SONG Chang-zheng, WANG Chao, XlE Sha, ZHANG Zhen-wen,

1 College of Enology, Northwest A&F University, Yangling 712100, P.R.China

2 Shaanxi Engineering Research Center for Viti-Viniculture, Yangling 712100, P.R.China

1. lntroduction

Canopy management includes a series of viticultural practices such as trellis system, vigor controlling, shoot trimming, cluster thinning and leaf removal, and they have been widely used throughout the world in grape production for many years (Osrecak et al. 2015; Song et al.2015; Sivilotti et al. 2016). They can change the canopy microclimate of grapevine by modifying sunlight exposure,vine temperature and air circulation and so on to affect the vine growth and quality of grape and subsequence wine(Duncan et al.1995; Reynolds et al. 1996; Tardaguila et al.2008).

Leaf removal around the fruit clusters has been commonly applied in cool climate viticultural regions around the world, and many studies have shown notable effects of such practice on the quality parameters of mature grape(Poni et al. 2013; Risco et al. 2014; Baiano et al. 2015; Song et al. 2015; Osre?ak et al. 2016). Timing and intensity of leaf removal are the most important factors that determine the final effect on grape quality (Intrigliolo et al. 2014;Sternad Lemut et al. 2015; Acimovic et al. 2016). Some researches have indicated that earlier leaf removal could effectively improve the quality of grape and wines (Intrigliolo et al. 2014; Sternad Lemut et al. 2015). On the other hand,other reports suggested that earlier onset of ripening that occurs often in the hot seasons could lead to unbalanced wines, and late apical leaf removal was a tool for delaying ripening and consequently modify the wine quality (Palliotti et al. 2013a, b; Poni et al. 2013).

The crop load of grape is another factor that closely related to the fruit quality and it influences the yield and quality in the next year to a large extent as well (Gil et al.2013). In the study by Palliotti et al. (2000), significant reduction of yield could be achieved when 40% or more clusters were thinned. Gil et al. (2013) reported that cutting 50% of the clusters at mid-veraison could reduce grape yield per vine by around 40%. In the study on Doonuri grape by Chang et al. (2015), the grapes with less 1.5 tons per 10 acres were best for producing high quality wine. As for the timing of thinning, previous study showed that cluster thinning at 8 weeks after flowering was best for improving grape quality (Kok 2011). Too early cluster thinning may lead to the reduction of photosynthetic rate and furtherly restricted the accumulation of sugar in the rest berries(Fanzone et al. 2011).

Due to the difference of cultivar, climate, vineyard location,rootstock and other factors, the same combination of leaf reamoval and cluster thinning protocol may lead to conflicting results. Therefore, determining appropriate protocol for the given cultivar growing in special grape region is vital for producing high quality grapes and wines. The semi-arid area Weibei Dryland is an important wine production region in northwestern China. Wine grapes grown there do not have to be buried to overwinter making this area a promising region for wine production. However, the warm climate and insufficient light exposure during berry ripening negatively affect the quality of grapes and wines. The aim of present study was to modify the microclimate of vine canopy by combination of leaf removal and cluster thinning, providing theoretical basis for grape and wine production in regions with similar ecological conditions around the world. According to results of previous researches (Kok 2011; Poni et al. 2013), both leaf removal and cluster thinning were performed before veraison (55 days after anthesis), and the orthogonal experiment with 2 factors of 3 levels were carried out in vintage of 2013 and 2014.

2. Materials and methods

2.1. Plant materials and field trial

The vineyard of red variety Vitis vinifera cv. Cabernet Sauvignon (CS) and white variety V. vinifera cv. Ugni Blanc(UB) were located in Kou Town and Beiyu Town in Jingyang County, Xianyang City, Shaanxi, China, respectively. The region belongs to the Weibei Plateau which characterized by a semiarid continental monsoon climate with 2 195.2 h of sunshine, 4 330°C of active accumulated temperature annually and an average frost-free period of 213 days per year. The average low temperature in winter and high temperature in summer were –6 and 34°C, respectively.And average annual precipitation was 548.7 mm.

