Dynamic changes in anthocyanin biosynthesis regulation of Cabernet Sauvignon (Vitis vinifera L.) grown during the rainy season under rain-shelter cultivation

Accepted Manuscript Dynamic changes in anthocyanin biosynthesis regulation of Cabernet Sauvignon (Vitis vinifera L.) grown during the rainy season under rain-shelter cultivation Bingbing Duan, Changzheng Song, Yimei Zhao, Yue Jiang, Pengbao Shi, Jiangfei Meng, Zhenwen Zhang PII: DOI: Reference:

S0308-8146(19)30081-0 https://doi.org/10.1016/j.foodchem.2018.12.131 FOCH 24108

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

30 June 2018 5 December 2018 31 December 2018

Please cite this article as: Duan, B., Song, C., Zhao, Y., Jiang, Y., Shi, P., Meng, J., Zhang, Z., Dynamic changes in anthocyanin biosynthesis regulation of Cabernet Sauvignon (Vitis vinifera L.) grown during the rainy season under rain-shelter cultivation, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2018.12.131

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Dynamic changes in anthocyanin biosynthesis regulation of Cabernet Sauvignon (Vitis vinifera L.) grown during the rainy season under rain-shelter cultivation Bingbing Duan a, Changzheng Song a, Yimei Zhao a, Yue Jiang a, Pengbao Shi a,b, Jiangfei Meng a,c,*, Zhenwen Zhang a,c,* a

College of Enology, Northwest A&F University, Yangling 712100, Shaanxi, China

b

College of Food Science & Technology, Hebei Normal University of Science & Technology,

Qinhuangdao 066600, Hebei, China c

Shaanxi Engineering Research Center for Viti-Viniculture, Northwest A & F University, Yangling

712100, Shaanxi, China

* Corresponding author: Jiangfei Meng, Zhenwen Zhang College of Enology Northwest A&F University No. 22 Xinong Road Yangling Shaanxi 712100, China Tel.: +86 29 87092107; Fax: +86 29 87092107 E-mail address: [email protected] [email protected]

Email addresses for all authors: [email protected] [email protected] [email protected] [email protected] [email protected]

1

Abstract The grapevine (Vitis vinifera L.) berry coloring mechanism in response to seasonal rain during grape ripening remains poorly understood. Therefore, anthocyanin biosynthesis regulation, dynamic changes in anthocyanin accumulation, biosynthetic enzyme activities, and related gene expression patterns were investigated in Cabernet Sauvignon grown under rain-shelter cultivation and open-field cultivation. Results showed that anthocyanin biosynthesis was strongly repressed during the rainy season. Environmental fluctuation from seasonal rain provoked metabolic responses in grapes, and there was significantly greater accumulation of most of anthocyanins, mainly the compositions of non-acylated and non-methylated, under rain-shelter cultivation; these findings indicate that rain-shelter cultivation may help improve tolerance to seasonal rain-induced stresses. Obvious resilience was observed in anthocyanins of open-field-cultivated grapes at harvest. Hierarchical cluster analysis indicated strong correlations between anthocyanin contents, CHI and DFR activities, and VvMYB5b transcriptional level. These findings provide novel insight into the crucial factors that directly modulate anthocyanin biosynthesis and consequently control grape coloration. Keywords: Grape, anthocyanins, rain-shelter cultivation, rainy season, anthocyanin biosynthesis Chemical compounds studied in this article Malvidin-3-O-glucoside (PubChem CID: 443652); Malvidin-3-O-(6-acetyl)glucoside (PubChem CID: 74977116); Malvidin-3-O-(trans-6-coumaryl)-glucoside 2

(PubChem CID: 72193651); Delphinidin-3-O-glucoside (PubChem CID: 443650); Cyanidin-3-O-glucoside

(PubChem

CID:

44256715);

Peonidin-3-O-glucoside

(PubChem CID: 443654); Peonidin-3-O-(6-acetyl)-glucoside (PubChem CID: 72193652);

Petunidin-3-O-glucoside

(PubChem

CID:

443651);

Petunidin-3-O-(6-coumaryl)-glucoside (PubChem CID: 44256965); L-phenylalanine (PubChem CID: 6140)

3

1 Introduction Anthocyanins are abundant vacuolar pigments derived from the flavonoid pathway and important health-promoting phytochemicals associated with scavenging of reactive oxygen species and reduction in risk of cardiovascular diseases (Castellarin et al., 2007). The anthocyanins in red wine are mainly originated from grapevine berries as well as wines fermented in the presence of red skins, and therefore anthocyanin composition and concentration of grapes is the major criterion used to judge the quality of red grapes and there resulting wine (Li, He, Wang, Li, & Pan, 2014; Sun, Pan, Duan, & Wang, 2015). The quantity and composition of anthocyanins in grapes mainly depend on varieties, environmental factors, and cultivation managements (Guan et al., 2016). Grapevine berries are constantly affected by a plethora of abiotic and biotic stimuli caused by environmental fluctuations during their development, which induces a wide range of morphological and biochemical adaptations that ameliorate the effects of these adversities (Degu, Ayenew, Cramer, & Fait, 2016; Matus, 2016). Anthocyanins may act as critical regulators under various stress conditions (Wang et al., 2018; Zorenc et al., 2017). Dynamic changes of anthocyanin biosynthesis is transcriptionally induced in response to elevated light conditions (Li, Guan, Hua, & Wu, 2013; Guan et al., 2016; Miao et al., 2016; Sun, Pan, Duan, & Wang, 2015), high temperature stresses (Degu et al., 2016; Lecourieux et al., 2017), and drought stress (Cáceresmella et al., 2017; Kyraleou et al., 2016). Nowadays, the major wine regions in China, which has a continental monsoonal 4

