Different light-response patterns of coloration and related gene expression in red pears (Pyrus L.)

Scientia Horticulturae 229 (2018) 240–251

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Research paper

Different light-response patterns of coloration and related gene expression in red pears (Pyrus L.)

MARK

Yang-fan Zhua,1, Jun Sub,1, Gai-fang Yaoa, Hai-nan Liua, Chao Gua, Meng-fan Qina, Bing Baia, ⁎ Shao-shuai Caia, Guo-ming Wanga, Run-ze Wanga, Qun Shub, Jun Wua, a Centre of Pear Engineering Technology Research, State Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China b Institute of Horticulture, Yunnan Academy of Agricultural Sciences, Kunming 650205, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Red pear Coloration Bagging Anthocyanin Light-response patterns

The coloration of fruit peel in red pear is influenced by light. However, the various responses to light among different varieties are still unstudied. In this study, the different light-response patterns of 27 pear cultivars under artificial light conditions were observed. The results revealed three types of coloration patterns: type-I cultivars showed no coloration; type-II cultivars showed significant coloration; and type-III cultivars showed faint coloration. Cultivars ‘Mantianhong’, ‘Fojianxi’, ‘Yunhongli 1′, and ‘Starkrimson’ were chosen as representative for further analysis of anthocyanin content and components as well as gene expression related to color. Four anthocyanin components were detected in different cultivars when exposed to artificial light and natural light, and the difference in anthocyanin content led to different colored phenotypes, with a similar response under different light conditions. The expression patterns of genes encoding enzymes indicated that ANS and UFGT were decisive genes involved in anthocyanin biosynthesis for red-skinned pears, and the varying expression of these genes led to coloration differences observed between light-response patterns. The expression patterns of transcription factors indicated that the MYB10, bHLH33, and WD40 genes are involved in differential regulatory mechanisms of anthocyanin biosynthesis and coloration patterns between light-response patterns. The expression of light-responsive genes indicated that the expressions of HY5, PhyA, COP1, DET1, and PIF3 genes are involved in the differential regulatory mechanisms of anthocyanin biosynthesis and coloration patterns between light-response patterns. The results provide an important reference for studying the molecular mechanism of light response and coloration in red pear and lay the foundation for future studies on red pear coloration research.

1. Introduction Pear is a major fruit crop in temperate regions of the world. China ranks as the leader in pear production, accounting for more than 60% of the total world output. Five kinds of commercial pear species are grown in China (Teng et al., 2004): Chinese sand pear (P. pyrifolia), Chinese white pear (P. bretschneideri), Xinjiang pear (P. sinkiangensis), Qiuzi pear (P. ussuriensis), and European pear (P. communis). Traditional Chinese cultivars have yellow, green, or russet-brown skin, with only a few redskinned cultivars (Wang et al., 1997). Red coloration varies between cultivars and species, as Asian pears mostly color at a late stage of fruit development, and are greatly affected by light and temperature. Conversely, some European pears color at the very beginning of fruit development, showing little effects from the environment. The red

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coloration of pear is the result of anthocyanin accumulation, and redskinned pears are increasingly popular in the market for their attractive appearance and the health-promoting benefits of anthocyanin. Post-harvest fruit bagging can significantly improve the coloration in various kinds of fruit, such as in peach (Jia et al., 2005), apple (Wang et al., 2015), grape (Signes et al., 2007), and pear (Huang et al., 2009). However, the coloration pattern of bagged fruit is different between red pear cultivars. Previous studies have proven that the concentration of anthocyanin accumulates rapidly under natural light within 10 days after removing bags in ‘Yunhongli No. 1′ and ‘Meirensu’, while the ‘Yunhongli No. 1′ has a higher potential anthocyanin biosynthesis (Huang et al., 2009). In addition, another study reported that ‘Mantianhong’ exhibited good red coloration in bagging treatments under postharvest artificial light, but no anthocyanin was detected in

Corresponding author. E-mail address: [email protected] (J. Wu). These authors have contributed equally to this work.

https://doi.org/10.1016/j.scienta.2017.11.002 Received 2 May 2017; Received in revised form 19 October 2017; Accepted 1 November 2017 0304-4238/ © 2017 Elsevier B.V. All rights reserved.

