248 E u r o p e a n J o u r n a l o f H o r t i c u l t u r a l S c i e n c e

Eur. J. Hortic. Sci. 81(5), 248–254 | ISSN 1611-4426 print, 1611-4434 electronic | http://dx.doi.org/10.17660/eJHS.2016/81.5.3 | © ISHS 2016

The anthocyanins component and the influence factors of contents in red flesh apple ‘Hong-Xun No.1’Yugang Zhang1,2, Ruixue Zhao3, Wenlian Liu1, Xiaohong Sun1,2, Suhua Bai1,2, Ya Xiang1 and Hongyi Dai1,2

1 College of Horticulture, Qingdao Agricultural University, Qingdao, China2 Qingdao Key Laboratory of Genetic Improvement and Breeding in Horticultural Plants, Qingdao, China3 Shandong Provincial Extension Station of Fruits and Tea, Jinan, China

Original article

2015) and also have the role of delaying aging and anti-ath-erosclerosis (Faramarzi et al., 2015). Such as peach (Rahim et al., 2014), grape (Asada et al., 2015), plum (Hernández-Herrero et al., 2015), black mulberry (Boranbayeva et al., 2014), red cabbage (Wiczkowski et al., 2015), berry (Veberic et al., 2015), red flesh apples (Zhang et al., 2013b) and so on, many plants are rich in anthocyanins.

The components and contents of anthocyanins are dif-ferent in different plant varieties (Veberic et al., 2015) and are changed in red cabbage during fermentation, storage and stewing (Wiczkowski et al., 2015). New techniques to isolate and purify anthocyanins from grapes and apples have been applied (Asada et al., 2015).

There are many effect factors in regulation of anthocya-nin biosynthesis and stability (Rahim et al., 2014; Liu et al., 2013). Storage could influence on anthocyanin degradation in black mulberry juice (Boranbayeva et al., 2014) and auxin, cytokinin and nitrogen could effect on anthocyanin biosyn-thesis in callus cultures of red-fleshed apple (Ji et al., 2015). The studies on anthocyanin metabolism and regulation have currently become a hot research spot; especially in deep studies on transcription factors MYB gene family (Gesell et al., 2014).

Red flesh apples contain high levels of anthocyanins and they have gained favorable popularity and attracted increas-

German Society for Horticultural Science

Summary Red flesh apple is rich in anthocyanins, known

as functional fruit. ‘Hong-Xun No.1’ apple is a new red flesh apple cultivar, which was taken as materi-al to detect the anthocyanins component of red flesh using ultra performance liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS/MS), and to determine the anthocyanins contents of different organs, developmental stages, harvested locations and tissue culture plantlets un-der different conditions of culture temperatures, and with different sugars in the media. Six components of anthocyanins and six components other flavonoids in red-flesh apple fruits were detected. The antho-cyanins contents were different under different con-ditions as follows: 1) The order of anthocyanin con-tents from high to low in different organs/tissues was fruit peel > fruit flesh > new leaves > flowers > mature leaves in Fresh Weight (FW). The anthocyanin con-tents reached the highest in the fruit peel and fruit flesh at 12 weeks after abloom; 2) The anthocyanin contents in fruit peel and fruit flesh of the apple fruit harvested in Xinjiang were much higher than those in the apple fruit harvested in Shandong province; 3) With the increase in culture temperature, the antho-cyanin contents in the leaves and stems of the tissue culture plantlets displayed a significantly decreasing trend; 4) Different sugars in the culture medium also had significant effects on anthocyanin contents and the order from high to low was sorbitol>fructose> galactose>glucose>sucrose > lactose > maltose after 14 days treatment at 15°C. The anthocyanin content of tissue culture plantlets cultured with sorbitol MS after 14 days at 15°C was up to 359.86 U g-1 FW, which was 7.8 times higher than that of sucrose MS in room temperature, and was 1.8 and 3.6 times higher than those of peel and flesh of 5-year old tree, respectively. These results have provided us with the basic data to effectively utilize and industrially produce anthocy-anin in the red flesh apple cultivar.

Keywordsred flesh apple, anthocyanins component, sugar, sorbitol, flavonoid

Significance of this studyWhat is already known on this subject?• Anthocyanin is an important type of isoflavonoids,

which has the anti-oxidant functions and also has the roles of delaying ageing. Red flesh apple is rich in anthocyanins, known as functional fruit. Temperature can influence the contents of red flesh apple.

