Vine vigor and cluster uniformity on Vitis vinifera L. seed procyanidin composition in a warm Mediterranean climate

Seed procyanidin composition of Vitis vinifera L. var. ˈCarignanˈ and ˈGrenacheˈ was analyzed to assess the impact of vintage climatology, plant vigor and bunch variability on the quality of grapes. This study was carried out over 2007 and 2008 vintages in Terra Alta denomination of origin (DO). This region is located in northeastern Spain and characterized by a Mediterranean climate with a continental tendency. Procyanidin composition of seeds from four vineyards was analyzed by rapid resolution liquid chromatography (RRLC-DAD-TOF/MS). Vintage, vigor and ripeness uniformity had an influence on the procyanidin concentration in seeds. Flavan-3-ol polymerization increased during the warm year, together with a notable dependence on the variety and vine vigor. In warmer years and low vigor, ˈGrenacheˈ seed composition is likely to be more vulnerable than ˈCarignanˈ. High levels of flavan-3-ol monomers and low polymerization characterized the seeds of the temperate year.

During the later stages of ripening, the extractable levels of PAs start to decline (Downey et al., 2003). Physiologically, the decreasing extractability of PAs, particularly from grape skins, represents a decrease in the bitterness and astringency of PAs in the berry and is likely a part of the seed dispersal strategy that includes sugar accumulation and anthocyanin biosynthesis in the berry (Downey et al., 2006). The decrease in extractable PAs during ripening is the result of polymerization of the PAs (Coombe & McCarthy, 2000;Fournand et al., 2006). However, the actual mechanism of what causes this decrease in extractable PAs has yet to be elucidated (Dixon et al., 2005;Lepiniec et al., 2006). It is well known that the grape growing region, location of vines in the vineyard, bunch position into the vine canopy, and berry position into the bunch generate some differences in the ripening rate (Smart et al., 1985;Haselgrove et al., 2000;Le Moigne et al., 2008) and affecting the wine quality. Several studies have focused on the different environmental changes, viticultural practices and berry composition at different stages of maturity, on the PAs content of grapes and on the wine phenolic composition (Harbertson et al., 2002;Cortell et al., 2005;Ristic et al., 2007;Cohen et al., 2008). However, there is a lack of research about procyanidin content from distal parts (top and bottom) of the bunch. Consequently, the aim of this study was to determine the level of the uniformity in procyanidin content of two different varieties cultivated under the effects of vigor and in two vintages in a warm and dry Mediterranean grape growing region (Terra Alta DO, northeastern Spain).

Site details
Terra Alta Denomination of Origin (DO) is located in the precoastal mountains, in the province of Tarragona (Spain). This region has a Mediterranean climate with a continental tendency characterized by temperatures varying sharply from day to night and from summer to winter and receiving very low annual rainfall. According to the heat summation method, based on temperature and developed by Amerine & Winkler (1944), Terra Alta can be classified as Climate Region IV. The typical soils of the region, known as panal (mixture of silt and limestone), belong to the Entisols order according to the American Soil Taxonomy (USDA, 1998).