climate, have challenging conditions for grape production and wine industry. Unfavorable abundant rainfall during ripening could lead to non-optimal accumulation of sugar, organic acid and phenolic compound including anthocyanin. (Gao et al., 2016; Li et al., 2014; Meng, Ning, Xu, & Zhang, 2012). Rain-shelter cultivation, a viticulture technique that employs an artificial microclimate, is an organic production system that improves grape yield and quality, and greatly reduces grape shattering and diseases in rainy regions (Li et al., 2014; Meng et al., 2012; Shi et al., 2017). Polyphenol content is closely related to micrometeorological alterations in vineyards that use plastic coverings in rain-shelter systems (Gao et al., 2016; Meng et al., 2012). Many reports revealed decreased contents of most anthocyanin compounds in grapes grown by rain-shelter cultivation owing to increases in ambient temperature and humidity, and decreases in photosynthetically active radiation (Meng et al., 2012; Shi et al., 2017). Additionally, rain-shelter cultivation contributed to delays in grape berry veraison and ripening (Matus, 2016). Li et al. (2014) reported that rain-shelters constructed with colorless polyethylene film, compared with open-field cultivation, increased soluble solid content and significantly enhanced major anthocyanin concentrations in grape berries. Rain-shelter cultivation improved the quality and phytochemical content of grapes grown in rainy wine-producing regions when combined with full sunlight at harvest, which depends mainly on seasonal variations and the characteristic of films (Meng, Ren, Yang, & Pan, 2018; Xu et al., 2014). These studies mainly focused on the effect of rain-shelter cultivation on biochemical components of berry and the relation between these components and 5

environmental factors. However, there were few reports about the mechanism of rain-shelter cultivation regulating these compounds accumulation from the point of the gene transcription or enzyme activity. The anthocyanins are synthesized as part of the flavonoid branch pathway and were initially recruited from the phenylpropanoid pathway by a series of enzymes in Vitis, such as phenylalanine ammonialyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate:-CoA ligase (4CL), chalcone isomerase (CHI), and dihydroflavonol 4-reductase (DFR), which are produced by structural genes and directly participate in the production of anthocyanins and other flavonoid compounds (Lillo, Lea, & Ruoff, 2008; Miao et al., 2016; Xu et al., 2014; Zorenc et al., 2017). In particular, parallel pathways downstream of F3′H and F3′5′H produce either cyanidin or delphinidin in the cytosol of red grape epidermal cells, which are then delivered into vacuoles, where they are visible as colored coalescences (Castellarin et al., 2007; Zorenc et al., 2017). Cyanidin and delphinidin can undergo chemical modifications such as glycosylation, methylation, and acylation by many different enzymes (Castellarin et al., 2007). Transcription factors can directly participate in regulating the expression of anthocyanin biosynthetic genes by binding to the promoters of the corresponding genes (Wang et al., 2018). Moreover, this regulation is mainly controlled by a ternary complex (MBW) that contains DNA binding R2R3-MYB, WD40-repeat, and basic helix-loop-helix proteins in several plant species, including petunia (Petunia hybrida), arabidopsis (Arabidopsis thaliana), and grapevine varieties (Vitis vinifera L.) (Hichri et al., 2010; Schaart et al., 2013; Sun et al., 2017). MYB regulators are master 6

candidate genes that regulate biosynthesis of anthocyanins (MYBA1/A2, MYB5B) and proanthocyanidins (MYBPA1/PA2) and fruit coloration (Cavallini et al., 2014; Wong et al., 2016). Collectively, knowledge of seasonal variations is critical to increase the levels of bioactive compounds to meet consumer demands for healthy products. However, the rain-shelter cultivation has often been applied without a systematic understanding of anthocyanin biosynthesis variation during the rainy season. Based on this, our objective was to evaluate the effects of rain-shelter cultivation and open-field cultivation during the rainy season on grape anthocyanin compositions and contents, anthocyanin biosynthesis-related enzyme and gene expression levels through grape ripening. This study was carried out based on the hypothesis that rain-shelter cultivation affected anthocyanins biosynthesis by altering levels of related gene expressions and enzyme activities, expecting to provide experimental evidence regarding the scientific utility of rain-shelter cultivation.

2 Materials and methods 2.1 Plant materials and treatments The experiment was conducted in a commercial vineyard of Vitis vinifera L. cv. Cabernet Sauvignon located in Jingyang County of Shaanxi Province (34°40′56′′ N, 108°38′53′′ E; elevation 440 m), China, during two consecutive growing years (the vintages of 2016 and 2017). The own-rooted vines in this vineyard, planted in 2010, were in a north-south orientation. The spacing distance was 0.8 m × 2.5 m. Vines were 7