What are the new findings?• The anthocyanin content of tissue culture plantlets

cultured with sorbitol MS after 14 days at 15°C was up to 359.86 U g-1 FW, which was 7.8 times higher than that of sucrose MS in room temperature, and was 1.8 and 3.6 times higher than those of peel and flesh of 5-year old tree, respectively.

What is the expected impact on horticulture?• The results described in this study have provided us

with the basic data and new way to industrially pro-duce anthocyanins through tissue culture method and effectively utilize in the red flesh apple cultivar, ‘Hong-Xun No.1’.

IntroductionAnthocyanins are an important type of isoflavonoids

(Treutter, 2006), which have the anti-oxidant and anti-can-cer functions (Nemś et al., 2015; Hernández-Herrero et al.,

V o l u m e 8 1 | I s s u e 5 | O c t o b e r 2 0 1 6 249

Yugang Zhang et al. | Anthocyanins component and influence factors of contents in apple ‘Hong-Xun No.1’

ing attentions (Liu et al., 2009; Nocker et al., 2012; Wang et al., 2012b; Faramarzi et al., 2015).

‘Hong-Xun No.1’ apple is a new cultivar selected from Xinjiang wild red-flesh apple (Malus sieversii f. neidzwetzkya-na [Dieck] Langenf.). Its leaves, flowers and fruits are all red in color and contain abundant anthocyanins. Thus, it is a pre-cious resource for the breeding of red flesh apples (Zhang et al., 2013b). ‘Hong-Xun No.1’ was taken as material to detect the anthocyanin components of red flesh, and to determine the anthocyanin contents of different organs, developmen-tal stages, harvested locations and tissue culture plantlets under different conditions of culture temperatures, aiming to determine the key factors that influence the anthocyanin contents and stability and to provide the theoretical basis for exploration and utilization of this natural plant pigment re-source.

Materials and methods

Experimental materialsThe fruits, young and mature leaves, flowers of red flesh

apple cultivar ‘Hong-Xun No.1’ were collected from the 5-year old apple trees grown in Jiaozhou Farm of Qingdao Agricultural University, Qingdao, Shandong province in Chi-na. The sample-collecting timings were the young leaves at leaf flushing period, the flowers at stage of full bloom, three top mature leaves of new shoots at young fruit setting pe-riod, fruits at 4, 8, 12, and 16 weeks after bloom. The fruits at the 16th week after bloom were also collected at Yining and Tacheng areas of Xinjiang province when the apples were ripe.

Extraction of anthocyanin and measurement of anthocyanin contents in red flesh apple

The fresh samples of one gram leaves, flowers, and fruits (peel and flesh together) were chopped into small pieces and immerged into 10 mL of 1% HCl-methanol mixture, and ex-tracted at 4°C in the refrigerator for 24 h by stirring, and then were filtered. The absorbance of the extracted solutions was measured with spectrometry (UV-2550, Shimadzu, Japan) at 553 nm and 600 nm. The changes in absorbance intensity of the extracted solution from 1 g of sample at A553nm and A600nm was used as the relative contents of anthocyanin, and the 1% of different absorbance value between A553nm and A600nm was defined as the 1 unit (U) and expressed as U g-1 FW (Liu et al., 2009).

The component detecting of anthocyanins and other flavonoids in red flesh apple

Using the ultra high pressure liquid chromatography tandem quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS/MS), the anthocyanins and other flavonoids of red-fresh apple total fruit were detected. Dionex Ultimate 3000 Nano LC System (Thermo Scientific, USA) was used for UPLC, which includes Symmetry C18 chromatographic column (150 mm × 2.1 mm, 5 μm). The gradient eluting conditions were as follows: 0–0.1 min, 5% A; 0.1–20.0 min, 5%–20% A; 20.0–22.0 min, 20–80% A; 22.1–35.0 min, 5% A; methanol as mobile phase A and 0.2% formic acid-water so-lution as mobile phase B; flow velocity is 0.3 mL min-1; mass spectrometric conditions: Bruker Maxis UHR-TOF MS mass spectrometer, the ESI ion source, scanning range 50–1,000 amu, electrospray ionization is ion acquisition mode (ESI+), capillary voltage of 4.5 kV, ion source temperature of 180°C, removal of solvent gas flow rate 6 L min-1.