Plant material and experimental design
The study was carried out in 2007 and 2008 vintages. It was conducted in 'Carignan' (Car) and red 'Grenache' (Gre) varieties, in which two different levels of vigor were considered, low (L) and high (H). In total, four combinations (vigor/variety) were established: L-Car (alt. 370 m), H-Car (alt. 305 m), L-Gre (alt. 236 m), and H-Gre (alt. 422 m). In order to classify vineyards into vigor levels, growth and yield variables were measured. Three plot replications of each vigor/variety combination were randomly distributed in the vineyards, with each elementary plot consisting of 30 vines, where each replication was used for sampling as described in the "Fruit sampling and analysis" section. Two vines of each replication were used for vigor measurements (growth, berry weight, yield, pruning weight, length of shoots and total leaf area) with a total of six vines per treatment. Total leaf area (measuring the length of the leaf main nerve) was calculated as described by Sánchez-de-Miguel et al., 2011). This previous characterization allowed us to classify the vineyards into two vigor levels (L and H).
All vineyards had a minimum slope, which ranged between 4% and 7%. Vine spacing in L-Car and H-Car was 1.4 m (between vines) × 2.8 m (between rows); and 1.2 m × 2.8 m in L-Gre and H-Gre. Ten-year-old vines were not irrigated. Plants were bush-trained and pruned to 5-7 buds in L-vigor plants and 9-11 buds in H-vigor plants. L-Gre treatment was Grenache and Carignan seed composition in a warm Mediterranean climate 773
Additionally, a HOBO weather station (www.onsetcomp.com) was installed in each treatment. The following meteorological data: minimum temperature (Tmin), maximum temperature (Tmax), mean temperature (Tm) and relative humidity (%RH), were recorded every 15 min to characterize the specif ic conditions of each vineyard. The weather stations were placed between two vines in the same row, with temperature and humidity data loggers close to the canopy. Meteorological data collected in each treatment allowed us to define the climatic conditions occurring along the growing season and thus to determine the conditions in every phenological grape stage: fruit set, veraison and harvest. Based on the data collected, we calculated the number of days with an average temperature higher than 35°C (daysTmax > 35°C) and higher than 40°C (days-Tmax > 40°C), and number of days with thermal amplitude higher than 20°C (daysThAamplitude > 20°C).

Fruit sampling and analysis
In order to analyze the pulp composition and phenolic maturity of the distal parts of the grapes during ripening, samples of four bunches from the three replications per vineyard were randomly collected from different positions in the canopy, with a total of 12 bunches per vineyard. Bunches were stored in plastic bags and kept refrigerated (3-4°C). Samplings were carried out approximately every week from veraison to harvest in order to have measurements at the same physiological stage, to compensate for the ripening delay between vineyards, getting comparable results among treatments.
Samples of each replication were divided in two parts (top and bottom half of the bunch) as previously described in Edo-Roca et al. (2013). For each part, a sample of 100 berries was used to determine the sugar level (degree Brix), acidity (g L -1 tartaric acid) and pH according to OIVV (1990); another sample of 300 berries was used to analyze the phenolic maturity according to the method described by Nadal (2010).

Sample seed extraction
Approximately 100 berries from each replicate were hand-pressed to separate the pulp, the skin and the seeds. Seeds were washed three times with Milli-Q water, dried on filter paper, lyophilized and crushed to finally obtain a fine powder. Methanol solution (50 mL) was added to 1 g of seed powder. Samples were stirred and ultrasonicated for 15 minutes to completed extraction, and then centrifuged at 8,000 rpm at 5°C for 7 min. The supernatants were combined and dried under a nitrogen stream for their subsequent analysis by HPLC. The resulting fraction was dissolved into 20% of methanol, 0.1% of formic acid (98%) and Milli-Q water and it was filtered through 0.22 mm PVDF filter. Finally, filtered samples were injected in RRLC-DAD-TOF/MS (rapid resolution liquid chromatography coupled with diode array detection and electrospray ionization time-of-flight mass spectrometry).

Instrumentation
Seed procyanidins were analyzed on a Rapid Resolution Liquid Chromatograph RRLC 1200 (Agilent Technologies, USA). The RRLC was coupled to a TOF mass spectrometer G6220A (Agilent Technologies) equipped with an electrospray interface. Detection was done by a DAD (diode array detector).

Chromatographic conditions
According to Valls et al. (2009) methodology, a volume of 3 µL of each sample was injected onto a Zorbax Eclipse Plus C18 column (Agilent Technologies). The phenolic compounds were identified according to their order of elution, the retention times of pure compounds (gallic acid, catechin, procyanidin dimer B2, dimer monogallate, procyanidin trimer C1 and epicatechin gallate) and their molecular masses. The analyses were performed between 280 and 306 nm wavelength.