The own-rooted vines were both planted in 2008 in north–south orientation. Spaces within and between the vine rows were 1.0 m×2.0 m for CS and 0.8 m×2.5 m for UB. Both of the varieties were moderately winter-pruned into bilateral cordon trellis, and water and fertilizer were controlled in conventional management ways.

At pre-veraison in late July (55 days after anthesis), 3 levels of basal leaf removal, 2, 4 and 6 leaves removed from base of the shoots, were conducted on the 2 varieties.Meanwhile, 3 levels of crop load by cluster thinning (HL, high crop load; ML, middle crop load; LL, low crop load) were set,which were 16, 33 and 66% of clusters thinned per plant.Among these treatments, 2 leaves removal and high crop load (2 HL, Table 1) was the most similar way with the local conventional management. Randomized block design with three replications was used, and each treatment replicate contained at least 15 vines.

2.2. Sample collection and analysis of index

At the optimum technological maturity of each year, which was judged by ratio of sugar and acid content, a 500-berrysample was randomly collected from each replicate. All samples were transported in low-temperature to laboratory immediately and stored at ?40°C prior to subsequent analysis.

Table 1 Treatments of canopy managements for wine grape Vitis vinifera cv. Cabernet Sauvignon (CS) and Ugni Blanc (UB)

2.3. Determination of basic physicochemical parameters and phenols in berry skins

Reducing sugar (RS) and titratable acidity (TA) were determined with Fehling reagent and sodium hydroxide titration, respectively, according to the National Standard of the People’s Republic of China (GB/T 15038-2006). The berry pH was measured using PB-10 pH meter (Sartorius,Gottingen, Germany).

Phenols extraction was performed according to Song et al. (2015a). Total phenols (TP) content was determined by using the Folin-Ciocalteu method (Jayaprakasha et al.2001). Total anthocyanins (TAC) content was determined by the pH-differential method (Meng et al. 2012a). Tannin(TAN) was determined by the methyl cellulose precipitation(MCP) method (Sarneckis et al. 2006). The measurements were performed in triplicate.

2.4. Determination of anthocyanin profile

The extraction of anthocyanins was performed according to the method previously reported (Liang et al. 2014). The chromatographic analyses of anthocyanins were performed in twice for each sample using Shimadzu LC-20 A HPLC system(Shimadzu, Kyoto, Japan). A volume of 2 mL of extract was injected into the system and eluted by using a Hibar RT Lichrospher SB-C18 column (250 mm×4.6 mm, 5 μm) and detected at 520 nm. The mobile phase included solvent A(water/acetonitrile/formic acid, 800/100/25, v/v) and solvent B (water/acetonitrile/formic acid, 400/500/25, v/v). The gradient elution conditions were as following: 0.00–45.00 min,0–70% B; 45.00–50.00 min, 70% B; 50.00–51.00 min, 70–0%B; 51.00–55.00 min, 0% B. The column was held at 35°C and was flushed at a flow rate of 1.0 mL min–1.

2.5. Determination of polyphenol compounds

The extraction procedure of polyphenol compounds was completed in dark condition. The dried sample skin (2.0 g)was added into 3 mL water and 25 mL ethyl acetate, and shaken for 30 min at 130 r min–1and 25°C. Then the supernatant was collected and filtered through 0.45-μm organic membrane into an evaporation flask. Extraction of the residue was repeated 3 times in the same conditions.Thereafter, 3 supernatants were combined and evaporated to dryness under vacuum at 30°C and re-dissolved in 10 mL of chromatographic methanol. The methanol extract was injected into a Shimadzu HPLC System and analyzed according to the previous method (Meng et al. 2012b).Polyphenol compounds were identified by comparing their retention times with those of pure standards and by spiking the samples with standard solutions. Quantification of the compounds was made by using the external standard method (Meng et al. 2012b). And the subclasses of polyphenol compounds were calculated. The measurements were performed twice for each sample.

