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Bioactive Constituents, Metabolites, and Functions
Changes of anthocyanin components biosynthesis in 'Summer Black' grape berries after the red flesh mutation occurred Kekun Zhang, Zhongjie Liu, Le Guan, Ting Zheng, Songtao Jiu, Xudong Zhu, Haifeng Jia, and Jinggui Fang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02150 • Publication Date (Web): 09 Aug 2018 Downloaded from http://pubs.acs.org on August 12, 2018
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Journal of Agricultural and Food Chemistry
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Changes of anthocyanin components biosynthesis in ‘Summer Black’
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grape berries after the red flesh mutation occurred
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Kekun Zhang1, Zhongjie Liu1, Le Guan1, Ting Zheng1, Songtao Jiu1, Xudong Zhu1, Haifeng Jia1,
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Jinggui Fang1*
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1
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People’s Republic of China
College of Horticulture, Nanjing Agricultural University, Nanjing City, 210095, Jiangsu Province,
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*Correspondence Author: Jinggui Fang
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Tel: 0086-025-84395217
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e-mail:
[email protected] 12 13
Kekun Zhang,
[email protected] 14
Zhongjie Liu, 2017204011@ njau.edu.cn
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Le Guan, 2017104011@ njau.edu.cn
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Ting Zheng,
[email protected] 17
Songtao Jiu,
[email protected] 18
Xudong Zhu,
[email protected] 19
Haifeng Jia,
[email protected] 20
Jinggui Fang,
[email protected] 21 22
Declarations of interest: none 1
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Abstract
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The coloring process of grape flesh is valuable for research and promotion for the high nutritional
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quality of anthocyanins. ‘Summer Black’ and it’s new red flesh mutant were used to analyze the
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changes of anthocyanins biosynthesis during grape berries development. Eighteen kinds of
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anthocyanins were detected in mature berries of the two cultivars, but the content of most 3'-,
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3',5'-substituted anthocyanins was higher in the skin of the mutant. Anthocyanins accumulation
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occurred simultaneously in the skin and flesh of the mutant, and their types and content were more
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abundant in the former. For the mutant, there were only CHS, OMT, MYBA3 and MYBPA1 at lower
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transcriptional level in the flesh during veraison when compared with these in the skin, which might be
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an important factor to limit the anthocyanins accumulation in the flesh. The occurrence of red flesh
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might be related the enhancement of anthocyanins biosynthesis in the whole berry.
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Keywords
Grape·Anthocyanins·LC-MS·Bud
Mutation·Multivariate analysis
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Introduction
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Anthocyanins are a class of flavonoids produced during secondary metabolism in plants, formed
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by the glycosidic linkage between anthocyanidins and glycosides. As the natural protectors for plants,
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they are widely found in vairious organs, such as roots, stems, leaves, flowers and fruits (Guan et al.
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2012; González-Villagra et al. 2017). With the strong antioxidant capacity, anthocyanins can also
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prevent the occurrence of cardiovascular disease in humans (Kruger et al. 2014). Anthocyanins are
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synthesized mainly through the phenylalanine and flavonoid metabolic pathway in plants, in which a
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variety of structural and regulatory genes involved. The cytoplasm is the initial place where
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anthocyanins appear in grapevine (Castellarin et al. 2007), and then they are transported to the
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vacuole for accumulation mediated by membrane vesicles (anthocyanoplasts, Zhao et al. 2010) and
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membrane transporters (Francisco et al. 2013). The red, purple or black color of grape berries is
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closely related to the type, content and proportion of individual anthocyanin components in the fruit
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(Jeong et al. 2006).
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The production of anthocyanins is closely regulated by MYB transcript factors (Li et al. 2015).