Treatment with different temperatures on tissue culture plantlets of ‘Hong-Xun No.1’

The uniformly grown tissue culture plantlets of ‘Hong-Xun No.1’ cultured in Murashige and Skoog (MS) media with 3% sucrose were selected, and randomly divided into four groups and were cultured at 5, 15, 25 and 35°C, respectively, in constant temperature incubator. The samples were col-lected at 5, 10 and 15 days after culture, respectively. The anthocyanin contents in leaves and stems were measured, as described above in red flesh apple.

Treatment with different types of sugars on tissue culture plantlets of ‘Hong-Xun No.1’

The tissue culture plantlets of ‘Hong-Xun No.1’ were di-vided into 14 groups, which were cultured in MS medium containing 3% of sorbitol, fructose, glucose, sucrose, galac-tose, lactose and maltose, respectively, and cultured at 15°C or 20°C condition. Samples were collected after two weeks. The anthocyanin contents of total plants in these treatment groups were measured, as described above in red flesh apple.

Statistical analysesEach biological experiment was repeated three times and

the experimental data were statistically analyzed by ANOVA using DPS7.05 software. The difference with P < 0.05 be-tween groups was regarded as statistically significant.

Results

The component detecting of anthocyanins and other flavonoids in red flesh apple fruits

Using the UPLC-Q-TOF-MS/MS the anthocyanins and oth-er flavonoids of red-fresh apple total fruit were detected (Fig-ure 1). According to the retention time of chromatographic peak, molecular ions, fragment ions, as well as the references (Wang et al., 2012a; Zhang et al., 2012), six components of anthocyanins and six components other flavonoids in red-flesh apple fruits were detected as myricetin, catechin, cy-anidin-3-O-anidin galactoside, kaempferol-3-O-rutinoside, kaempferol derivative, cyanidin-3-O-succinylarabinoside, pelargonidin-3-glucosidechloride, cyanidin-3-O-arabino-side, cyanidin-3-O-glucoside, quercetin, delphinidin-3-acetyl rutinose acyl-5-glucoside, quercetin derivative (Table 1).

The effects of different organs, different developmental stages and different harvested locations on anthocyanin contents of ‘Hong-Xun No.1’

The new leaves, young sprouts, fruit peel, fruit flesh and flowers of 5-year old tree ‘Hong-Xun No.1’ were all red or pink in color (Figure 2 A–D). However, anthocyanin contents among these organs were not similar and the order of their anthocyanin contents from high to low was fruit peel > fruit flesh > new leaves > flowers > mature leaves (Figure 2 E, F). For the fruit, the anthocyanin contents in apple fruit at dif-ferent sampling times in Qingdao area of Shandong province were also different. The anthocyanin contents gradually in-creased with continuing development and ripening of the fruits. At the early 8 weeks after bloom, the flesh, the core and the peel of fruit were all red in color. The part of flesh close to peel was white in color (Figure 2 D1, D2). At 12 weeks after bloom, fruit had fully developed and became ripen. At this time, the anthocyanin contents in fruit peel and fruit flesh reached the highest level and the fruit flesh all became red in color (Figure 2 D3). Thereafter, at 16 weeks after bloom, anthocyanin contents in fruit peel and fruit flesh displayed a

250 E u r o p e a n J o u r n a l o f H o r t i c u l t u r a l S c i e n c e

Yugang Zhang et al. | Anthocyanins component and influence factors of contents in apple ‘Hong-Xun No.1’

decreasing trend. However, the anthocyanin contents in peel and flesh of the fruit of ‘Hong-Xun No.1’ at 16 weeks after bloom harvested at Yining (Figure 2 D5) and Tacheng (Fig-ure 2 D6) areas of Xinjiang province were much higher than those harvested in Qingdao area of Shandong province, and the color of these fruits harvested at Xinjiang province were deeply red, smooth and brighten than those harvested at Shandong province.

Effects of different temperatures on anthocyanin contents in ‘Hong-Xun No.1’

Temperature significantly influenced anthocyanin con-tents. Anthocyanin contents in both leaves and stems signifi-cantly decreased with the rise in temperature (Table 2). The color of the leaves and stems of the tissue culture plantlets grown in constant temperature incubator at 5°C was obvi-ously red but the red color became lighter with the rise in temperature. At 35°C, the coloring of the tissue culture plantlets was the lightest. Their growth was poor and some leaves became shriveled. At 15 days after culture, anthocya-nin contents in leaves were hardly detected. Regardless at any temperatures, anthocyanin contents in leaves and stem

increased with the prolonged duration of temperature treat-ment, i.e., at a given temperature, the longer the treatment was the higher accumulation of anthocyanin was (Table 2).