2.6. Determination of volatile compounds

Fifty frozen berries were pounded to pieces in a mortar,and the obtained residue was homogenized with a chilled pulverizer after the seeds was removed. After 2.5 h standing, the homogenate was centrifuged at 8 000 r min–1for 20 min at 4°C. The supernatants (10 mL) was transferred into a 15-mL sample vial, which contained 2 g NaCl and 50 μL 0.234 g L–12-octanol, and then the SPME extraction and GC-MS analysis were conducted according to the methods in previous research (Zhang et al. 2013; Song et al. 2015b).The 2-octanol was used as internal standard substance for quantification. The volatile compounds were identified by comparing mass spectra of the sample with those from pure standards injected in the same conditions, and with those found in the NIST2.0 MS Library Database or in the literatures. The quantification of most volatile compounds was achieved using internal standard quantification method.For the quantification of those had no pure reference compounds, the quantification was achieved using the calibration graphs of the compounds with the most similar chemical structure according to formula and chemical character (Tao et al. 2008). Afterwards, contents of volatile compound subclasses were calculated.

2.7. Statistical analysis

Significant differences were assessed with one-way analysis of variance (ANOVA), and statistical differences between the means were evaluated using least significant difference(LSD) analysis at the P=0.05 level. For experimental variables, two-way ANOVA was applied with leaf removal and cluster thinning as the principal factors. Statistical data processing was performed using the software SPSS Statistics 20.0 for Windows (IBM, Armonk, NY, USA).

3. Results

3.1. General quality parameters of mature grape

Sugar, acids and phenols are the primary parameters that determine the general quality of grape. Among the leaf removal and cluster thinning treatments, the treatment 2 HL was most similar with the local conventional management.Therefore, treatment effects could be found by comparing the parameters with that of 2 HL.

The effect of comprehensive canopy managements on general quality parameters of mature grape in two consecutive years are showed in Table 2 (2013) and Table 3 (2014). In the year of 2013, the treatments did not significantly affect the content of RS in grape berry of CS.The total acid content under all other treatments decreased compared with treatment 2 HL, while no evident difference was found among the pH of berry juice in different treatment groups. The content of TP, TAN and TAC in most treatments was higher than that in 2 HL, and the most effective treatments were 2 ML, 4 ML and 4 LL. In the year of 2014,except for the significant enhancement in 2 ML and 4 LL,most treatments had no pronounced effect on RS content.The 2 ML, 4 LL, 6 HL and 6 ML reduced the content of TA,and the result was consistent with the notably higher pH of berries in these groups. Although the entire effects of these managements on general quality were not quite the same between the two years, promoting effects of 4 LL in both years were still notable. According to the results of two-way ANOVA, significant effects of both leaf removal and cluster thinning on contents of TP and TAC were found in both years.

As for UB, differences of RS contents among treatments were found, while the influence by treatments was not regular. Most treatments reduced the TA, which was also agreed well with the higher pH in the same treatments.Additionally, TP and TAN was also notably higher in most treatments than that in 2 HL in 2013. Due to the climate factors, such as more rain, the RS content in 2014 was generally lower than that in 2013, while the promoting effect was more significant for most treatments in this year. Similar influence was obtained on TA and pH in the second year.Whereas, the effects on TP and TAN in 2014 were not as notably as in 2013. The two-way ANOVA showed that the effects of leaf removal on TP and cluster thinning on RS were highly significant in both years.

It was worth noting that the comprehensive canopy managements could effectively influence the general qualityof mature grape in both years. Therefore, in 2014, further analysis was conducted on the profiles of monomeric anthocyanins, non-anthocyanins phenolic compounds and volatile compounds of mature berries in those treatment groups.

Table 2 General quality parameters of mature Vitis vinifera cv. Cabernet Sauvignon (CS) and Ugni Blanc (UB) berries (2013)

Table 3 General quality parameters of mature Vitis vinifera cv. Cabernet Sauvignon (CS) and Ugni Blanc (UB) berries (2014)

3.2. Anthocyanins profiles of CS grape

As shown in Table 4, a total of 9 monomeric anthocyanins compounds were detected. The concentration of total monomeric anthocyanins ranged from 5.37 to 12.48 mg g–1. The relative content of total monomeric anthocyanins in all treatments was not coincided with that of TAC using the pH-differential method. The reason might be the different expression methods. TAC was directly expressed as milligrams of cyanidin-3-monoglucoside equivalence per gram of dry berry skin (mg ME g–1DW), while actually the molecular weights of different primitive anthocyanidins were quite different, and the pH-differential method could not distinguish the different primitive anthocyanidins and whether they were acylated. Besides, leaf removal had more significant effects on the contents of monomeric anthocyanins compounds.

