Hujing’. The transcripts of all these genes were more abundant in light treated-fruit than that in control peaches irrespective of the cultivars (Fig. 6). . Discussion In the white cultivar ‘Hujing’ in present study, less or no carotenoids were observed in the skin or flesh, however, the transcripts for all carotenoid biosynthetic genes investigated in this study were detected, which indicated that the transcriptional regulation of these genes was not the major reason for the low level of carotenoids in the white peaches. It is well documented that the biosynthetic or degradative enzyme involved in carotenoid metabolism can regulate carotenoid accumulation (Nisar et al., 2015).In peach fruit, PpCCD4 

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Similarly, higher expression of CmCCD4 was related to the much less carotenoid accumulation in white chrysanthemum petals (Ohmiya, Kishimoto, Aida, Yoshioka, & Sumitomo, 2006). Our results confirmed that the levelof PpCCD4 transcripts in white fleshed-cultivar ‘Hujing’ was higher than in ‘Jinli’, the yellow one regardless of fruit skin and flesh 2 suggested that the biosynthetic carotenoids were gradually degraded by the highly active PpCCD4 during fruit development, further resulting into almost undetectable carotenoids levels in the white fleshed-peaches. Together with the previous reports, our results here demonstrated that the differential expression of PpCCD4 plays a determining role in the carotenoid content in white 

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in watermelon (Ly, Li, Liu, Gu, & Zhao, 2015). In addition, it must be pointed that higher transcripts of PpPDS were also observed from the early fruit stage (S2) to ripening (S5) in the pulp of yellow peaches in comparison with ‘Hujing’. In citrus, the expression of PDS gene was revealed to link with b-carotene accumulation directly (Ha, Kim, Park, Lee, & Cho, 2007). High concentration of carotenoids was related to the high PDS transcripts as well in pepper (Ha et al., 2007). These results ombined to suggest that the increased of PpPDS in the pulp in ‘Jinli’ might lead to the high level of carotenoids in this cultivar to some extent. Cyclisation of lycopene is a critical regulatory point of branching in carotenoid synthesis (Fraser & Bramley, 2004; Taylor & Ramsay, 2005). LCYB and CHYB play important roles in regulating the biosynthesis of b-carotene, lutein, zeaxanthin and 

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kiwifruit, (Ampomah-Dwamena et al., 2012). In squash fruit, high transcript level of CHYB was observed in the high carotenoid cultivars in comparison with the low carotenoid ones (Nakkanong,Wang, & Zhang, 2012). In this study, PpLCYB expression in ‘Jinlipulp and peel was more strongly expressed than that in the white cultivar at the late development stages, possibly contributing to b- carotene accumulation in ‘Jinli’. Meanwhile, the higher expression evels of PpCHYB in flesh and skin of ‘Jinli’ compared with‘Hujing’ might partially explain the higher levels of lutein, zeaxanthin and -cryptoxanthin. In contrast, although the transcripts of PpCCD4 showed an increasing trend during ‘Jinli’ ripening, its expression level was much lower than in ‘Hujing’. Therefore, the lower rate of carotenoid degradation with CCD4 gene may be another factor for the increased carotenoid accumulation in 

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complete parallel, with the expression pattern of certain genes such as FPPS and GGPS in the methylerythritol phosphate pathway and PpLCYB and PpCHYB in the carotenoid biosynthetic pathway during fruit development. However, in terms of the flesh in yellow fleshed-fruit, in addition to the higher transcripts of the ‏تنام عتتمطق‎ genes, the higher expression of PpPDS and lower transcripts of PpCCD4 genes, as compared to the white cultivar, also coincided with the higher level of carotenoids, suggesting that both of the two genes might be involved in carotenoid accumulation in pulp of yellow fleshed-fruit during maturation. It is noteworthy that total carotenoids in the flesh in yellow peaches showed higher levels compared with those in the peel during development, althouch the qene exnrescion pnrofile in the flesh was similar to 

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During storage, carotenoid accumulated in yellow fleshedpeaches, however, no significant change was observed in the white cultivar. Similarly to the white fleshed-peaches during maturation, in the white peaches during storage, the discrepancy between carotenoid level and expression profile of carotenoid biosynthetic genes indicated that high PpCCD4 gene expression during storage might be also concomitant with the low level of carotenoids in ‘this accession. However, the pattern of changes in gene expression in yellow fleshed-peaches during storage was different from that in fruit during development. As compared with the white cultivar, besides the higher transcription of PpCHYB and lower expression of PpCCD4 gene, the yellow fruit also experienced higher transcripts of PpPSY and PpZDS, two important genes for the linear carotenoid formation, which was also probably responsible for fs PO BRR Se ES SA ‏تن اه‎ roa! Es angina! ‏و رم سا رت‎ i Bek as pe ‏اه 1۳۷۲۵ با وا هت‎ 

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Blue light irradiationenhanced the expression levels of genes involved in the carotenoid biosynthesis in sprouts of Tartary buckwheat (Tuan et al., 2013). Blue light treatment could also induce carotenoid accumulation via regulating the expression of carotenoid biosynthetic genes in the juice sacs of Satsuma mandarin and Valencia orange (Zhang et al., 2012, 2015). In yellow-fleshed peaches, blue light treatment during storage not only induced carotenoid level but also brought about important changes in the gene expression involved in carotenoid metabolism. Blue light up-regulated the expression of all the genes analysed with a lesser extent on PpCCD4, which indicated that as compared to the control fruit, the much more increased carotenoid synthesis rate could result into the higher carotenoid content in blue light-treated fruit. With respect to white peaches, it is noticeable that the expression of all the genes analysed was stimulated by blue light as well, however, no significant difference on carotenoid composition and content was 

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In conclusion, accumulation of carotenoids was observed in peel and flesh of yellow fleshed-peaches during fruit maturation. The expression of PpFPPS, PpGPPS, PpLCYB and PpCHYB might be contributed to the carotenoid accumulation in the peel of this accession during fruit maturation. Besides these four genes, PpPDS might also play an important role in carotenoid biosynthesis in the pulp. However, in the white cultivar, the transcript of PpCCD4 was likely to be the major determinant in carotenoid content. Our results also showed blue light was effective in inducing the carotenoid accumulation in yellow fleshed-peaches after harvest. Results on expression profiles of genes in carotenoid metabolism indicated that the much more increased carotenoid synthesis rate induced by blue light could result into the higher carotenoid content in treated fruit. 

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Acknowledgments This study was supported by the National Natural Science Foundation of China (31371866 and 31571905), the Natural Science Foundation of Zhejiang Province (LQ15C200004) and Natural Science Foundation of Ningbo (2015A610262). 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.foodchem.2016. 07.085. 

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