Effects of different types of sugars on anthocyanin contents of ‘Hong-Xun No.1’ tissue culture plantlets

Tissue culture plantlets of ‘Hong-Xun No.1’ were cul-tured at 15°C or 20°C with MS medium containing sorbitol, fructose, glucose, sucrose, galactose, lactose and maltose, re-spectively. It can be seen from Figure 3 that different types of sugars had significantly different effects on the color and the anthocyanin contents in the tissue culture plantlets of ‘Hong-Xun No.1’. The order of the anthocyanin contents af-fected by different types of sugars from high to low was fructose > sorbitol > galactose > glucose > maltose > sucrose >lactose at 20°C after 14 days of treatment (Figure 3C). This order was consistent with corresponding changes in color (Figure 3A). At 15°C after 14 days culture, the anthocyanin contents and the red color of the leaves gradually increased by 3–5 times in comparison to culture at 20°C (Figure 3B, D). The order of the anthocyanin contents affected by differ-ent types of sugars from high to low was sorbitol > fructose

Figure 1. The total ion chromatogram of red flesh apple fruit.

8

12

11

10 98 7 6 5

4 3

2 1

0 5 10 15 20 25 30 35 40 Time [min]0.0

0.5

1.0

1.5

2.0

6x10Intens.

yuanye-lc-ms2-5ul-20150401_RA2_01_2062.d: TIC +All MS yuanye-lc-ms2-5ul-20150401_RA2_01_2062.d: TIC +All MSn

FIGURE 1. The total ion chromatogram of red flesh apple fruit.

Table 1. The component analysis of anthocyanins and other flavonoids in red flesh apple fruit by UPLC-Q-TOF-MS/MS.

Peak number Retention time(min)

Molecular ions[M+] (m/z)

Fragment ions[M+] (m/z) Tentative identification

123456789101112

2.1 2.211.111.116.117.318.719.721.723.523.629.6

319.10289.09449.17447.26489.10519.22461.20419.19449.20303.05465.23505.34

317.08289.09287.06285.05285.20287.99271.11287.03287.06303.05303.05301.15

MyricetinCatechin

Cyanidin-3-O-anidin galactosideKaempferol-3-O-rutinoside

Kaempferol derivativeCyanidin-3-O-succinylarabinosidePelargonidin-3-glucosidechloride

Cyanidin-3-O-arabinosideCyanidin-3-O-glucoside

QuercetinDelphinidin-3-acetyl rutinose acyl-5-glucoside

Quercetin derivative

V o l u m e 8 1 | I s s u e 5 | O c t o b e r 2 0 1 6 251

Yugang Zhang et al. | Anthocyanins component and influence factors of contents in apple ‘Hong-Xun No.1’

> galactose > glucose > sucrose > lactose > maltose at the 14th day after treatment (Figure 3D). The anthocyanin content of tissue culture plantlets cultured with sorbitol in MS me-dium after 14 days at 15°C was up to 359.86 U g-1 FW, which was 7.8 times higher than that of sucrose in MS medium at room temperature, and was 1.8, 2.8, 2.9, and 3.6 times higher than those of peel (199.92 U g-1 FW, 16 weeks after bloom), new leaves (128.52 U g-1 FW), flower (124.09 U g-1 FW), flesh (99.96 U g-1 FW, 16 weeks after bloom) of 5-year old tree, respectively. The leaf color of the tissue culture plantlets cul-tured with fructose or sorbitol was significantly deeper than that of the plantlets cultured with lactose or maltose (Figure 3A, B).

DiscussionRed flesh apple’s health care function mainly depends on

the contents and types of anthocyanins. Different varieties of same plant usually contain different contents and compo-

nents of anthocyanins. Nine anthocyanins were found in red pulp of ‘Zihong 1’ apple assayed by UPLC-PAD-/MS/MS, of which four kinds of anthocyanins were detected as known, and other five anthocyanins could not be determined tem-porarily (Wang et al., 2012a). In this study, six components of anthocyanins in red-flesh apple ‘Hong-Xun No.1’ fruits were detected as cyanidin-3-O-anidin galactoside, cyanidin-3-O-succinylarabinoside, pelargonidin-3-glucosidechloride, cyanidin-3-O-arabinoside, cyanidin-3-O-glucoside, and del-phinidin-3-acetyl rutinose acyl-5-glucoside. Only three types of anthocyanins were the same as ‘Zihong 1’ apple, of which cyanidin-3-anidin galactoside and cyanidin-3-glucoside were putatively the main components of anthocyanins in red flesh apple.

