Experimental site and plant material

The experiment was conducted during three consecutive years (2014, 2015, and 2016) in a vineyard planted on a clay loam soil with Richter 110 rootstock grafted in 2001 to Vitis vinifera L. cv. ˈTempranilloˈ. The vineyard was located in the experimental fields of the Instituto de Investigaciones Agrarias La Orden-Valdesequera belonging to the Centro de Investigaciones Científicas y Tecnológicas de Extremadura (CICYTEX), Junta de Extremadura (38° 51' N; 6° 40' W; 186 m a.s.l.). Vine spacing was 2.5 m between rows and 1.2 m within each row (3333 vines ha^-1). They were trained as bilateral cordons (Royat), and winter pruning was to leave six spurs per vine with two buds per spur.

Two irrigation treatments were tested. The first was a rainfed control, in which the vines were not irrigated (NIr). In the second treatment, irrigation (Ir) started when the stem potential reached 0.6 MPa (Williams & Baeza, 2007), as measured at midday using a pressure chamber (Model Soil Moisture Crop, Santa Barbara, CA, USA), and it was maintained over the course of grape development at amounts needed to replace 100% of crop evapotranspiration (ET_c). This last parameter was determined with a weighing lysimeter located in the experimental vineyard (Picón-Toro et al., 2012). Irrigation started on June 9^th 2014, May 13^th 2015, and June 15^th 2016. The total amount of water applied in the irrigation treatments, as well as the values of the reference evapotranspiration (ET₀), ET_c, and rainfall were determined from fruit set to veraison and from veraison to harvest.

For the two irrigation regimes, crop load was controlled by shoot thinning. High crop load vines were subjected to a "low shoot-thinning" (LT) treatment, with 12 shoots vine^-1 (vines were pruned in winter to six spurs with two buds each). Some vines were subjected to a low crop load treatment, adjusting them to 6 shoots vine^-1 to constitute the "high shoot-thinning" (HT) treatment. This pruning adjustment was done on April 23^rd 2014, April 24^th 2015, and May 3^rd 2016, corresponding to phenological stage 12 (Eichhorn & Lorenz, 1977). Therefore, four treatments were applied as the result of the combination of irrigation and shoot thinning practices: NIr-LT (non-irrigation and low thinning), NIr-HT (non-irrigation and high thinning), Ir-LT (irrigation and low thinning), and Ir-HT (irrigation and high thinning).

The experiment had a split-plot design, with four sub-plots corresponding to each treatment. The sub-plots were divided into four replicate blocks, comprising six rows of 18 vines each one. The surrounding perimeter was taken as vine guards, with neither the two outermost rows of each sub-plot nor the two outermost vines of each row being used for sample collection.

A selection criterion was set for grape sampling in or-der to minimize berry heterogeneity. During berry deve-lopment and the first stages of ripening, samples were selected on an equatorial diameter basis (Table 1). On the other hand, during the later ripening stages (Table 1), the berries were classified in accordance with their soluble solids content as determined by their density in different NaCl solutions (Carbonell-Bejerano et al., 2012). Altoge-ther, berries were collected at eight stages of development corresponding to different days after anthesis (DAA) (May 11^th 2014 and 2015, and May 23^rd 2016): (1) pre-veraison corresponding to berries 4-6 mm in diameter (10 DAA); (2) pre-veraison corresponding to berries 7-8 mm in diameter (18 DAA); (3) pre-veraison corresponding to berries 10-11 mm in diameter (30 DAA); (4) onset-veraison corres-ponding to berries 11-12 mm in diameter (45 DAA); (5) mid-veraison corresponding to berries 12-13 mm in dia-meter when grapes are coloured at 50 % (65 DAA); (6) end-veraison corresponding to berries completely coloured (approximately 20 ^oBrix, density 120-140 g L^-1 NaCl) (79, 86 and 73 DAA in 2014, 2015 and 2016 seasons, respecti-vely); (7) first harvest when the berries of at least one of the treatments had reached to commercial ripening (23-24.5 ^oBrix, density 150-170 g L^-1 NaCl) (93, 93 and 80 DAA in 2014, 2015 and 2016 seasons, respectively); and (8) se-cond harvest when the berries of the rest of the treatments had reached to commercial ripening (23-24.5 ^oBrix, density 150-170 g L-1 NaCl) (100, 100 and 87 DAA in 2014, 2015 and 2016 seasons, respectively). Berries reaching firstly to commercial ripening were from HT treatment in 2014 and 2015, and from Ir treatment in 2016.