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‘Cascade’ (Qian et al., 2013), indicating that pear cultivar coloration varies in response to light exposure. However, little research has been conducted to systematically analyze coloration patterns in response to the light under artificial and natural conditions. Anthocyanins are water-soluble phenolic compounds in higher plants that are responsible for purple, blue, and red colors (Castellarin et al., 2007; Mano et al., 2007; Yamagishi et al., 2010; Zifkin et al., 2012). Anthocyanin accumulation is controlled by enzyme genes and transcription factors (Holton and Cornish, 1995), including seven enzyme genes: phenylalanine ammonia lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), dihydroflavonol 4-reductase(DFR), anthocyanidin synthase/leucoanthocyanidin dioxygenase (ANS/LDOX), and UDP-glucose: flavonoid 3-Oglucosyltransferase (UFGT). However, DFR, ANS, and UFGT also act downstream of the anthocyanin pathway and are closely related to anthocyanin biosynthesis. In addition, the transcription factors MYB, bHLH, and WD40 have been reported to regulate anthocyanin biosynthesis (Allan et al., 2008). On the other hand, some light-responsive genes play important roles in photomorphogenesis and anthocyanin biosynthesis. For example, light-activated phytochrome A and B promote photomorphogenesis in Arabidopsis (Sheerin et al., 2015). In addition, cry1 and GPA1 signaling genetically interact in hook opening and anthocyanin biosynthesis in Arabidopsis (Fox et al., 2012). Moreover, HY5 is a transcription factor of the bZIP family that combines directly with a light-induced gene promoter; HY5 abundance directly correlates with the degree of photomorphogenesis (Osterlund et al., 2000). COP1 binds to photomorphogenic transcription factors and is degraded by the 26S proteasome in the dark; blue light, red light, and far-red light negatively regulate COP1 (Sanchez-Barcelo et al., 2016). In addition, overexpression of MycDET1 enhances the COP1 phenotype to regulate photomorphogenesis (Ly et al., 2015), and PIF3 binds to the Gbox of the light-regulated gene promoters; the short hypocotyls and the open cotyledon phenotype of the PIF mutant confirmed that the PIF protein acts as a repressor for global light morphogenesis (Smirnova et al., 2011). However, there are few studies to reveal the molecular mechanism of anthocyanin biosynthesis and accumulation for red pear cultivars with different light-response patterns. In order to uncover the different light-response patterns of red pear cultivars, we investigated the coloration of 27 pear samples under the same artificial light conditions, which divided the light response pattern of red pears into three types. Moreover, gene expression levels in three types of red pear helped to identify key genes that control the development of coloration. The results will lay a foundation for further molecular mechanism study of differential coloration of pear and provide useful techniques for improving the coloration of red pears to meet the demands of consumers.

Table 1 Pear cultivars used for coloration study under artificial light. Cultivars

Code

Region

Species

Fojianxi Youhongxiao Hongzhimuyang

1 2 3

Xingcheng

P. bretschneideri Rehd

Yunhongli 1 Mantianhong Meirensu

4 5 6

Kunming

P. pyrifolia Burm Nakai

Hongxiangsu

7

Zhengzhou

P. pyrifolia Burm Nakai

Miduxiaohongli Hongyun 2

8 9

Wuhan

P. pyrifolia Burm Nakai

Mantianhong Hongsucui

10 11

Nanjing

P. pyrifolia Burm Nakai

Mantianhong Honghuoba Meirensu

12 13 14

Xingcheng

P. pyrifolia Burm Nakai

Hongxiansu

15

Beijing

P. pyrifolia Burm Nakai

Nanhong Nanguo

16 17

Yingkou

P. ussuriensis Msxim

Guanhongxiao Hongbalixiang Shanli24

18 19 20

Xingcheng

P. ussuriensis Msxim

Starkrimson Dr. Jules Guyot Clapp’s Liebling Clapp’s Favorite

21 22 23 24

Beijing

P. communis Linn

Starkrimson Clapp’s Favorite Damaliesi

25 26 27

Xingcheng

P. communis Linn

Table 2 The artificial light conditions in this study. Lamp type

Light intensity

Temperature

Daylight lamp UV-B lamp

4300 lx 50 lx

17 °C

Table 3 The natural light conditions in this study. Light components

Light intensity

Temperature

UV-A UV-B Visible light

More than 100000 lx at ground level on a sunny day, 50—500 lx on a cloudy day

15 ∼ 35 °C

2. Methods and methods 2.2. Fruit color measurement 2.1. Plant materials and experimental treatments Fruit skin color was measured using a colorimeter (CR-400, Minolta, Japan) on the eight colored parts of the fruit, which represented overall fruit color; the meter provided CIE L*, a*, and b* values. L* represents the relative lightness of color in the range of 0–100, being low for dark color and high for light color. Both a* and b* scales extended from −60 to 60. Negative a* values indicate greenness, whereas positive values indicate redness; b* is negative for blueness and positive for yellowness. These values were then used to calculate the hue angle degree (h° = arctangent[b*/a*]), where 0° = red-purple, 90° = yellow, 180° = bluish green and 270° = blue (McGuire, 1992). Two fruits were used for each measurement.