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MYBA1 and MYBA2, located on chromosome 2, determined the coloring of grape skins (Kobayashi er
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al. 2004). MYBA1 can additionally trans-activate 3AT, producing the acylated anthocyanins with
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aliphatic and aromatic acyl groups in grape skins (Rinaldo et al. 2015). Recently, Matus et al. (2017)
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found that MYBA6 and MYBA7 on chromosome 14 also showed regulatory activities on the
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biosynthesis and transportion of anthocyanins in the coloring process. But compared with MYBA1,
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MYBA6 and MYBA7 have a weaker regulatory effect on F3'5'H gene, and mainly affects the coloring
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of vegetative organs. Anthocyanins biosynthesis pathway is also affected by the external environment.
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High temperature and dark conditions could repress the skins coloring (Azuma et al. 2012). UV-B, 3
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with a shorter wavelength, affects the biosynthesis of flavonols and anthocyanins by changing the
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transcription level of F3'H, F3'5'H and OMT2 (Martinez-Luscher et al. 2014). Modification of
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diurnal temperature can change the accumulation of proanthocyanidins and fruit coloration speed by
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affecting the expression of key genes in the flavonoid metabolic pathway (Cohen et al. 2012).
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Moderate water deficit promotes the accumulation of secondary metabolites, such as anthocyanins,
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esters, in grapevine or wines (Ju et al. 2018).
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In grape, berry skin is the main organ enriched of anthocyanins. There are also a few varieties,
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termed as teinturiers, not only accumulate anthocyanins in berry skin and flesh, but also in the peduncle,
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inflorescence axis and leaves (Jeong et al. 2006; Guan et al. 2012). Berry skin and flesh differed in
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anthocyanin composition and accumulation profile in teinturier varieties (Castillo-Muñoz et al. 2009;
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Castellarin et al. 2011), and these traits are heritable. In the ‘Alicante Bouschet’, the original teinturier
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variety, anthocyanins were originally found in the vicinity of the style in flesh where the synthesis of
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anthocyanins gradually progresses from the top to the basal part of the peduncle and the skin is finally
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colored (Castellarin et al. 2011). Limited by germplasm and the complex regulation mechanism, the
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mechanism of activation and regulation of flesh coloration is not clear.
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‘Summer Black’ (3n, V. vinifera× V. labrusca) is a Japanese seedless grape cultivar and currently
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cultivated broadly in China, with a farther genetic distance to already known teinturier varieties. As the
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grape berries grow, its skin appears purple-black with no anthocyanins accumulated in the flesh. Its red
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flesh sport was recently discovered in Chinese vineyard, which had similar tissues to ’Summer Black’
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except the flesh. Bud mutation is a common variation in the natural growth of fruit trees. Benefiting
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from its stability in clone propagation, bud mutant varieties become favorable resources to reveal the
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growth mechanism of fruit varieties (Kobayashi et al. 2004, Pelsy et al. 2015). In the study of 4
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Castellarin et al. (2011), ‘Alicante Bouschet’ started coloring when the TSS reached about 4, but this
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new mutant cultivar began to color when the TSS reached about 7, indicating that the activation and
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regulation mechanism of anthocyanins pathway in these two cultivars are different, it’s necessary to
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study the changes of anthocyanin components biosynthesis in grape berries of ‘Summer Black’ after the
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bud mutation.
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With higher nutritional quality, the market of the red-flesh table grapes is promising. However,
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few researches have been carried out on the anthocyanins components biosynthesis in the skin and
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flesh of table grapes from V. vinifera× V. labrusca cultivars. So the goal of this study is to identify the
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anthocyanin components in the mutant flesh and the key biosynthesis steps involved in the coloring
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process, by systematically comparing the anthocyanins and transcript profiles of anthocyanins
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biosynthesis pathway during berries development. The results would be helpful for better developing
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agronomic techniques to increase grape anthocyanins content, and revealing the regulatory path of
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flesh coloring.