Mass spectrometry
The ionization of the compounds was carried out by electrospray in negative mode. Nitrogen was used as a drying gas and also as a nebulizing gas at an inlet pressure of 60 psi and a temperature of 350°C. Analyses were carried out in scan mode from 100 to 1,600 m/z.

Mean degree of polymerization (mDP)
The flavanol fraction was estimated according to mDP = [Σ(Ni · Ui)]/Nt, where Ni is the amount of flavanols of each group (i.e., monomers, dimers B, dimers gallate and trimers), Ui is the number of elementary units in each group of flavanols (i.e., 1, 2, or 3 for monomers, dimers, and trimers, respectively), and Nt is the amount of total flavanols in the sample (= Ni). The method used to calculate mDP has been previously described by González-Manzano et al. (2006).

Statistical analysis
Analysis of the variance (ANOVA) was conducted using SPSS 19.0. Significant differences were identified by Tukey's test. Factorial multivariate analysis results show the effects of vigor and uniformity separately as well as interaction among them (p ≤ 0.1; p ≤ 0.05; p ≤ 0.001).

Climatic characterization and effect on vine development
Annual and 10-year meteorological data of the experimental site is shown in Table 1 To better understand how vintage climatology affected the maturation of vineyards, three periods were defined: I, II and III; where I refers to the period between fruit set and veraison; II from veraison to advanced ripeness (one week prior to harvest); and III is the last stage of ripening (the last week before harvest) ( Table 2). Vineyards of L-Car and L-Gre had lower %RH than H-Car and H-Gre during the grape growing season for both years. In general, temperatures were higher in L-vigor treatments than in H-vigor treatments. Tm was higher in L-Car than in H-Car treatment during the summer season (periods I, II and III); but in L-Gre, Tm was higher in periods II and III (from veraison to harvest date). L-Car registered lower thermal amplitude (ThAmplitude) than H-Car, contrary to the treatments of 'Grenache'.
Comparing varieties, 'Grenache' phenology stages happened earlier than the 'Carignan' phenology (Table 3). Actually, the length of the growing period (from bud break to leaf drop) was longer in 'Carignan' than in 'Grenache'. Dates of phenological phenomena occurred later in 2008 than in 2007 for both cultivars.
Veraison showed a delay in 2008 in both varieties, although the duration of the period was the same. 'Carignan' showed a clear delay on harvest date in 2008. Phenology stages were found to start sooner in low vigor vineyards than in high vigor vineyards for both vintages and in both varieties.
The results of growth and yield variables (berry weight, yield, pruning weight, the length of shoots and the total leaf area) verify clearly the higher vigor of the vineyards previously selected in our research trial ( Table 4). The ANOVA (p ≤ 0.05) of vine vigor variables showed that the most vigorous grapevines grew and produced consistently more than the weakest.   I  II  III  I  II  III  I  II  III  I  II
Concerning 'Carignan', L-Car plants showed higher PAs concentration at the beginning of ripening (period II) than the H-Car plants for both vintages, even if the total procyanidins did not vary at the end of matura-tion. PAs for L-Car and H-Car followed clearly a twoslope pattern decrease. Both in 2007 and in 2008, during the first two weeks after veraison the slope was higher than in the following weeks (Figs. 3a and 3b). Until the end of ripeness the slope showed low and even values. In 2007, 'Grenache' followed the same pattern than L-Car and H-Car and in 2008 patterns of L-Gre followed showed two markedly different slopes (Figs. 3a and 3b). The main difference can be shown in the H-Gre/2008 treatment, where the diminution of total PAs along the maturation followed a unique pattern (m H-Gre = -0.19).