Fig. 1 showed the percentages of 5 individual anthocyanidins in total content of monomeric anthocyanins and acylation percentages. Malvidin and its derivatives were the most abundant anthocyanidins, and they took a percentage ranging from 74.39% (4 LL) to 82.19%(4 HL). Previous study showed that shading treatment could increase the percentage of malvidin and its derivatives(Downey et al. 2004), the lower percentage in treatments the higher intensity of leaf removal, which agreed well with that conclusion. Particularly, the lower malvidin percentage in 4 LL could also well explain the lower concentration of total monomeric anthocyanins, due to its higher molecular weights and more acylated derivatives. Petunidin and peonidin were the second abundant anthocyanidins of which the percentages ranged from 3.53 to 5.41% and from 8.62 to 12.94%, respectively. The other anthocyanidins, delphinidin and cyanidin, together accounted for a small percentage.The results were similar with that of other studies (Lee and Skinkis 2013; Feng et al. 2015). Notably, significant effectsof leaf removal and cluster thinning on most anthocyanidins were found.

Table 4 The concentration of monomeric anthocyanins in Vitis vinifera cv. Cabernet Sauvignon (CS) berries (2014) (mg g–1 skin DW)1)

Fig. 1 Percentages of 5 anthocyanidins (A) and acylation percentages (B) in Cabernet Sauvignon (CS) berries in 2014. Dps,delphinidin; Cys, cyanidin; Pts, petunidin; Pns, peonidin; Mvs, malvidin. HL, high crop load; ML, middle crop load; LL, low crop load. P-values of the ANOVAS of leaf removal (LR), cluster thinning (CT) and their interactions (LR×CT) are indicated. ＊, P＜0.05; ＊＊,P＜0.01; ＊＊＊, P＜0.001; ＊＊＊＊, P＜0.0001; ns, not significant. Different letters for the same anthocyanidin indicate a statistically significant difference at 5% level. Bars indicate the standard deviation of anthocyanidin percentages.

Fig. 1-B showed a notable influence of canopy managements on the acylation percentages of anthocyanidins. Non-acylated anthocyanins were the most abundant in all samples, which took percentages from 54.85 to 61.00%. It was worth noting that among the same leaf removal treatments, the percentages of cinnamylated anthocyanins decreased with the decrease of grape crop load. On the other hand, the percentages of non-acylated anthocyanins were lower in higher crop loading treatments.

3.3. Non-anthocyanin phenolics

As for the influence of canopy managements on the accumulation of non-anthocyanin phenolics, similar treatment effect was found on the two wine grape varieties(Table 5). Regardless of leaf removal treatments, the total content was higher in lower crop loading groups. Cluster thinning showed more significant effects than leaf removal.The highest three treatments were 4 LL, 6 LL and 4 ML for CS and 6 ML, 6 LL and 4 LL for UB, respectively. Flavonols were the dominate phenolics among the five categories. And its relative contents among different treatments were identical with that of TP. The accumulation of benzoic acids and cinnamic acids had different responses to the treatments for different varieties. In general, the mean and low crop loads enhanced the synthesis of benzoic acids in CS and cinnamic acids in UB as well. Interestingly, the content of flavanols had opposite responses in the two varieties. The lower crop load increased the flavanols contents in CS while decreased that in UB. Stilbenes were in trace amount for all samples,but promoting effect of lower crop load could still be noted for both varieties.

3.4. Volatile compounds

The content of volatile compound subclasses in the two wine grape berries were showed in Table 6. The total concentration of the volatile compounds in CS ranged from 925.34 μg L–1(2 HL) to 1 685.98 μg L–1(4 HL), and the treatments of 4 leaves removal had higher concentrations on average. On the other hand, cluster thinning had no notable effect on the total concentrations. Acids, alcohols and esters were 3 dominating volatiles, which took a percentage over 95% of the total volatiles in each sample. It is worth noting that the middle level’s treatments (2 LL, 4 HL, 4 ML, 4 LL and 6 HL) were beneficial for the synthesis of the three volatiles. Among these treatments,4 HL had quite higher concentrations of acids, alcohols, esters and owned the highest total concentration. The total variety numbers of volatiles ranged from 20 (4 ML and 6 ML) to 30(2 LL and 4 LL) (Appendix A), and the 3 treatments that had the highest total concentrations also had the most abundant volatile varieties (2 LL, 4 HL and 4 LL). Acids, alcohols and esters were also the volatiles that contained the most varieties. And the amount of those compounds determined the entire complexity of the volatile compounds to a great extent.