To explore the different coloration patterns of bagging fruit under postharvest artificial light conditions, 27 red pear samples were chosen from 7 regions in China (Table 1). Sixty fruits of uniform size and growing positions were selected for bagging treatment with doublelayered paper bags (the outer layer is yellow outside and black inside, and the inner layer is red) for 40 days after full bloom, and 40 wellexposed fruits were used for the control group. The fruit were harvested 10 days before commercial maturity and then immediately transported to the laboratory. The fruits of uniform size were selected as experimental material. There were 3 fruits in every treatment. After 0, 3, 6, and 9 days of exposure, fruit of each treatment (Table 2) per period were taken, peeled, and stored in a −80 °C refrigerator. All treatment fruits were placed in an illumination incubator with optimum light and temperature,24 h of exposure per day (Sun et al., 2014); the conditions of artificial light and natural light are listed in Table 2 and Table 3.

2.3. Determination of the anthocyanin content Anthocyanin measurements were performed according to the 241

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Table 4 Correlation analysis of anthocyanin content and genes related to anthocyanin. Anthocyanin

DFR

ANS

UFGT

MYB10

bHLH33

WD40

HY5

PhyA

COP1

DET1

PIF3

Mantianhong Fojianxi Yunhongli 1 Starkrimson

0.158 0.386 0.756** 0.597*

0.162 0.776** 0.848** 0.542

−0.059 0.775** 0.886** 0.624*

−0.043 0.758** 0.773** −0.287

−0.602* 0.424 −0.113 0.394

−0.614* 0.775** 0.776** 0.189

−0.548 0.680* 0.674* 0.123

0.134 0.330 0.761** −0.588*

0.371 −0.022 0.308 −0.002

−0.028 −0.439 −0.325 0.076

0.053 0.068 0.631* 0.483

All data are means based on three replicates. Levels of significance: * = p ≤ 0.05; ** = p ≤ 0.01.

Fig. 1. The coloration of 27 red pear samples during artificial light illumination. A: Bagged fruit, B: Debagged fruit, C: Non-bagged fruit. 0: 0 days of exposure, 9: 9 days of exposure. The corresponding varieties for numbers 1–27 are shown in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

performed at 25 °C (laboratory temperature). Two microliters were used as injection volume, using partial loop mode for sample injections.

method described by (Huang et al., 2009), with modifications. Briefly, approximately 0.5 g of sample was ground to fine powder in liquid nitrogen and extracted with 5 ml of cold extraction solution (0.1% HCl in methanol) at 4 °C for 24 h. Samples were then centrifuged at 4 °C and 12000 rpm for 20 min, after which the supernatant was transferred to a clean tube. Absorbance was measured using a spectrophotometer (UV1800, MAPADA) at 510 nm.

2.5. qRT-PCR analysis of anthocyanins and genes related to the light response The primers used to amplify anthocyanins and genes related to the light response are listed in Table 4. Total RNA was extracted from fruit peels of three representative coloration patterns using a Plant Total RNA Isolation Kit Plus (Fuji, Chengdu). The concentration of RNA was measured using a NanoDrop 2000 spectrophotometer (NanoDrop 339 Technologies, Wilmington, DE, USA). Equal amounts of total RNA from all of samples used for cDNA synthesis were collected using the PrimeScript™ RT Reagent Kit (Perfect Real Time; TaKaRa). Then, 20 μl of system solution, including 10 μl of LightCycler 480 SYBR GREEN I Master (Roche, USA), 0.5 μl of diluted cDNA, 0.4 μl of each primer, and 8.7 μl of nuclease-free water, was used for qRT-PCR (TaKaRa SYBR PrimeScript RT-PCR Kit for Perfect Real Time). The reaction conditions of qRT-PCR were as follows: 45 cycles of 94 °C for 3 s, 60 °C for 30 s, and 72 °C for 10 s. The average threshold cycle was calculated per