Seed procyanidin composition at harvest
'Grenache' accumulated more flavan-3-ol monomers and oligomers than 'Carignan'. The level of mo-Grenache and Carignan seed composition in a warm Mediterranean climate 779  nomeric flavan-3-ols in temperate/2008 vintage was higher than in warm/2007 (Table 6). Consequently, the mDP was higher in the warm than in the temperate vintage for both varieties. Results of seed composition in 'Carignan' showed that the percentage of monomers in 2008 was twice the relative amount of 2007. In both years, the percentage of monomers was higher in H-Car than in L-Car. On the other hand, dimers gallate were higher in L-Car/2008 than in H-Car/2008. The concentration of total PAs was significantly smaller in L-Car/2008 than in H-Car/2008. In warm vintage/2007, total concentration of PAs did not statistically vary between the vigor treatments. Within vigor, the mDP index showed no statistically different values between L-Car and H-Car in both vintages in spite of the tendency of the less vigorous treatment (L-Car) to achieve higher polymerization than the more vigorous. The mDP value was close to 2, indicating the predominance of dimers over trimeric and monomeric forms, also indicated by the highest percentage of dimers (Table 6).
In contrast to 'Carignan', the proportion of monomers in high vigor 'Grenache' (H-Gre/2007) was reduced significantly to about half of the amount of that of low vigor (L-Gre/2007; Table 6). The percentage of dimers B and dimers gallate doubled the percentage of monomers in the seeds of H-Gre/2007, and were significantly higher than those measured in L-Gre. The total procyanidins attained in H-Gre/2007 were signi-ficantly lower and, as expected, achieving an mDP higher than in L-Gre/2007. In 2008, mDP in 'Grenache' was about 1.50 and there were no statistical differences between the low and high vigor. Thus the amount of monomers and dimers did not vary in the 'Grenache' seeds in 2008.

Seed procyanidin composition from distal parts of bunch at harvest
Regarding the uniformity in seed ripeness of each of the berries within a bunch (Tables 7 and 8), it was found that the relative amounts in seed flavan-3-ol monomers and procyanidin oligomers of the distal parts were significantly variable in the warm and dry (2007) vintage but not in the temperate (2008).
The seed composition of 'Carignan' was not completely uniform in 2007 (Table 7). In L-Car/2007, statistical differences were evident in the percentage of monomers, which was lower in top side than in bottom seeds. Opposite, in H-Car/2007, the top seeds reached twice the percentage of monomers and higher total PAs than the bottom seeds. On the other hand, the percentage of dimers gallate was lower in top seeds than in bottom in H-Car/2007. Nevertheless, the mDP was statistically equal for both sides in all treatments. In temperate vintage, both top and bottom seeds ripened evenly.  Table 6. Pulp composition (°Brix and TA), procyanidin content (monomers, dimers B, dimers gallate and trimers) and total PAs (proanthocyanidins as the sum of monomers, dimers and trimers) and mDP (mean degree of polimerization) in seeds at harvest according to vintage and vigor effect for 'Carignan' and 'Grenache' Seed ripeness of 'Grenache' showed similar heterogeneity between distal parts in 2007 (Table 8), as well as 'Carignan' (Table 7). H-Gre/2007 top side showed a notably higher relative percentage of monomers than the bottom side, oppositely to L-Gre/2007. The same pattern was also observed in the total PAs concentration. The percentage of dimers gallate was higher in top seeds than those in bottom in L-Gre/2007 opposi-Grenache and Carignan seed composition in a warm Mediterranean climate 781  tely to H-Gre/2007. The mDP did not vary among the seeds from the same bunch.

Effect of vintage, vigor and uniformity on seed procyanidin composition
Factorial multivariate analysis demonstrated that the vintage was the most influential factor on the seed composition (data not shown). Furthermore, there were two factors (vigor and uniformity) that were influential to the composition of the seed. Therefore, the factorial multivariate analysis was also made with vigor (low and high), uniformity (top and bottom) and their interaction (Vigor * Uniformity) in each vintage for both varieties (Table 9).

Carignan
In the warm vintage 2007, vigor had major effects (p ≤ 0.001) on concentrations of monomers and mDP (Table 9). In addition, we found in 2007 a slight (p ≤ 0.05) impact by vigor on dimers gallate and total PAs content. The uniformity had major effects on monomers, dimers, total PAs and mDP, and a slight effect on trimers (p ≤ 0.1). However, the interaction of Vi-gor * Uniformity significantly affected all variables. In 2008, the importance of vigor was more pronounced than in 2007. Conversely, uniformity had no significance on dimers B and trimers. Moreover, the interaction of both factors in this vintage (2008) was smaller on dimers B (p ≤ 0.05) and trimers (p ≤ 0.1).