trained to a bilateral cordon, trellised to a vertical shoot positioning system and pruned to two buds per spur. All other viticultural practices followed the local standards. The experimental vines were selected from 12 rows in the experimental field and divided into two groups. Vines in Group 1 were grown using rain-shelter cultivation, and the shelter was built along with vine rows before the rainy season (one week before veraison in this study). The rain-shelter was 2.2 m high, 1.7 m wide and covered with 0.05 mm colorless and transparent polyethylene film having a light transmittance of 87% (T1). Group 2 vines were cultured in an open field without the polyethylene film (Control; T2). Treatment and control conditions each occurred on six rows, containing 125 vines per row. Each condition was established in triplicate (two rows per replicate). To achieve homogeneity, each row was divided into seven plots, from which grapes were collected at seven sampling dates. The plots were arranged in a completely randomized design. Each replicate was randomly sampled from 60 grape clusters (from both sides of the canopy; 5 grapes from different areas of each cluster) from 30 plants, for a total of 300 grapes. Sampling was scheduled at 8-d intervals to span grape development from veraison to commercial harvest (i.e., 0, 8, 16, 24, 32, 40, and 48 days after veraison). The grapes were collected at the same time of day (9–11 AM) and immediately stored at −80°C for subsequent analyses. Additionally, the temperature and humidity levels around grape bunches were recorded using a RC-4 automatic recorder (Jinchuang Co., Jiangsu, China). 2.2 HPLC analysis of anthocyanin compounds 8

The extraction and analysis of anthocyanins from the grape skins were performed according to previously published methods (Meng et al., 2012; Shi et al., 2017). The skins of triplicate samples from 100 grapes were peeled, ground in liquid nitrogen, and freeze-dried in an FD5-series vacuum FREEZE DRYER (GOLD SIM, Newark, NJ, USA). The powder was accurately weighed (0.5000 g) and then suspended in 5 mL of 1% (v/v) methanoic acid in methyl alcohol. The resulting suspension was ultrasonicated in water (< 28℃) for 10 min, shaken in darkness for 30 min, centrifuged at 12,000 g for 5 min, and then the supernatant was collected. The precipitate was re-extracted with the same solvent (5 mL) three times, and the supernatants were pooled and concentrated using a CentriVap® Concentrator (LABCONCO, Kansas, Missouri, USA). The residue was dissolved with 10 mL mobile-phase solvent (A:B; 9:1), and the suspension was passed through a 0.45 μm polypropylene syringe filter (Jinteng, Tianjing, China) for a quantitative HPLC analysis. The anthocyanin analysis was performed using an Agilent 1100 Series & Agilent Technologies 1100 series LC/MSD (Agilent Co., Santa Clara, CA, USA), reversed-phase column (Kromasil100-5C18, 250 × 4.6 mm i.d., 5 μm; Restek Co., Bellefonte,

USA).

The

mobile

phase

included

solvent

A (water/formic

acid/acetonitrile, 92:2:6, v/v/v) and solvent B (water/formic acid/acetonitrile, 44:2:54, v/v/v), establishing the following gradient: 1–18 min, 10%–25% B; 18–20 min, 25% B; 20–30 min, 25%–40% B; 30–35 min, 40%–70% B; 35–40 min, 70%–100% B. The column temperature was 50℃, and the flow rate was 1.0 mL min−1. The injection 9

volume was 30 μL. The anthocyanin content was detected at 525 nm. Quantitative determinations were carried out using the external standard method with malvidin-3-O-glucoside. 2.3 Enzyme extractions and activity analyses PAL and CHI were extracted based on a previous description (Lister, Lancaster, & Walker, 2015) with slight modifications. Briefly, grape skins (1.0 g) were ground in 50 mM precooled borate buffer (pH 8.3), containing 5 mM β-mercaptoethanol, 1 mM EDTA-Na2, 5% glycerinum, 5% polyvinylpyrrolidone (PVP). C4H, 4-CL, F3H, and DFR were extracted according to the method of Kong et al. (2017) and Xu et al. (2014), in which grape skins (1.0 g) were homogenized in 200 mM pre-chilled Tris-HCl buffer (pH 7.5), including 0.1 M dithiothreitol, 5 mM β-mercaptoethanol, 25% glycerol, and 10% PVP. The homogenate was filtered and centrifuged at 10,000 g for 15 min at 4°C, and then the supernatant was used as a source of crude enzymes for assaying enzyme activity levels. PAL was determined in reaction mixtures containing 0.1 M borate buffer (pH 8.8), enzyme extract and 20 μM L-phenylalanine (Blank instead of ultrapure water). After incubation at 30℃ for 60 min, the reaction was stopped by adding 6 M HCl. The method of Lister et al. (2015) was used to assay CHI activity. CHI was assayed in a buffer containing 50 mM Tris-HCl buffer (pH 7.6) with 18.5 μM 2-ethoxyethanol, enzyme extract and 0.1% chalcone. After incubation, the reaction was stopped by adding 0.1mL 75% trichloroacetic acid. C4H was measured using the method described by Cao, Hu, Zheng, & Lu (2010). The enzyme-responsive incubation 10