2.4. Detecting anthocyanin content by UPLC UPLC analyses were applied according to the method described by (Novakova et al., 2006) with modifications using Waters Acquity Ultra Performance Liquid Chromatographic system (Waters, Prague, Czech Republic) with a PDA detector, cooling autosampler and column oven enabling temperature control of the analytical column. The data were collected and processed using the chromatographic software Empower. A special analytical column was connected to this UPLC system. Second-generation X-Terra sorbent loaded into an Acquity UPLC BEH C18 (2.1 mm × 50 mm, 1.7 μm) was used as a stationary phase. UPLC analyses utilized flow rates of 0.4 ml*min−1. All analyses were 242

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Fig. 2. The hue angle of bagged fruits of 27 red pear samples under artificial light treatment. C: control; T: artificial light treatment. The corresponding varieties for numbers 1–27 are shown in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

sample, Pyrus tubulin served as the internal control, and relative expression levels were calculated with the 2−ΔΔCt method described by Livak (Livak and Schmittgen, 2001).

3. Results

2.6. Statistical analysis

Nine days after bag removal, fruits exhibited different degrees of coloration change under artificial light conditions (Fig. 1). Based on the phenotype of fruit coloration, the 27 red pear cultivars were classified into three light-response patterns: type I, in which there was no obvious coloration (12 pear cultivars: ‘Clapps Liebling’, ‘Hongxiangsu’, ‘Nanhongli’, ‘Mantianhong’, ‘Meirensu’, ‘Damaliesi’, ‘Hongbalixiang’, ‘Honghuoba’, ‘Hongzhimuyang’, ‘Mantianhong’, ‘Youhongxiao’ and

3.1. Varying responses of red pears under postharvest artificial light conditions after bag removal

The data were analyzed using SPSS 18.0 (SPSS, Chicago, IL, USA) for ANOVA and the least significant difference test to compare means at the 95% confidence level.

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Fig. 3. The heat map of anthocyanin content of bagged fruits of 27 red pear samples under artificial light treatment. C: Control fruits; T: artificial light treatment. 0: 0 days of exposure, 9: 9 days of exposure. The corresponding varieties for numbers 1–27 are shown in Table 1. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

‘Shanli24′); type II, in which there was obvious coloration (‘Fojianxi’ and ‘Yunhongli 1′); and type III, in which the fruit coloration fades slightly, (‘Starkrimson’ from both Xingcheng and Beijing). In addition, some red pear samples exhibited sunburnt phenomena such that the fruit surface turned black under artificial light, which included the cultivars ‘Nanguoli’, ‘Hongsucui’, ‘Mantianhong’, ‘Hongxiangsu’ (collected in Zhengzhou), ‘Yunhong 2′ and ‘Miduxiaohongli’. It is possible that fruits suffer damage from sudden intense light after bag removal that results in partial burns and black coloration. Based on the hue angle value of the color difference, there were significant differences on the 9th day of exposure to light conditions in ‘Fojianxi’, ‘Yunhongli 1′, ‘Mantianhong’, ‘Meirensu’, and ‘Starkrimson’ cultivars (Fig. 2). On the other hand, anthocyanin content increased significantly after 9 days of light treatment in ‘Fojianxi’ and ‘Yunhongli 1′, while anthocyanin content decreased in ‘Starkrimson’ (Fig. 3).

Fig. 4. The fruit coloration of each light-response pattern under artificial light and natural light during illumination. A: Bagged fruit, B: Debagged fruit, C: Non-bagged fruit. 0: 0 days of exposure; 3: 3 days of exposure, etc.

light conditions, but the coloration turned red when exposed to natural light for a long exposure. With increasing time after debagging, the hue angle values of color difference were significantly reduced in fruits whose color turned red, such as ‘Fojianxi’ and ‘Yunhongli 1′ in artificial light, and all of these decreased in natural light (Fig. 5). Fruit under natural light conditions showed lower hue angle values than those under artificial light, indicating higher anthocyanin content under natural light conditions and fruit that look more red. Bagging fruits induced anthocyanin accumulation after bag removal and exposure to light. The anthocyanin content in ‘Fojianxi’ and ‘Yunhongli 1′ clearly increased after debagging in artificial light, but in natural light not only ‘Fojianxi’ and ‘Yunhongli 1′ but also ‘Mantianhong’ and ‘Starkrimson’ showed increased anthocyanin. The speed of anthocyanin accumulation under natural light conditions was faster than that under artificial light (Fig. 6).