The stability of anthocyanins is the prerequisite for its ef-fective utilization. In the present study, we investigated the effect factors of different organs, development stages and harvested locations, temperature, types of sugars on the con-

Figure 2. Effects of different organs, developing stages and harvested locations on anthocyanin contents of ‘Hong-Xun No.1’. A. new leaves; B. flower; C. mature leaves; D. fruits (1–4) at 4, 8, 12, and 16 weeks after abloom harvested from Qingdao area, Shandong province; fruits at 16 weeks after abloom harvested from Yining (5) and Tacheng (6) areas, Xinjiang, China; E. anthocyanin contents in flower and leaves; F. anthocyanin contents in fruits.

9

FIGURE 2. Effects of different organs, developing stages and harvested locations on anthocyanin contents of ‘Hong-Xun No.1’. A. new leaves; B. flower; C. mature leaves; D. fruits (1–4) at 4, 8, 12, and 16 weeks after abloom harvested from Qingdao area, Shandong province; fruits at 16 weeks after abloom harvested from Yining (5) and Tacheng (6) areas, Xinjiang, China; E. anthocyanin contents in flower and leaves; F. anthocyanin contents in fruits.

252 E u r o p e a n J o u r n a l o f H o r t i c u l t u r a l S c i e n c e

Yugang Zhang et al. | Anthocyanins component and influence factors of contents in apple ‘Hong-Xun No.1’

tents and stability of anthocyanin. Different organs, developmental stages and harvested lo-

cations have relatively greater impacts on the anthocyanin contents of cultivar ‘Hong-Xun No.1’. Generally, the antho-cyanin contents in the fruit in the ripening stage are higher than those in leaves and flowers. We observed that the an-thocyanin contents in fruit peel were higher than those in fruit flesh. It can be explained by several reasons. Firstly, this is probably due to the reason that the water content in fruit peel is lower than that in fruit flesh. Secondly, this may be also due to the reason that anthocyanin is synthesized in the epidermis cells underneath fruit peel. With the continu-ing development of fruit, the anthocyanin synthesis gradu-ally increased, and reached the optimal level at the ripening process and thereafter, decreased. This is consistent with the results observed by Wang (2012b). This is presumably due

to the reason that the production of ethylene is closely re-lated to the synthesis of anthocyanin by mediating the activ-ity of UFGT (Liu et al., 2012). There were large variations in anthocyanin contents in apple fruit at 16 weeks after bloom which were harvested at different locations. The anthocya-nin contents in fruit of ‘Hong-Xun No.1’ harvested in Yinin and Tacheng areas in Xinjiang province were much higher than those in fruit of ‘Hong-Xun No.1’ harvested in Qingdao area of Shandong province. Xinjiang is located at the north-west part of China. The temperature difference between day time and night time is large, which is favorable for the accu-mulation of soluble solids, and more soluble solids influence the accumulation of anthocyanin (Liu et al., 2012). Thus, the color of fruit and fruit flesh is deeply red, smooth and bright.

Temperature is another important factor influencing anthocyanin biosynthesis. Generally, lower temperature is

Table 2. Effects of different temperatures and cultivated days on anthocyanin contents of leaves and stems of tissue culture plantlets of ‘Hong-Xun No.1’ cultured in MS media with 3% sucrose.

Cultivateddays (d)

5°C (U g-1 FW) 15°C (U g-1 FW) 25°C (U g-1 FW) 35°C (U g-1 FW)Leaves Shoots Leaves Shoots Leaves Shoots Leaves Shoots

5 43.13c 92.08c 22.78c 29.67c 17.21b 26.21c 0 11.25b10 198.89b 133.05b 72.77b 86.93b 20.30b 37.46b 0 11.46b15 468.78a 363.09a 158.56a 165.29a 37.76a 46.55a - 19.78a

Note: Means within a row followed by the different letter represent significant difference at the 0.05 level (n=3).