The total concentrations of the volatile compounds in UB ranged from 362.79 μg L–1(4 HL) to 1 923.99 μg L–1(6 LL),and the treatments of 6 leaves removal (6 HL, 6 ML and 6 LL) had higher concentrations on average. Different from CS, aldehydes other than acids were the most abundant volatiles of most UB samples. For each subclass of volatiles,the concentrations were quite different among the treatments,and both leaf removal and cluster thinning had significant effects on most subclasses of volatiles. Additionally, similar effects were found on the volatile variety of UB. Namely,samples under the middle level’s treatments owned more varieties of volatiles (Appendix A). Same with CS, 2 LL, 4 HL and 4 LL were also the treatments that had the most volatile varieties for UB.

4. Discussion

Grapevine canopy management is a vital practice for improving quality of vine, berry and subsequent wine, and it has provoked many studies around the world. However,inconsistent results were obtained probably due to the different management operations, local climatic conditions, cultivars and even rootstocks (Feng et al. 2015). Hence, searching for an optimum management mode is still very important for wine grape production.

In present study, no evident influence of the treatments on sugar content was found, which was similar to the results obtained by other researchers (Chorti et al. 2010; Tardaguila et al. 2010; King et al. 2012). The result suggested that six at most basal leaf removal was enough to support grape berry development and ripening in all crop loading levels.

Regarding to TA, previous study showed that cluster thinning at both pre-flowering and lag-phase of berry growth could result in low titratable acidity and high pH (Gatti et al. 2012).Others showed that defoliated vines, without cluster thinning,produced the lowest total titratable acidity in grapes (Bergqvist et al. 2001; Almanza-Merchán et al. 2011). Bavaresco et al.(2008) concluded that acidity was affected by leaf removal differently depending on meteorological conditions and cultivar.The present study indicated that these treatments of canopy managements in the region of Weibei Dryland could reduce TA in berries. This effect is meaningful for wine production in regions where the grape acidity is commonly high.

The quantitative and qualitative profiles of anthocyanins largely determine the grape and wine color. Many researches showed that leaf removal and cluster thinning can enhance the TAC content in both grape skin and subsequent wine(Gatti et al. 2012; Tardaguila et al. 2012; Sternad Lemut et al.2013; Intrigliolo et al. 2014; Matsuyama et al. 2014), which was mainly attributed to the increased sunlight exposure and temperature (Chorti et al. 2010; Feng et al. 2015). Similar result was obtained in this study on CS. Additionally, cluster thinning and leaf removal had different effects on individual compounds. The work of Filippetti et al. (2005) showed that cluster thinning reduced tri-substituted anthocyanins(Dp-3-glucoside, Pt-3-glucoside and Mv-3-glucoside) and increased di-substituted anthocyanins (Pn-3-glucoside and Cy-3-glucoside). And according to another research (Sternad Lemut et al. 2013), early leaf removal showed a reduction in peonidin glucoside but an increase in all other individual compounds. The changed leaf/fruit ratio among different treatments, together with the changed sun exposure and surrounding temperature, could promote either biosynthesis or degradation of anthocyanins (Fanzone et al. 2011).Therefore, it is reasonable that different combinations of leaf removal and cluster thinning treatments would result in different effects on monomeric anthocyanin profiles. In the work by Matsuyama et al. (2014), the overexpression of flavonoid 3′,5′-hydroxylase (F3′5′H) in the leaf removal grape skins suggested that leaf removal contributed to the accumulation of delphinidin-based anthocyanins in grape skin. Present study also showed evident increase of delphinidin-based anthocyanins (2 ML, 4 ML, 4 LL and 6 ML) in most treatments, indicating the advantage of proper treatments for producing dark-colored red wine.