3.2. Light-response patterns of red pears under postharvest artificial light and natural light conditions To explore the coloration characteristics of pear fruits from different species, we chose representative cultivars of each light-response type, including ‘Mantianhong’ collected from Kunming as type I, ‘Fojianxi’ and ‘Yunhongli 1′ as type II, and ‘Starkrimson’ (P. communis) from Xingcheng as type III. The coloration characteristics of the light-response types are different under postharvest artificial light and natural light conditions (Fig. 4). The skins of ‘Mantianhong’, ‘Fojianxi’, and ‘Yunhongli 1′ were yellow before treatment, whereas the peel of ‘Starkrimson’ was pink. In ‘Mantianhong’, coloration was not obvious after bag removal under artificial light conditions, but coloration was obvious under natural light conditions. In ‘Fojianxi’ and ‘Yunhongli 1′, debagged fruit did turn red under artificial light, but the coloration was better under natural light conditions. In ‘Starkrimson’, color faded slightly under artificial

3.3. Anthocyanin component analysis of red pears under artificial light and natural light conditions To explore the difference in anthocyanin components in debagged fruit and non-bagged fruit in terms of different light response-types of cultivars, anthocyanin components were detected by UPLC. When the 244

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Fig. 5. The hue angle of each light-response pattern under artificial light and natural light during illumination. A: Bagged fruit, B: Debagged fruit, C: Nonbagged fruit. 0: 0 days of exposure; 3: 3 days of exposure, etc.

exposure in the cultivar of ‘Starkrimson’, which might be due to the instability of peonidin-3-galactoside. On the other hand, four components were detected in non-bagged fruit in all red-skinned cultivars. The coloration of fruits under natural light was different from that under artificial light conditions. There were four anthocyanin components detected in ‘Yunhongli 1′ at 3 days of natural light exposure, and they were detected in ‘Mantianhong’ and ‘Fojianxi’ after 6 days of natural light as well. However, in ‘Starkrimson’, anthocyanin component accumulation was initially slightly lower but then increased significantly. In addition, four components were detected in non-bagged fruit under natural light conditions, which was the same for debagged fruit after bag removal. On the other hand, the content of anthocyanin components in all cultivars was higher under natural light than under artificial light, as

bag was not removed from fruit under artificial light conditions, no anthocyanin component was detected in cultivars of type I or type II (Fig. 7), as the bag blocked the radiation, and fruits had not received light since the fruit-bagging stage. However, four anthocyanin components were detected in the ‘Starkrimson’ cultivar at the same time. This finding shows that there was no anthocyanin accumulation at the young stage of fruit development in Asian pear, whereas anthocyanins began to accumulate during the fruit-setting stage in European pear. In addition, four anthocyanin components, including cyanidin-3-galactoside, cyanidin-3-glucoside, cyanidin-3-arabinoside, and peonidin-3-galactoside, were detected in cultivars ‘Fojianxi’ and ‘Yunhongli 1′ at 9 days of artificial light exposure, whereas only one component was detected in the cultivar ‘Mantianhong’ at that time. Furthermore, the anthocyanin components changed from four to three after 9 days under artificial 245

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Fig. 6. The anthocyanin content of each light-response pattern under artificial light and natural light during illumination. A: Bagged fruit, B: Debagged fruit, C: Non-bagged fruit. 0: 0 days of exposure; 3: 3 days of exposure, etc.

were determined using real-time PCR in different cultivars belonging to the three light-response types during artificial exposure stages (Fig. 8), to have uniform light conditions for the different pear species. Among the three light-response types, ‘Mantianhong’ showed no significant correlation between anthocyanin content and structural gene expression due to no obvious coloration (Table 4). In addition, for ‘Fojianxi’ and ‘Yunhongli 1′, the expression of genes related to color in the artificial light treatment was much higher than that of the control. Significant correlations between anthocyanin content and the structural genes ANS and UFGT were identified (Table 4), indicating that ANS and UFGT are the key structural genes of anthocyanin biosynthesis in cultivars of type II. Moreover, DFR and UFGT genes were significantly correlated with anthocyanin content in the cultivar ‘Starkrimson’ (Table 4), indicating that DFR and UFGT are important structural genes for anthocyanin biosynthesis in cultivars of type III.

the light component and light intensity were different between artificial light and natural light conditions. In artificial light conditions, the light components were UV-B and incandescent light, and the average light intensity was 4300 lx. In natural light conditions, the main light components were UV-A, UV-B, and visible light. The average light intensity was generally greater than 100000 lx on a sunny day. These results indicated that differences in light intensity and composition between natural and artificial conditions affect the intensity of red coloration in pear. 3.4. Effects of artificial light on the expression of genes related to anthocyanins 3.4.1. Expression profiles of anthocyanin biosynthesis structural genes The expression profiles of structural genes, including DFR, ANS, and UFGT, which act downstream in the anthocyanin biosynthesis pathway, 246