Figure 3. Effects of seven kinds of sugar present in MS media on anthocyanin contents of ‘Hong-Xun No.1’. A. phenotypes of tissue culture plantlets of ‘Hong-Xun No.1’ in MS media with seven kinds of sugar at 20� after 14 days cultivated; B. pheno-types of tissue culture plantlets of ‘Hong-Xun No.1’ in MS media with seven kinds of sugar at 15� after 14 days cultivated; C. anthocyanin contents of ‘Hong-Xun No.1’ leaves in MS media with seven kinds of sugar at 20� after 14 days cultivated; D. anthocyanin contents of ‘Hong-Xun No.1’ leaves in MS media with seven kinds of sugar at 15� after 14 days cultivated.

10

FIGURE 3. Effects of seven kinds of sugar present in MS media on anthocyanin contents of ‘Hong-Xun No.1’. A. phenotypes of tissue culture plantlets of ‘Hong-Xun No.1’ in MS media with seven kinds of sugar at 20℃ after 14 days cultivated; B. phenotypes of tissue culture plantlets of ‘Hong-Xun No.1’ in MS media with seven kinds of sugar at 15℃ after 14 days cultivated; C. anthocyanin contents of ‘Hong-Xun No.1’ leaves in MS media with seven kinds of sugar at 20℃ after 14 days cultivated; D. anthocyanin contents of ‘Hong-Xun No.1’ leaves in MS media with seven kinds of sugar at 15℃ after 14 days cultivated

V o l u m e 8 1 | I s s u e 5 | O c t o b e r 2 0 1 6 253

Yugang Zhang et al. | Anthocyanins component and influence factors of contents in apple ‘Hong-Xun No.1’

favorable for the fruit coloring and anthocyanin biosynthe-sis and its stability whereas higher temperature accelerates anthocyanin degradation. Freezing temperatures influenced susceptibility of blackberry flowers (Takeda and Glenn, 2016). The results obtained from this study also confirm this point. In a majority of studies, the anthocyanin stabil-ity was measured under different temperatures after being extracted. At high temperature, anthocyanin in the extracted solution of apple (Wang et al., 2012a) was found to be rapidly degraded. In this study, the tissue culture plantlets of ‘Hong-Xun No.1’ were cultured at 5, 15, 25 and 35°C, respectively (Table 2), within constant temperature incubators and the changes in their color and anthocyanin contents were moni-tored. We found that either in leaves or in stems, the changes in anthocyanin contents displayed a same significantly de-creasing trend with the increase in temperature.

The anthocyanidins combine with various monosaccha-rides via glycosidic bonds to form anthocyanin (Aron et al., 2008) and anthocyanin biosynthesis is also influenced by the exogenous sugars. For instance, it has been observed that Arabidopsis thaliana growing in the medium containing su-crose accumulated high levels of anthocyanins (Ohto et al., 2001). Yang et al. treated A. thaliana seedlings with several combinations of sucrose and fructose and glucose. Their re-sults indicated that sucrose at 60 mmol L-1 significantly in-creased anthocyanin contents in A. thaliana seedlings (Yang et al., 2011). In this study, we observed that all seven exog-enous sugars had different effects on the color and anthocya-nin contents of ‘Hong-Xun No.1’ tissue culture plantlets. The coloring effects of sorbitol, fructose and glucose were the most noticeable, followed by the effects of sucrose, galactose, lactose and maltose. These results are consistent with those reported by Zhang et al., who observed that spraying 2% of fructose solution effectively enhanced the synthesis and ac-cumulation of anthocyanins in the peel of ‘Hongtayang’ pear fruit (Zhang et al., 2013a). Sugars can enhance the coloring of fruit because sugars are premise materials of anthocyanin synthesis pathway, also could act as the messenger molecules and activate or suppress the expression of certain genes and thus, regulate anthocyanin accumulation (Solfanelli et al., 2006; Smeekens et al., 2000).

ConclusionsLow temperature of 15°C and MS medium with sorbitol

are more favorable for the accumulation of anthocyanins of ‘Hong-Xun No.1’ tissue culture plantlets than those are in room temperature and other type sugars, which was up to 359.86 U g-1 FW, and was 7.8 times higher than those of su-crose MS in room temperature, and was 1.8 and 3.6 times higher than those of peel and flesh of 5-year old red flesh ap-ple tree, respectively.

The results described in this study have provided us with the basic data and a new way to industrially produce antho-cyanins through tissue culture method and effectively utilize the red flesh apple cultivar, ‘Hong-Xun No.1’.

AcknowledgmentsThis work was supported by China Agriculture Research

System Foundation (CARS-28), National Natural Science Foundation of China (31372032), Taishan Scholar Construc-tive Foundation and Qingdao Scientific Research Foundation (15-9-2-99-nsh).