system contained 2 mM β-mercaptoethanol, and 0.5 mM NADPH in 50 mM phosphate buffer, enzyme extract, and 0.2 mM trans-cinnamic acid. The mixture was incubated for 1 h at 37°C. 4CL was determined using the method of Knobloch & Hahlbrock (1977). The 4CL assay was performed at 37°C for 1 h in an assay mixture containing 0.8 mM ATP, 7.5 mM MgCl2, and 38 M CoA in 100 mM Tris-HCl buffer (pH 7.5), enzyme extract, and 0.2 mM p-coumarate. F3H and DFR were assayed according to the method of Katsu et al. (2017) by monitoring the change in enzyme activity, equal to a change of 0.01 per hour. The reaction mixture consisted of 50 mM Tris-HCl (pH 7.5), 5 mM NADPH, crude enzyme extract, and naringenin or dihydroquercetin/dihydromyricetin. Reaction tubes were incubated at 30°C for 60 min. One unit of enzyme was expressed as U mg−1·h−1 protein. Protein was assayed with bovine serum albumin as the standard (Miao et al., 2016). 2.4 Total RNA and cDNA preparation and RT-PCR analysis Total RNA was isolated from ground grape skin tissues using a General Plant Total RNA Extraction Kit (Bioteke, Beijing, China) and purified with RNase-free DNaseI (Bioteke) to remove any contaminating gDNA. The integrity of the RNA was determined using a Bioanalyzer 2100 (Agilent). Total RNA concentration, values of A260/A230 and A260/A280 were measured using a Spectrophotometer (BioDrop μlite, Cambridge, England, UK) and further verified by 1% formaldehyde-agarose gel electrophoresis. First-strand cDNA was synthesized using HiScript II Q RT SuperMix for qPCR (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The qPCR primers were synthesized by the Biotech Co., Ltd (Shanghai, China) and 11

checked in advance by melting curve analysis and electrophoresis to avoid dimer formation and unspecific amplifications. The primers are detailed in Supplemental Table S1. The real-time quantitative PCR (qPCR) reaction system (20 μL) was prepared using a 2 × SYBR® real-time PCR Premixture kit (Bioteke), which consisted of 10 μL of 2 × Premix, 0.5 μL of each primer (10 μM), 2 μg·μL−1 of diluted cDNA, and the appropriate volume of double-distilled H2O. Subsequently, the expression analysis of three replicates was performed using the CFX96 Real-Time PCR Detection system (BIO-RAD, Hercules, CA, USA). The thermal cycling conditions were an initial denaturation at 94°C for 2 min, followed by 42 cycles of amplification (denaturation at 94°C for 15 s and annealing/extension at 60°C for 30 s). The VvActin gene was chosen for the normalization of gene expression based on its relatively constant expression throughout grape ripening. Expression levels were calculated based on the 2−ΔΔCT method (Livak & Schmittgen, 2001) using the T2 grape skins of ‘Cabernet Sauvignon’ from the first sampling date in 2017 as the reference sample. 2.5 Statistical analysis Data were expressed as mean ± standard deviation (SD) of triplicate experiments, and a one-way analysis of variance (ANOVA) was carried out to establish significant differences at P < 0.01 (**), P < 0.05 (*). All statistical analyses were performed using the SPASS.20.0 program (SPSS Inc., Chicago, IL, USA). Figures were generated with Origin Pro 2016 (OriginLab, Northampton, MA). Hierarchical cluster analysis was using R-3.5.1 software. 12

3 Results and discussion 3.1 Modulation of rain-shelter cultivation on grape anthocyanins In grapevine berries, the changes in sugar and acid are well described, with the reducing sugar concentrations accumulating as ripening progresses, and the titratable acid concentrations decreasing (Supplemental Fig. S1). Anthocyanins accumulate simultaneously in grapes, and the production of a wide variety of anthocyanin compounds are further modified through glycosylation, methylation, and acylation events (Lecourieux et al., 2017). In this paper, 18 individual anthocyanin compounds were detected through HPLC analysis in ripe grapes from two consecutive vintages (Table 1). Principal component analyses (PCAs) are particularly useful for simplifying and visualizing data sets and help to identify potential correlations in the underlying data sets. In 48 days after veraison (DAV; i.e., at harvest), Mv was the largest constituent in both years, followed by Mv-acet and tMv-coum. These three anthocyanins were predominant in the reddest ‘Cabernet Sauvignon’ grapes (Supplemental Fig. S2). While PCA models are unsupervised and elucidate the maximal variation in the data, Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA) models are supervised prediction and regression methods that identify the variables statistically contributing to, and the optimal models for, treatment and sampling vintage discriminations (Young et al., 2016). OPLS-DA multivariate statistics outlined Mv, Mv-acet, tMv-coum, Pn-acet and Pt-coum as the most discriminant anthocyanins (Supplemental Fig. S3). Compared with T2, the total anthocyanins, Mv and Mv-acet, decreased by 12.53%, 12.06%, and 8.42% after 13

treatment with T1 in 2017, respectively. However, the effect was not significant in 2016. Variations in the composition and content of anthocyanins were closely correlated with micrometeorological alterations in vineyard resulting from the plastic covering under rain-shelter cultivation (Meng et al., 2012). The inter-annual variations of climate caused intermittent rainfall from veraison to harvest in 2016; however more stressful continuous rainfall conditions occurred during ripening in 2017. It was important that there was full sunshine the day before harvest with a lower relative humidity and higher temperature (Supplemental Fig. S4). Our results were consistent with previous studies in which lower humidity levels, appropriate light conditions and the surrounding air temperature could promote anthocyanin biosynthesis (Li, et al, 2013; Kyraleou et al., 2016; Xu et al., 2014). Significant seasonal variations in the total anthocyanin content (TAC) and anthocyanin components (i.e., the composition of glycosylation, acylation, and methylation patterns) of skin extracts during ripening in the two consecutive years were shown in Fig. 1. The TAC increased from veraison to 16 DAV in 2016 and to 24 DAV in 2017, after which it mildly strengthened and even decreased. The TAC was strongly weakened by the unfavorable seasonal rain, while significantly upturned at 48 DAV, particularly in T2 (especially in 2017). On the contrary, T1 significantly ameliorated the condition to protect the anthocyanin regulatory mechanism, and also delayed the completion of maturation (Li et al., 2014; Shi et al., 2017). There was a consistent result that high air temperature and humidity levels were detrimental to anthocyanin accumulation (Bergqvist, Dokoozlian, & Ebisuda, 2001; Degu et al., 14