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Materials and methods
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Plant materials
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The experiments were carried out at the Baima Experimental Vineyard, Nanjing Agricultural
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University (31°36′N, 119°10′E). The vines were under the same routine management. Four-year-old
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‘Summer Black’ and its mutant cultivar were selected as test materials. The original mutant vine was
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derived from mutant shoots on ‘Summer Black’, and then the new mutant vine produced a large
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number of cuttings by means of vegetative propagation. Some vines grown from these cuttings were
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used for our experiments. Twenty vines of each variety were chosen and the berry samples (three
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biological replicates) were collected at the following four developmental stages: i) DS1, the early stage
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of fruit enlargement (20 days after anthesis); ii) DS2, initiation of veraison (10% of the berries began
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to change color); iii)DS3, full veraison (90% of the berries completed the color changing); iv) and
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DS4, maturity (TSS and TA tended to be stable). Samples were randomly collected and the location of
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vines and clusters were also considered. After weighing, berries were separated into skins and flesh,
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and then were immediately frozen in liquid nitrogen individually. All samples were stored at -80 ° C for
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the subsequent analysis.
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Color observation of grape flesh and measurement of quality indexes
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After collecting the grape berry samples at various developmental stages, a portion of fresh fruits
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were selected for flesh color observation. Grape berries were transversely and longitudinally cut along
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the central axis respectively. Then a thickness of about 5 mm cut slice along the longitudinal section
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and the cross section were observed under the microscope. The vertical and horizontal diameter of the
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grape fruit was measured using an automatic vernier caliper, Total soluble solids (TSS) was measured
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using a portable hand-held dialyzer (PAL-1, Japan), and the titratable acidity was titrated with 0.1 mol /
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L NaOH.
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Anthocyanin profiling
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Anthocyanins were extracted according to Guan et al. (2016). The extracted anthocyanin solute
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was analyzed by LC–MS system (G2-XS QT, Waters). Aliquot of 2 µL solution was injected into the
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UPLC column (2.1×100 mm ACQUITY UPLC BEH C18 column containing 1.7 µm particles) with a
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flow rate of 0.4 mL/min. Buffer A consisted of 0.1% formic acid in water, and buffer B consisted of
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0.1% formic acid in acetonitrile. The gradient for Buffer B was 5% Buffer B for 2 min, 5–95% 15 min, 6
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and 95% for 2 min. Mass spectrometry was performed using electrospray source in both positive and
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negative ion mode with MSe acquisition mode, with a selected mass range of 50–1200 m/z. The lock
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mass option was enabled using leucine-enkephalin (m/z 556.2771for positive ion mode, 554.2615 for
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negative ion mode) for recalibration. The ionisation parameters were the following: capillary voltage
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was 2.5 kV, collision energy was 40 eV, source temperature was 120°C, and desolvation gas
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temperature was 400°C. Data acquisition and processing were performed using Masslynx 4.1.
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Malvidin-3-O-glucoside (Extrasynthese, Genay, France) was used as common external standard and
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other anthocyanin components were quantified by it.
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Total RNA isolation, cDNA synthesis and gene expression analysis
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Total RNAs were isolated using a CTAB method according to Guan et al. (2016). Then the first
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strand cDNA was synthesized from 1 µg total RNA with P1[an oligo(dT)20 primer], P02(a random
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primer) and Superscript Ⅲ RNase H-RT kit from Invitrogen (Carlsbad,CA) according to the
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manufacturer’s instruction. The cDNA was diluted at 1:10 for RT-PCR.
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RT-PCR was carried out using the CFX96 Real-Time PCR Detection system (Bio-Rad, Hercules,
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CA, USA). Reaction mixes volume was 10 µL, which included 5 µL of SYBR Green Supermix
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(Bio-Rad), 2 µL of diluted cDNA, and 0.2 µL of each primer. Each pair of qRT-PCR primers were
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validated by cloning and sequencing of the RT-PCR product with this pair of primers. The efficiency of
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each primer pair was quantified using a PCR product serial dilution. All biological samples were
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assayed in technical triplicates. Ubiquitin and EF1γ were used as internal standards and for normalizing
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the expression. Expression levels were calculated based on the 2-△△Ct method with each lowest
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expressed sample chosen as a reference .
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Statistical Analysis
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Statistical analysis was performed with the software SAS 9.2 (Inc. Cary, NC, USA). Differences
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between genotypes and tissues for a giving sampling date were analyzed by a two-way ANOVA
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followed by the Duncan's multiple comparison test at P