ReferencesAron, P.M., and Kennedy, J.A. (2008). Flavan-3-ols: nature, occur-rence and biological activity. Molecular Nutrition & Food Research 52, 79–104. http://dx.doi.org/10.1002/mnfr.200700137.

Asada, T., Koi, Y., and Tamura, H. (2015). New technique to isolate anthocyanins from delaware grapes by forming an aluminium com-plex using a Discovery DPA-6S. Food Chemistry 166, 10–16. http://dx.doi.org/10.1016/j.foodchem.2014.05.100.

Boranbayeva, T., Karadeniz, F., and Yılmaz, E. (2014). Effect of stor-age on anthocyanin degradation in black mulberry juice and con-centrates. Food Bioprocess. Technol. 7, 1894–1902. http://dx.doi.org/10.1007/s11947-014-1296-8.

Faramarzi, S., Pacifico, S., Yadollahi, A., Lettieri, A., Nocera, P., and Pic-colella, S. (2015). Red-fleshed apples: old autochthonous fruits as a novel source of anthocyanin antioxidants. Plant Foods Hum. Nutr. 70, 324–330. http://dx.doi.org/10.1007/s11130-015-0497-2.

Gesell, A., Yoshida, K., Tran, L.T., and Constabel, C.P. (2014). Char-acterization of an apple TT2 type R2R3 MYB transcription fac-tor functionally similar to the poplar proanthocyanidin regulator PtMYB134. Planta 240, 497–511. http://dx.doi.org/10.1007/s00425-014-2098-y.

Hernández-Herrero, J.A., and Frutos, M.J. (2015). Influence of ru-tin and ascorbic acid in colour, plum anthocyanins and antioxidant capacity stability in model juices. Food Chemistry 173, 495–500. http://dx.doi.org/10.1016/j.foodchem.2014.10.059.

Ji, X.H., Wang, Y.T., Zhang, R., Wu, S.J., An, M.M., Li, M., Wang, C.Z., Chen, X.L., Zhang, Y.M., and Chen, X.S. (2015). Effect of auxin, cyto-kinin and nitrogen on anthocyanin biosynthesis in callus cultures of red-fleshed apple (Malus sieversii f. niedzwetzkyana). Plant Cell Tiss. Organ. Cult. 120, 325–337. http://dx.doi.org/10.1007/s11240-014-0609-y.

Liu, J., Wei, J.L., Liu, M.Y., Song, Y., Feng, S.Q., Wang, C.Z., and Chen, X.S. (2012). The relationships between the enzyme activity of antho-cyanin biosynthesis, ethylene release and anthocyanin accumulation in fruits of precocious apple cultivars. Acta Horticulturae Sinica 39, 1235–1242.

Liu, X.J., Feng, B.C., Feng, S.Q., Wang, H.B., Shi, J., Wang, N., Chen, W.Y., and Chen, X.S. (2009). Studies on anthocyanin biosynthesis and ac-tivities of related enzymes of ‘Ralls’ and its bud mutation. Acta Hor-ticulturae Sinica 36, 1249–1254.

Liu, Y.L., Che, F., Wang, L.X., Meng, R., Zhang, X.J., and Zhao, Z.Y. (2013). Fruit coloration and anthocyanin biosynthesis after bag removal in non-red and red apples (Malus×domestica Borkh.). Molecules 18, 1549–1563. http://dx.doi.org/10.3390/molecules18021549.

Nemś, A., Peksa, A., Kucharska, A.Z., Sokoł-Łetowska, A., Kita, A., Drozdz, W., and Hamouz, K. (2015). Anthocyanin and antioxidant activity of snacks with coloured potato. Food Chemistry 172, 175–182. http://dx.doi.org/10.1016/j.foodchem.2014.09.033.

Nocker, S.V., Berry, G., Najdowski, J., Michelutt, R., Luffman, M., Forsline, P., Alsmairat, N., Beaudry, R., Nair, M.G., and Ordidge, M. (2012). Genetic diversity of red-fleshed apples (Malus). Euphytica 185, 281–293. http://dx.doi.org/10.1007/s10681-011-0579-7.

Ohto, M.A., Onai, K., and Furukawa, Y. (2001). Effects of sugar on vegetative development and floral transition in Arabidopsis. Plant Physiol. 127, 252–261. http://dx.doi.org/10.1104/pp.127.1.252.