2016; Gao et al., 2016), while re-exposure to sunlight results in a greater resilience to metabolic alterations in anthocyanin synthesis at the post-veraison stage in red grape clusters. In this study, the major anthocyanins were Mv, Mv-acet, and tMv-coum through grape ripening, which accounted for 75%–80% of the TAC. The amplitudes of variation for individual anthocyanins were similar under both T1 and T2, whereas almost all anthocyanin significantly increased in concentration under T1 during the rainy season. Acylated and non-acylated anthocyanins represented approximately 50% of the TAC. The composition of non-acylated forms was significantly greater and predominant when anthocyanins were modified under T1 compared with under T2 during the rainy season. The ratios of 3′5′-substituted/3′-substituted and methylated/non-methylated anthocyanins (nine-fold) were constant at all sampling times after veraison. The average composition of non-acylated and non-methylated anthocyanins was obviously greater under T1, which was probably because they easily produced other metabolites from unstable structures when there was unexpected fluctuation of seasonal rain under T2; this finding was not completely consistent with those of a previous study, which found that rain-shelter cultivation did not alter the anthocyanin composition (Li et al, 2014). The positive effects of rain-shelter cultivation on fruit coloring occurred during the rainy season. In contrast, rain-shelter cultivation had a negative effect on anthocyanin accumulation during dry weather at harvest. This finding indicated that plentiful light, appropriate temperatures, and lower ambient humidity were important 15

environmental factors for anthocyanin accumulation, particularly for forming grape color at maturation. Consistent with our results, other researchers have also found that temperature, humidity, and sunlight exposure affect apples (Ubi et al., 2006), grapevine berry (Degu et al., 2016; Li et al., 2014; Meng et al., 2012), and strawberry (Miao et al., 2016) coloring. Additionally, Bergqvist et al. (2001) found that sufficient sun irradiation during the late ripening period of the grape developmental process contributed most to anthocyanin content. However, frequent climatic fluctuations during the ripening period were the primary negative factors that influenced anthocyanin biosynthesis. For example, non-acylated forms of anthocyanin decreased in the rainy season, but increased 20.53% under open-field cultivation and decreased 7.88% under rain-shelter cultivation at harvest in 2017, respectively. 3.2 Effects of rain-shelter cultivation on enzyme activities The action of many related enzymes in the phenylpropanoid pathway might contribute to the accumulation of anthocyanins in grape skins (Sun et al., 2017; Xu et al., 2014). In the present study, the activities of six pivotal enzymes, PAL, C4H, 4CL, CHI, F3H, and DFR, were investigated in grape skins during ripening (Fig. 2). PAL is a critical enzyme in the development of the color and flavor of ‘Cabernet Sauvignon’ grapes that is used to channel carbon catalyzed phenylalanine to cinnamic acid in phenylpropanoid metabolism (Cao et al., 2010; Xu et al., 2014). Compared with T2, the T1-treated samples first had lower PAL activity levels, and then it significantly increased throughout ripening (around 40 DAV), followed by a non-significant difference at 48 DAV. Thus, PAL activity was affected by rain-shelter cultivation, 16

which slowed the stimulation caused by the high ambient humidity and temperature that occurred during the rainy season. The activities of C4H, 4CL and CHI exhibited similar dynamic changes. C4H and 4CL that modify cinnamic acid are used to produce p-coumaroyl-CoA, the flavonoid precursor (Miao et al., 2016). The C4H activity had a greater continuity level and reached a maximum level at 32 DAV. It had a greater activity under T1 than T2 after 24 DAV in 2016 but showed no significant difference in 2017. After CHI, the flavonoid pathway diverges into side branches leading to different classes of flavonoids, including anthocyanins (Zorenc et al., 2017). The T1 markedly increased the activities of 4CL and CHI, which continued at greater levels throughout grape ripening after 24 DAV, while the differences between T1 and T2 diminished in a minority of sampling dates. F3H belongs to the 2-oxoglutarate-dependent dioxygenases family and is essential for flavonol synthesis in Vitis. It is a critical enzyme in controlling cyanidin derivatives (Kumar & Yadav, 2013). Nevertheless, no significant activity changes were observed at any sampling date in both years, with the exception of only a few samples in which the activity of F3H under T1 was significantly greater than that of T2. DFR is the conclusive enzyme that can catalyze three substrates, dihydrokaempferol,

dihydroquercetin

and

dihydromyricetin,

committed

to

anthocyanin and proanthocyanidin biosynthesis (Katsu et al., 2017). Compared with under T2, DFR activity increased by 31.96% in T1 at the last sample periods in 2016, while no significant differences were found in 2017. The TAC and DFR activity 17

showed increasing patterns during fruit ripening, with a slight decreasing tendency to different extents during the rainy season of a corresponding vintage. Additionally, compared with T2, most enzyme activities in T1 showed slightly inhibitive effects during the early sampling stage and have substantial positive impacts during the rainy season. It may be possible to confirm that the variations in these enzyme activities were to the result of the microclimate around grape clusters during the rainy season (Gao et al., 2016). 3.3 Effects of rain-shelter cultivation on gene expression levels Numerous