Previous researches have indicated that leaf removal could increase the content of phenolics in grape and resultant wine, maintaining the complexity and balance of wine body(Tardaguila et al. 2012; Intrigliolo et al. 2014; Osrecak et al.2015; Song et al. 2015). Similar results were obtained in this study, and the effect was more evident on UB. In addition,cluster thinning also effectively enhanced the accumulation of phenolics according to the results. Chang et al. (2015)suggested that as cluster thinning increased, total polyphenol and total anthocyanin contents increased as well with earlier maturity. Another research on Cabernet Franc showed that cluster thinning was effective only in one of the experimental years (Zhuang et al. 2014). The difference was probably caused by the variance of cultivars and climate conditions.Phenolic acids are the most abundant phenolic compounds in grape juice and wine of white grapevine cultivars (Bubola et al. 2012), the results of UB also agreed with this view.Leaf removal has been shown to improve the accumulation of hydroxycinnamics (Bubola et al. 2012; Diago et al. 2012),and the concentration of phenolic acids in grape and wine of white cultivars could be managed by altering the timing of leaf removal according to the desired wine style (Bubola et al. 2012). In present study, the combination of mean level of leaf removal and cluster thinning was the most favor to the accumulation of phenolic acids. Flavones are important phenolic components especially for red cultivars. Our and others’ studies showed a notable influence for this subclass of phenolics (Sternad Lemut et al. 2011; Bubola et al. 2012;Diago et al. 2012). As for flavanols and stilbenes, previous studies showed that leaf removal could positively affect their content in grapes and resultant wines (Bavaresco et al.2008; Osrecak et al. 2015). Present study also showed a pronounced effect of cluster thinning on content of these compounds, particularly for flavanols.

The accumulation of free and glycosylated aroma compounds commonly occurs at the advanced stages of ripening when sugar increase has already slowed down(Coombe and McCarthy 2000), so the maturity of grape greatly influences its aromatic character. According to Poni et al. (2013), limiting crop load could cause earlier ripening while leaf removal could delay this procedure. Therefore,proper combination of cluster thinning and leaf removal could be beneficial for the maturity and aromatic character of grape. Other researches also showed the effects of leaf removal and/or cluster thinning on grape and wine aroma(Borghezan et al. 2011; Feng et al. 2015; Song et al. 2015).Research by Song et al. (2015) concluded that canopy management by improving sun exposure promoted the formation of varietal aroma compounds like monoterpenes and C13-norisoprenoids, and another study (Feng et al.2015) also indicated that cluster zone leaf removal could positively influence the content of beta-damascenone in mature grape. We got the similar results in present study on the white grape variety UB. Although many studies have shown significant effects of canopy managements on modifying wine aroma, systematical study about the influence on grape aroma is still limited. In this research,pronounced effects of leaf removal and cluster thinning on aroma content and variety in grape were found.

5. Conclusion

Orthogonal experiment was conducted to study the effect of leaf removal and cluster thinning on grape quality in Weibei Dryland of China in 2013 and 2014. The results showed that the treatments had no influence on content of RS in both varieties. The TA, by contrast, was generally decreased by leaf removal and cluster thinning. Similar to previous researches, accumulations of TP, TAN and TAC were promoted by most treatments. Content and percentage of monomeric anthocyanins in Cabernet Sauvignon grape were also influenced by leaf removal and cluster thinning.Leaf removal decreased the percentage of malvidin and its derivatives. Meanwhile, among cluster thinning treatments,the middle and low crop loads led to larger percentages of petunidin, peonidin, delphinidin and cyaniding. Besides,cinnamylated anthocyanins decreased with the intensity of cluster thinning. As for non-anthocyanin phenolics, similar treatment effect was found on the two grape varieties. The middle degree of leaf removal and cluster thinning enhanced the accumulation of phenolic acids most. Moreover,pronounced increase of flavanols and stilbenes contents were found with the increase of cluster thinning. Additionally, the treatments also had notable effects on content and variety of aroma compounds in both grape varieties, particularly for the white grape cultivar Ugni Blanc.

Acknowledgements

This work was supported by the China Agriculture Research System for Grape Industry (CARS-29-zp-06).

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