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Fig. 7. The effects of bagging treatments and different light types on the anthocyanin content of various components of each light-response pattern during illumination of pear fruits. (A): Mantianhong, (B): Fojianxi, (C): Yunhongli 1, (D): Starkrimson.

coloration of red pears, and light is one of the most important environmental factors affecting anthocyanin biosynthesis. The anthocyanin biosynthesis pathway has been extensively studied in pears, and some related genes have also been identified (Feng et al., 2010; Fischer et al., 2007; Yang et al., 2013). However, few physiological and molecular studies have been conducted to distinguish different light-response types in different red pear cultivars. During fruit production, it is easy to observe that varying responses to light lead to differential coloration of pear fruits, and bagging of fruit at early stages and debagging near the ripening stages can quickly promote coloration (Huang et al., 2009). In this paper, 27 red-skinned pear accessions were used to detect the variation in responses to light at near-ripening stages of fruit after debagging, and three major light-response types were detected: low increase, significant increase, or decrease in anthocyanin content in pear skins. Previous reports have shown that ‘Mantianhong’ developed color well under artificial light exposure (Qian et al., 2013; Sun et al., 2014). In this study however, the coloration of ‘Mantianhong’ was not obvious under artificial light exposure, which might be due to differences in the light conditions. In Qian’s experiment, fruits were kept in plant growth chambers with overhead lights (Hangzhou Zeda Instruments Co.Ltd., AGC-D002Z, Hangzhou, China) at 17 °C. UV-B and white light were generated by two UV lamps (PHILIPS PL-S 9W/12 RS, 290–315 nm, Amsterdam, Holland) and four fluorescent tubes (FSL T8 36W/765, Foshan, Guangdong, China), respectively. In our experiment, fruits were kept in a light incubator (Shanghai CIMO Medical Instrument Manufacturing Co.Ltd., GZX-300BS-Ⅲ, Shanghai, China) at 17 °C. UV-B and white light were generated by two UV lamps (Shoude 8W, 302–310 nm, Nanjing, Jiangsu, China) and sixteen fluorescent tubes (Mulinsen 21w, Zhongshan, Guangdong, China), respectively. ‘Fojianxi’ and ‘Yunhongli 1′ were strongly light-sensitive and showed obvious coloration after treatment, while ‘Starkrimson’ showed a color decrease because of its early coloration, before the bagging treatment. Another reason might be the effect of post-ripening traits in European pear. In addition, four components of anthocyanins were consistent between artificial light and natural light conditions among the different light-response types. In other words, certain artificial light conditions replicate natural light to promote coloration of pear fruit. In addition, we found that anthocyanin degraded in ‘Starkrimson’ (light-response

3.4.2. Expression profiles of transcription factors The expression profiles of transcription factors MYB10, bHLH33, and WD40 were determined using real-time PCR during artificial light exposure stages in pear cultivars belonging to the three light-response types (Fig. 9). In ‘Mantianhong’, the anthocyanin content and transcription factors bHLH33 and WD40 were significantly negatively correlated (Table 4). However, the anthocyanin content was significantly positively correlated with MYB10 and WD40 in ‘Fojianxi’ and ‘Yunhongli 1′ (Table 4), indicating that MYB10 and WD40 are important transcription factors of anthocyanin biosynthesis in cultivars of type II (Table 4). There was no significant correlation between anthocyanin content and transcription factor expression levels in ‘Starkrimson’ (Table 4). These results indicated that different transcription factors regulate the coloration of different light-response types of red pear fruit. 3.4.3. Expression profiles of light-responsive genes The expression profiles of known light-responsive genes, including HY5, PhyA, COP1, DET1, and PIF3, were determined using real-time PCR during artificial light exposure in cultivars belonging to the three light-response types of pear fruit (Fig. 10). Different light-responsive genes regulate the coloration in different light responses. In the cultivar ‘Mantianhong’, there was no significant correlation between anthocyanin content and expression levels of light-responsive genes (Table 4). However, the anthocyanin content and HY5 showed a significant positive correlation in cultivars ‘Fojianxi’ and ‘Yunhongli 1′ (Table 4), indicating that HY5 is an important transcription factor of anthocyanin biosynthesis in cultivars of type II. In addition, there also was a significant positive correlation between anthocyanin content and PhyA and PIF3 in ‘Yunhongli 1′ (Table 4). However, the anthocyanin content and PhyA were significantly negatively correlated in ‘Starkrimson’ (Table 4). 4. Discussion 4.1. Differences in coloration between three light-response types Anthocyanin is the most important component determining the 247

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Fig. 8. The expression profiles of the enzyme genes in three light-response patterns at different stages of artificial light exposure. Error bars are the SE of three replicates.