anthocyanin-

and

anthocyanin-precursor-related

genes

were

significantly affected by rain-shelter cultivation during ripening (Fig. 3). In red grapes, VvPAL plays a critical role in regulating PAL by catalyzing phenylalanine into cinnamic acid in phenylpropanoid biosynthesis pathways (Boss, Davies, & Robinson, 1996; Miao et al., 2016). In this paper, the expression of VvPAL in grape skins treated with T1 was significantly up-regulated compared with under T2 after 32 DAV, especially at harvest. The expression was significantly lower in T1-treated grapes from veraison to 24 DAV in 2017, while no significant difference was observed between T1 and T2 before 32 DAV in 2016. The expression levels of VvC4H and Vv4CL experienced a similar declining trend under T1 and T2 throughout grape ripening. VvC4H and Vv4CL expression levels were greater under T1 after veraison and subsequently, the opposite result was obtained at harvest in 2016. The next vintage showed a consistent result, although slightly significant differences were observed in both T1 and T2. 18

Precursors of the flavonoid pathway are initially recruited from the phenylpropanoid pathway by a small family of chalcone synthases (i.e., CHS1, CHS2 and CHS3) and enter the flavonoid pathway (Castellarin et al., 2007). The expression levels of VvCHS3 in T1 was lower than in T2 before 24 DAV; however, the expression was significantly greater in T1 compared with T2 by 32-40 DAV. A rally in VvCHS3 transcript level was observed under T1 at harvest in 2017. It is consistent with light-treated grapes also maintain greater expression levels of biosynthetic enzymes and transcription factors (Sun et al., 2017). A similar pattern was observed for the VvCHI1 transcripts. Anthocyanin hydroxylation is mediated by F3′H and F3′5′H activities, which regulate the pathway branching point that addresses precursors to the parallel synthesis of di- or tri-substituted anthocyanins in plants, respectively (Bogs, Ebadi, Mcdavid, & Robinson, 2006; Sun et al., 2015). The early biosynthesis-related genes VvF3′H and VvF3′5′H had lower expression levels under T1 at the onset of the sampling period, followed by a gradual increase, with their levels being greater under T1 than T2 (a clear rebound in VvF3′H at harvest in 2017). However, this expression trend of VvF3′5′H was not found in 2017. The VvF3H2 and VvDFR genes were observed to respond identically to rain-shelter cultivation in this study. Compared with T2, the T1 in this study resulted in lower expression levels at earlier stages, but greater expression levels were observed from 16 DAV to 40 DAV in the successive vintages. However, both VvF3H2 and VvDFR exhibited low expression levels thereafter in 2016. 19

The UFGT is the critical anthocyanin-specific enzyme that catalyzes proanthocyanidin (i.e., the glycosylation of unstable anthocyanidin aglycones) into pigmented anthocyanins (Boss et al., 1996; Castellarin et al., 2007). According to previous studies, proanthocyanidins are localized throughout the fruit tissue and gradually decrease during fruit development (Schaart et al., 2013) owing to members of the GST protein family that participate in complicated trafficking from cytosol to vacuole and sequestration of anthocyanin (Castellarin et al., 2007; Zorenc et al., 2017). In the vital step of anthocyanin biosynthesis, the VvLDOX and VvUFGT transcript levels under T1 were greater than under T2 in 2016, and they showed the same tendencies during the rainy season in 2017. Thereafter, T1 in particular led to significantly lower expression levels in 2017, while no significant difference was observed at harvest in 2016. Thus, the increases in VvLDOX and VvUFGT transcript levels at maturation had positive effects on anthocyanin accumulation. Compared with the VvUFGT gene, a hysteretic tendency of VvGST transcripts was also observed in grapes, and remained undifferentiated in the last phase. The general regulators of the flavonoid pathway are MYB loci, including VvMYB5a (also named VvMYBCS1) and VvMYB5b, which are involved in modulating the several branches of the flavonoid pathway in grapevine (Cavallini et al. 2014; Deluc et al., 2006). VvMYBPA1 and VvMYBPA2 specifically control proanthocyanin synthesis (Sun et al., 2015). To determine the levels of transcription factors for controlling anthocyanin biosynthesis in seasonal rain during grape ripening, the expression levels of VvMYB5a, VvMYB5b, VvMYBPA1, and VvMYBPA2 were 20

measured (Fig. 4). Among the transcription factors, a phasic fluctuation in the expression level of VvMYB5a was measured in 2016, while a relatively steady trend was obtained throughout grape ripening in 2017. The VvMYB5a transcripts had a high similarity to the profiles of VvF3′H and VvF3′5′H. Consistent with this result, Cavallini et al. (2014) reported that the up-regulation of MYB5a may influence flavonoid/phenylpropanoid more than anthocyanin biosynthesis, especially the F3′H and F3′5′H branches in Vitis. The expression level of VvMYB5b significantly increased during all development stages in grapes under T1 and T2, indicating that its expression is positively regulated at anthocyanin biosynthesis. Grapes under T1 had a slightly greater VvMYB5b transcript level during 16-40 DAV in 2016 and increased with alternating fluctuations during the rainy season in 2017. No significant differences were exhibited between T1 and T2. The increased expression levels of two well-characterized proteins MYBPA1 and MYBPA2 have been reported, which are positive regulators in proanthocyanidin synthesis, in grapes from water- and heat-stressed plants (Cáceresmella et al., 2017; Lecourieux et al., 2017). In our results, the expression of VvMYBPA1 had a profile that resembled that of VvMYBPA2, which plays a key role in negatively regulating the metabolic levels of anthocyanins during grape ripening. They were greater in skins at veraison in both years, and then continuously declined to barely detectable levels under both T1 and T2 in 2017. The seasonal fluctuations affected the expression levels of VvMYBPA1 and VvMYBPA2 in rain stress level-dependent manner, with greater values under T1 during 16-40 DAV, while expression levels of VvMYBPA1 in 21