Grenache
The most important factor after the vintage effect was vigor, affecting all variables in both vintages. In the warm vintage, uniformity did not demonstrate influence neither dimers B nor trimers. The same result was found for the trimers in 2008. However, the interaction (Vigor * Uniformity) statistically affected all variables in both vintages.
compared with the pulp, which achieved acceptable values of °Brix, regardless of vigor (Table 6). In 2007, the high vigor conditions favored the reduction of monomers, causing an increase in mDP. Therefore, in warm vintage, the seeds ripened differently depending on the vigor effect, with the high vigor and warm vintage (H-Gre/2007) presenting the best conditions to mature. 'Grenache' (L-Gre and H-Gre) reached the same level of PAs at harvest of 2008 (L-Gre, 8.06 mg g -1 and H-Gre, 7.82 mg g -1 ) but not in 2007 (L-Gre, 6.66 mg g -1 and H-Gre, 4.08 mg g -1 ). In the L-Gre treatment, Tmax in period II of 2007 exceeded 35°C for 6 days; whilst in the H-Gre treatment the temperatures remained milder during ripening (periods II and III) (Table 4). Consequently, the accumulation of a relatively low concentration of monomers in 2007 suggests that the warm vintage conditions and high vigor, in the case of 'Grenache', allowed the optimum ripening of the seed. In contrast, the warm vintage in the low vigor vines revealed unripe seeds, indicating sensitivity in this variety during extreme climate conditions, when the high maximum temperatures and low relative humidity were registered in this vineyard during ripening. The different pattern observed in 'Grenache' and 'Carignan' let us to consider a viticultural management specifically for each variety in order to improve the seed ripeness.

Seed procyanidin composition from distal parts of bunch at harvest
In the current study, it was found that in the warm and dry vintage, top seeds ripen better in the low vigor conditions (L-Car/2007 and L-Gre/2007), yielding the lowest levels of monomers measured in this study. In contrast, in high vigor (H-Car/2007 andH-Gre/ 2007), the seeds of the bottom side were more mature at harvest (Tables 7 and 8). Moreover, the differences between the °Brix of distal parts of the bunch did not always correspond to a greater difference in levels of monomers and oligomers of the seeds in the top and bottom half of a bunch.

Effect of vintage, vigor and uniformity on seed procyanidin composition
According to the results from the multivariate factorial analysis (Table 9), regardless of the vintage, vi-gor is the most influential factor on the maturation of 'Grenache' seeds. Instead, seed ripeness for 'Carignan' does not depend on vigor when the vintage is warm. In warm vintage, the seeds of 'Carignan' were more influenced by the effect of the interaction Vigor * Uniformity than each factor individually.

Conclusions
Based on the current study it is clear that the evolution of total seed procyanidins from veraison to harvest depends primarily on the diminution of flavan-3ol monomers. The kinetics of this evolution depends on the variety, vintage and vigor. During seed maturation, monomers diminish faster in low vigor vines. At harvest, procyanidin content in Grenache remains always higher than in Carignan. Under temperate vintage conditions, both 'Carignan' and 'Grenache' maintain a high level of flavan-3-ol monomers and a low polymerization, suggesting that the seeds have not fully matured. Under warm conditions (very high temperatures and drought) and in high vigor vines, seed procyanidin polymerization was favored only in 'Grenache'. Seed ripeness depends first on the vintage and to a lesser extent on the vigor, for both 'Carignan' and 'Grenache'. Procyanidin seed composition from the distal parts of the bunch also depends on vine vigor and vintage. In temperate vintage (2008), seeds ripped homogeneously in both parts of the bunch. In warm vintage (2007), the top seeds ripened better in the low vigor, whereas bottom seeds ripened better in the high vigor.