Rahim, M.A., Busatto, N., and Trainotti, L. (2014). Regulation of an-thocyanin biosynthesis in peach fruits. Planta 240, 913–929. http://dx.doi.org/10.1007/s00425-014-2078-2.

Smeekens, S. (2000). Sugar-induced signal transduction in plants. Annual Review of Plant Physiology & Plant Molecular Biology 51, 49–81. http://dx.doi.org/10.1146/annurev.arplant.51.1.49.

254 E u r o p e a n J o u r n a l o f H o r t i c u l t u r a l S c i e n c e

Yugang Zhang et al. | Anthocyanins component and influence factors of contents in apple ‘Hong-Xun No.1’

Solfanelli, C., Poggi, A., Loreti, E., Alpi, A., and Perata, P. (2006). Sucrose-specific induction of the anthocyanin biosynthetic path-way in Arabidopsis. Plant Physiol. 140, 637–646. http://dx.doi.org/10.1104/pp.105.072579.

Takeda, F., and Glenn, D.M. (2016). Susceptibility of blackberry flow-ers to freezing temperatures. Eur. J. Hortic. Sci. 81(2), 115–121. http://dx.doi.org/10.17660/eJHS.2016/81.2.5.

Treutter, D. (2006). Significance of flavonoids in plant resistance: a review. Environmental Chemistry Letters 4, 147–157. http://dx.doi.org/10.1007/s10311-006-0068-8.

Veberic, R., Slatnar, A., Bizjak, J., Stampar, F., and Mikulic-Petkovsek, M. (2015). Anthocyanin component of different wild and cultivated berry species. Food Science and Technology 60, 509–517.

Wang, Y., Chen, X.S., Liu, D.L., Wang, C.Z., Song, Y., Chen, X.L., and Zhang, Y.M. (2012a). Antioxidant activity and anthocyanins analysis of pulp in ‘Zihong 1’ red-flesh apple. Acta Horticulturae Sinica 39, 1991–1998.

Wang, Y.L., Zhang, Y.M., Feng, S.Q., Song, Y., Xu, Y.T., Zhang, Y.P., and Chen, X.S. (2012b). The mechanism of red coloring difference be-tween skin and cortex in Malus sieversii f. neidzwetzkyana (Dieck) Langenf. Scientia Agricultura Sinica 45(13), 2771–2778.

Wiczkowski, W., Szawara-Nowak, D., and Topolska, J. (2015). Chang-es in the content and component of anthocyanins in red cabbage and its antioxidant capacity during fermentation, storage and stewing. Food Chemistry 167, 115–123. http://dx.doi.org/10.1016/j.food-chem.2014.06.087.

Yang, S.H., Wang, L., Mu, C., Wang, X., He, J.H., Zhao, J.Y., and Wang, L.S. (2011). Anthocyanin biosynthesis regulated by sucrose in Arabidop-sis thaliana seedling. Chinese Journal of Biochemistry and Molecular Biol. 27, 364–369.

Zhang, J., Wang, L.S., Gao, J.M., Xu, Y.J., Li, L.F., and Li, C.H. (2012). Rapid separation and identification of anthocyanins from flowers of Viola yedoensis and V. prionantha by high-performance liquid chromatography–photodiode array detection–electrospray ionisa-tion mass spectrometry. Phytochemical Analysis J. 23, 16–22. http://dx.doi.org/10.1002/pca.1320.

Zhang, Q., Yang, J., Wang, L., Wang, S.K., Li, X.G., and Zhang, S.L. (2013a). Correlation between soluble sugar and anthocyanin and ef-fect of exogenous sugar on coloring of ‘Hongtaiyang’ pear. Journal of Fruit Science 30, 248–253.

Zhang, Y.G., Zhu, J., and Dai, H.Y. (2013b). Morphological characteris-tic and pollination compatibility of a new red flesh apple, Hongxun No.1. Research on Crops 14, 199–204.

Received: Jun. 7, 2016Accepted: Sep. 13, 2016

Addresses of authors: Yugang Zhang1,2,*, Ruixue Zhao3, Wenlian Liu1, XiaohongSun1,2, Suhua Bai1,2, Ya Xiang1 and Hongyi Dai1,2

1 College of Horticulture, Qingdao Agricultural University, Qingdao, China

2 Qingdao Key Laboratory of Genetic Improvement and Breeding in Horticultural Plants, Qingdao, China

3 Shandong Provincial Extension Station of Fruits and Tea, Jinan, China

* Corresponding author; E-mail: zyg4458@163.com