2017 was observed an exception. 3.4 Hierarchical cluster analysis Anthocyanin biosynthetic reactions and their regulation in grapes do not occur separately, but usually involve different enzymes and genes that interact with and regulate each other, and finally lead to changes in the end-products. The anthocyanin biosynthesis-related enzymes and genes were observed in the above analysis, and many were positively or negatively regulated in response to the rainy season during grape ripening. The heatmaps and hierarchical cluster analyses of the compositional changes of anthocyanins, and associated enzymes and genes in the anthocyanin biosynthesis pathway showed a clear grouping pattern (Fig. 5). The data of relative anthocyanin contents, enzyme activities, and gene expression levels represented by color segments at the corresponding locations are listed in Supplemental Table S2. In the dendrogram, all anthocyanins clustered together, which indicated that the dynamic changes of various anthocyanin compositions were strongly consistent with total anthocyanins concentrations during grape ripening under both T1 or T2; among them, non-acylated

and

non-methylated

anthocyanins

were

mainly dispersed

in

anthocyanins remote locations, compared with other anthocyanins. The results showed that non-acylated and non-methylated anthocyanins were highly sensitive to the ambient fluctuations. However, rain-shelter cultivation could decrease the fluctuation from seasonal rain and to protect the unstable structures of anthocyanin by the heatmaps analyses. This was consistent with anthocyanin composition changes on during grape ripening. 22

In addition, strong correlations were found between anthocyanins, enzymes (CHI and DFR), and a transcription factor (VvMYB5b) in grape skins under T1 (Fig. 5 A). Crucial enzymes (DFR, CHI, PAL, C4H, and 4CL), transcription factor (VvMYB5b), and biosynthesis-related gene (VvPAL) were positively associated with total anthocyanin content changes and the composition of anthocyanin in grape skins, and they behaved as major regulators of anthocyanin biosynthesis during ripening (Fig. 5 B). In addition, the expression levels of anthocyanin biosynthesis-related genes (VvPAL VvF3′5′H, VvF3H2, VvLDOX, VvUFGT, VvDFR, and VvGST, ect.) was positively associated with anthocyanin changes to a certain extent. This tendency was observed in a previous study, which showed that PAL, 4CL, CHI, and DFR activities were correlated with the flavonoid biosynthesis pathway in Arabidopsis leaves (Saito et al., 2013), and DFR is the primary enzyme is closely associated with anthocyanin synthesis regulation in fruits (Kou et al., 2019). 4 Conclusion In this study, the metabolite profiles and expression levels of enzymes and genes in the anthocyanin biosynthesis pathway were analyzed in ‘Cabernet Sauvignon’ grapes under rain-shelter cultivation and open-field cultivation during berry ripening. The magnitudes of characterized metabolite fluctuations were shown, and anthocyanin accumulation was suppressed during the rainy period. Compared with open-field cultivation, grapes cultivated under rain-shelter cultivation had greater anthocyanin contents in the rainy season; however, greater resilience was observed under open-field cultivation at harvest. Overall, rain-shelter cultivation protected the 23

non-acylated and non-methylated anthocyanin compositions, which confirmed the importance of rain-shelter cultivation on stabilizing anthocyanin structure in response to the rain season. Furthermore, total anthocyanin and various anthocyanin concentrations were positively associated with CHI and DFR, and the transcription factor VvMYB5b under the two viticultural measures. The relative expression levels of anthocyanin biosynthesis-related genes, such as VvPAL, VvF3′5′H, VvF3H2, and VvGST & etc., were also positive roles. We hypothesize that cultivation with rain-shelter plastic films will be particularly important for the grape growers and winemakers prior to the rainy season; then, berries should be exposed to full sunlight at pre-harvest. This approach might be a useful supplemental cultivation practice that enhances anthocyanin content to produce optimal wine color. Conflict of interest The authors declare no conflict of interest. Acknowledgements This work was supported by the National Key Research and Development Program of China (2018YFD0201300) and the China Agriculture Research System from the National Technology System for Grape Industry (CARS-29-zp-6) financially. The experiments were finished in the Key Laboratory of Viticulture and Enology, Ministry of Agriculture, China.

Conflict of interest The authors declare no conflict of interest.