However, type II and the type III are different light-response types: the anthocyanin content of type II was significantly correlated with ANS, whereas that of cultivars of type III was significantly correlated with DFR. Similar phenomena were also found in other studies in pear. In a previous study, the anthocyanin content was significantly correlated with ANS and UFGT during fruit development of six cultivars, including oriental pear, occidental pear, and interspecific hybrid pear (Yang et al., 2015). In addition, the expression of DFR and ANS were up-regulated with increasing exposure time in ‘Yunhongli 1′, whereas opposite expression trends were reported for ‘Zaobaimi’ (Zhang et al., 2010). Moreover, the transcriptional levels of all eight genes, including DFR, ANS, and UFGT, were significantly up-regulated in the fruit skin of ‘Yunhongli No. 1′ and ‘Meirensu’ pears after bag removal (Yu et al., 2012). Compared with different light-response types, cultivars belonging to light-response type II had high expression levels of DFR, ANS, and UFGT with increasing artificial light exposure, and the expression of ANS and UFGT was significantly correlated with anthocyanin content (Table 4). Thus, ANS and UFGT are important enzyme genes in anthocyanin biosynthesis in pear.

type III) under artificial light but increased under natural light after a long time of exposure. Previous studies have reported that light can promote anthocyanin biosynthesis under postharvest exposure in red Chinese sand pears and apples but not in European pears (Marais et al., 2001; Zhang et al., 2012); this could be due to the existence of the acrylic layer in between the fruit and light source, which might absorb UV-B light (Qian et al., 2013). However, our results suggested that long exposure to natural light can break the limits of the acrylic layer to achieve the purpose of coloring for European pear. In addition, fruit color developed with excess photosynthates present as a source for the synthesis of the anthocyanins. This might also be a reason for the big differences between low artificial light level and saturated natural light conditions (Merzlyak and Chivkunova, 2000). 4.2. Expression of related enzyme genes involved in anthocyanin biosynthesis There are seven structural genes in the biosynthesis pathway of anthocyanins. We selected three of these genes, ANS, UFGT and DFR, located downstream of the anthocyanin biosynthesis pathway, for further analysis. The expression of structural genes was verified under artificial light exposure with three different light-response types. Since UFGT is the last gene in the anthocyanin biosynthesis pathway, the anthocyanin contents of cultivars of type II and type III were significantly correlated with the expression of UFGT (Table 4).

4.3. Expression of related transcription factors involved in anthocyanin biosynthesis Anthocyanin biosynthesis has been reported to be regulated by three transcription factors: MYB, bHLH, and WD40. The regulatory 248

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Fig. 9. The expression profiles of transcription factors in three light-response patterns at different stages of artificial light exposure. Error bars are the SE of three replicates.

(Rahim et al., 2014). In addition, MrMYB1 and MrbHLH1 overexpression in tobacco confirms the important role of MrMYB1MrbHLH1 in the synthesis of anthocyanins of Chinese bayberry (Liu et al., 2013). Moreover, co-expression between MYB10 and bHLH33 was reported in occidental pear but not in oriental pear (Yang et al., 2015). In this study, the anthocyanin content did not show a significant correlation with bHLH33 in cultivars of type II and type III but showed a significant negative correlation in cultivars of type I (Table 4). This result indicates that bHLH33 is not an important transcription factor that regulates anthocyanin biosynthesis in pears under different types of light responses. MrWD40-1 regulates anthocyanin biosynthesis with MrMYB1 and MrbHLH1 in Chinese bayberry by forming ternary complexes (Liu et al., 2013). Another study showed that a WD40 protein, MdTTG1, interacts with bHLH to regulate the accumulation of anthocyanins in apple (An et al., 2012). In this study, the content of anthocyanin was positively correlated with the expression level of WD40 in cultivars of type II but was negatively correlated with cultivars of type I, and anthocyanin content did not show a significant correlation with WD40 in cultivars of type III (Table 4). This result indicated that WD40 plays an important role in anthocyanin biosynthesis in pear. Different results in the light response between type I and type III might be due to the faint coloration of cultivars.