24

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Figure Captions Fig. 1 The content and composition of anthocyanin in grapes under rain-shelter cultivation (T1) and open-field cultivation (T2) during ripening. 31

A, The total of anthocyanin content in 2016. B, The average composition of anthocyanin in 2016. C, The total of anthocyanin content in 2017. D, The average composition of anthocyanin in 2017. Each value is shown by mean ± SD. Vertical bars represent the SD of the means. Different asterisks show significantly different at p<0.01, **; p<0.05, *. The blank column represent the rain-shelter cultivation, the dark grey column represent the open-field cultivation. Fig. 2 The activity of PAL, C4H, 4CL, CHI, F3H and DFR in grapes under rain-shelter cultivation (T1) and open-field cultivation (T2) during ripening. Fig. 3 The relative expression of anthocyanin biosynthetic genes in grapes under rain-shelter cultivation (T1) and open-field cultivation (T2) during ripening. Fig. 4 The relative expression of transcription factors in grapes under rain-shelter cultivation (T1) and open-field cultivation (T2) during ripening. Fig. 5 Hierarchical clustering analyses of differential compositions of anthocyanin and anthocyanin biosynthesis-related enzymes and genes for grape during ripening. A, Rain-shelter cultivation (T1). B, Open-field cultivation (T2). The abscissa indicates the sampling time; the ordinate indicates the differential anthocyanins, enzymes and genes. Red and blue segments indicate a relatively high and low expression levels, respectively, for each anthocyanin, enzyme or gene. The relative data represented by color segments at the corresponding locations are listed in Supplemental Table S2. Supplemental Fig. S1 Berry characterization: concentration of the reducing sugar and titratable acid in grape berries during ripening. 32

Supplemental Fig. S2 Unsupervised PCA of all individual anthocyanins in ripe grape. A, Scores plot for the respective samples. B, Loadings plot for the measured variables. C, Biplot for main variation and samples. Supplemental Fig. S3 Supervised OPLS-DA of individual anthocyanins in ripe grape. A, Scores plot for the respective samples. B, Feature importance for the measured variables. Supplemental Fig. S4 Characterization of the microclimate: the parameters of daily air temperature and relative humidity around the grape cluster of experimental period.

33

Table1 Individual anthocyanin profiles of ripe grape berries under rain-shelter cultivation (T1) and

Anthocyanins (mg g-1 skin dry weight (DW))

[M+]/[M-H]−

2016

2

(Frag.MS m/z)

T1

2017 T2

T1

T2

Delphinidin-3-O-glucoside (Dp)

465(303)

0.929±0.007

0.934±0.011

0.604±0.010

0.867±0.018 *

Cyanidin-3-O-glucoside (Cy)

449(287)

0.097±0.001 *

0.088±0.001

0.114±0.002

0.208±0.005 *

Petunidin-3-O-glucoside (Pt)

479(317)

0.873±0.004

0.873±0.010

0.486±0.002

0.646±0.010 *

Peonidin-3-O-glucoside (Pn)

463(301)

0.940±0.001

0.935±0.012

0.842±0.008

1.212±0.011 *

Malvidin-3-O-glucoside (Mv)

493(331)

8.750±0.017

8.902±0.093

5.870±0.084

6.675±0.032 *

Delphinidin-3-O-(6-acetyl)-glucoside (Dp-acet)

507(303,465)

0.177±0.001

0.179±0.002

0.154±0.001

0.216±0.001 *

Cyanidin-3-O-(6-acetyl)-glucoside (Cy-acet)

491(287,449)

0.140±0.000

0.148±0.001

0.093±0.001

0.120±0.005 *

Petunidin-3-O-(6-acetyl)-glucoside (Pt-acet)

521(317,479)

0.278±0.001

0.278±0.003

0.003±0.003

0.008±0.001

Delphinidin-3-O-(6-coumaryl)-glucoside (Dp-coum)

611(303,465)

0.021±0.009

0.039±0.000

0.027±0.003

0.030±0.002

Peonidin-3-O-(6-acetyl)-glucoside (Pn-acet)

505(301,463)

0.568±0.003

0.591±0.007

0.450±0.006

0.562±0.016 *

Malvidin-3-O-(6-acetyl)-glucoside (Mv-acet)

535(331,493)

6.250±0.011

6.471±0.066

3.917±0.006

4.247±0.055 *

Peonidin-3-O-(6-caffeoyl)-glucoside (Pn-caff)

625(301,463)

0.019±0.001 ** 0.014±0.001

0.005±0.001

0.010±0.002 *

Malvidin-3-O-(6-caffeoyl)-glucoside (Mv-caff)

655(331,493)

0.162±0.001 ** 0.128±0.003

0.066±0.001

0.071±0.006

Petunidin-3-O-(6-coumaryl)-glucoside (Pt-coum)

625(317,479)

0.090±0.001

0.098±0.002 * 0.034±0.005

0.032±0.001

Peonidin-3-O-(cis-6-coumaryl)-glucoside (cPn-coum)

609(301,463)

nd

0.001±0.000 * 0.024±0.004

0.020±0.001

Malvidin-3-O-(cis-6-coumaryl)-glucoside (cMv-coum)

639(331,493)

0.119±0.001

0.135±0.003 * 0.128±0.004

0.130±0.007

Peonidin-3-O-(trans-6-coumaryl)-glucoside (tPn-coum)

609(301,463)

0.273±0.003

0.278±0.006

0.300±0.267

0.310±0.008

Malvidin-3-O-(trans-6-coumaryl)-glucoside (tMv-coum) 639(331,493)

2.366±0.015

2.510±0.041 * 1.619±0.065

1.481±0.023

Total anthocyanins

22.042±0.024

22.601±0.264 14.733±0.003 16.843±0.193

Values are means of duplicate determination ± S.D. nd, means not detected. Different asterisks in each row show

significantly different (p<0.01, **; p<0.05, *).

34

Anthocyanin biosynthesis regulation was evaluated in rain-shelter cultivated grapes. Anthocyanin biosynthesis was suppressed during the rainy season. Obvious resilience was observed under open-field cultivation at harvest. Anthocyanin biosynthesis was strongly associated with VvMYB5b and crucial enzymes.

35

36

37

38

39

40