functions of the core transcription complex involving MYB, bHLH, and WD40 on the anthocyanin gene have been validated in petunia (Albert et al., 2011). A previous study reported that pyMYB10 plays an important role in regulating anthocyanin biosynthesis of Pyrus pyrifolia cv. ‘Aoguan’ (Feng et al., 2010). In addition, MYB10 also has similar reports in apple, suggesting that this gene plays an important role in the regulation of anthocyanins (Chagne et al., 2007). However, previous research from our lab revealed that the MYB10 transcription factor is not a key gene regulating the coloration of pear fruit. The expression level of MYB10 in two occidental pear cultivars is consistent with the variation of anthocyanin content (Yang et al., 2015). In this study, the expression level of MYB10 in type-II cultivars had a significant positive correlation with anthocyanin content. This finding shows that MYB10 plays an essential role in anthocyanin biosynthesis in pear. However, anthocyanin content was not correlated with the expression level of MYB10 in cultivars of type I or type III, which may be the reason that the coloration of the cultivars of type I and type III was not obvious. The transcription factor bHLH associates with related MYB and WD40, which form a protein complex to regulate the synthesis of anthocyanins (Davies et al., 2012; Feller et al., 2011; Hichri et al., 2011). In peach, the overexpression of MYB10/bHLH3 increases the production of anthocyanins by up-regulating NtCHS, NtDFR, and NtUFGT 249

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Fig. 10. The expression profiles of light-responsive genes in three light-response patterns at different stages of artificial light exposure. Error bars are the SE of three replicates.

photoreaction in Arabidopsis (Chen et al., 2006; Bernhardt et al., 2006). In the present study, there was no significant correlation between the expression level of DET1 and the anthocyanin content in cultivars of different light-response types, which shows that DET1 may not play an important role in anthocyanin biosynthesis in pear. The different expression levels of the light-responsive genes resulted in different anthocyanin biosynthesis patterns among cultivars of different light-response types.

4.4. Expression of related light-responsive genes involved in anthocyanin biosynthesis Light is one of the most important environmental factors in anthocyanin biosynthesis and plays an important role in regulating plant growth and development (Deng and Quail, 1999; Jiao et al., 2007). Many light-responsive genes are involved in the synthesis of anthocyanins. In Arabidopsis, PhyA, a far-red photoreceptor, and PhyB, a red photoreceptor, promote the accumulation of anthocyanins under their respective light conditions (Cashmore et al., 1999; Rockwell et al., 2006). Also, PhyA interacts with PhyB and inhibits the activity of COP1, which is a negative regulator of photomorphogenesis in Arabidopsis (Deng et al., 1991; Deng et al., 1992). In our study, the expression level of PhyA was positively correlated with anthocyanin content only in ‘Fojianxi’, but COP1 did not show negative regulation with anthocyanin content in different light-response types. This result is different from those of the previous reports, which might be due to the different regulatory mechanisms of anthocyanin biosynthesis for different species. PIF3 is the first bHLH family member and can activate PhyA and PhyB (Fujimori et al., 2004; Huq and Quail, 2002), while in our study, there was no significant correlation between the expression level of PIF3 and the anthocyanin content in different light-response types. This finding indicates that PIF3 might not play an important role in anthocyanin biosynthesis of pear. Other transcription factors have been reported to be involved in this network, such as HY5 of the bZIP family. HY5 was the first protein identified as a target of COP1. Previous studies using modified chromatin immunoprecipitation techniques have shown that HY5 is a high-level regulator of photomorphogenesis (Lee et al., 2007). In this study, we found that the anthocyanin content was significantly positively correlated with HY5 in cultivars of type II, which indicates that HY5 plays an important role in anthocyanin biosynthesis in pear. In addition, other proteins regulate photomorphogenesis through interaction with other substances. For example, DET1, DDB1, and COP1 combine with CUL4 to regulate the

5. Conclusion The coloration of 27 red pear samples varied under different light exposure conditions after bag removal near the ripening stage of fruit. Based on coloring patterns, the fruit can be divided into three lightresponse types. Four anthocyanin components were detected in all three light-response patterns, and the components of anthocyanins under artificial light were the same as those under natural light conditions. In addition, the different expression of structural genes, transcription factors, and light-responsive genes led to the different coloration patterns of pear cultivars. The potential mechanism behind anthocyanin biosynthesis is complex, and further research is needed to understand how these light-responsive genes interact with each other and determine the coloration of pear fruit. Competing interests The authors declare that they have no competing interests. Authors’ contributions YGF, ZYF and LHN conceived and designed the experiments. ZYF carried out the data analysis, ZYF, SJ, QMF, BB, CSS, WGM, and WRZ performed the experiments. ZYF wrote the paper. CG and WJ participated in revising the final manuscript. WJ and SQ managed the 250

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experiments. All authors have read and approved the final manuscript.

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