Berries were carefully picked from random plants on each date. They were picked from the central part of sou-th-oriented clusters early in the morning, and then trans-ported to the laboratory in an ice cooler at 4 °C. The berries were then cut at the base of the pedicel, rinsed, and dried. Finally, they were weighed (model M-Prove AY-412 ba-lance, SARTORIUS) to determine average berry weight, and frozen at -40 °C for later phenolics extraction.

Extraction of phenolic compounds

The phenolic extracts were obtained using entire gra-pes following the procedure described by Singleton & Rossi (1965) with some modifications. Samples were homogenized in extraction solvent (methanol 80%) in a 1:4 ratio (w:v). After 30 min stirring, the liquid extract was separated from the solid residues by centrifugation at 3000 rpm for 15 min (Centrifuge Eppendorf 5810R, Hamburg, Germany). The extraction procedure was repeated thrice, the final extract resulting in a mixture of the three supernatants. This was filtered through a 0.45 µm nylon membrane and stored in a freezer until assay for total phenolics, anthocyanin, and proanthocyanidin contents. One extraction was performed for each experimental block.

Table 1. Sampling criteria for berry collection during 'Tempranillo' grape development.

Determination of phenolic compounds

Total phenolics content

The extracts total phenolics contents were determined by the Folin-Ciocalteu colorimetric method (Singleton & Rossi, 1965) with some modifications. Briefly, 1 mL of diluted extract was oxidized with 5 mL of Folin-Ciocalteu reagent (10%) and neutralized with 4 mL of Na₂CO₃ solution (7.5%). After mixing and keeping the samples at 50 °C (water bath) for 5 min, their absorbances were read at 760 nm against a buffer blank in a UV/VIS spectrophotometer (Biomate 6UV-Vis, THERMO SCIENTIFIC). A calibration curve was constructed using gallic acid standard solutions (0–100 mg L^-1). The total phenolics content was expressed as mg of gallic acid equivalent (GAE) per g fresh weight. All extracts were assayed in triplicate and the results were expressed as means ± standard errors.

Proanthocyanidin content

The proanthocyanidin content of the grape extracts was determined by vanillin-HCl assay as described by Broadhurst & Jones (1978). Briefly, 0.5 mL of diluted extract was mixed with 3 mL of vanillin solution in methanol (4%) and 1.5 mL of HCl. The reaction mixture was incubated for 15 min at room temperature. Absorbance was measured at 500 nm against a buffer blank without vanillin in a UV/VIS spectrophotometer. A calibration curve was prepared using catechin standard solutions (0–335 mg L^-1). The proanthocyanidin content was expressed as mg of catechin equivalents per g fresh weight. All extracts were assayed in triplicate and the results were expressed as means ± standard errors.

Anthocyanin content

The monomeric anthocyanin content of the grape extracts was measured using a modified pH differential method (Boyles & Wrolstad, 1993). Diluted extract was mixed thoroughly with 0.025 M potassium chloride buffer of pH 1 in a 1:2 ratio (v:v). Other amount of diluted extract was similarly mixed with a sodium acetate buffer of pH 4.5, stirred, and left to stand for 15 min for the reaction to take place and stabilize. The absorbances at 510 nm (wavelength of maximum absorbance) and 700 nm were measured with a UV/VIS spectrophotometer against buffer blanks at pH 1.0 and pH 4.5. The absorbance readings were converted to total mg of malvidin 3-glucoside (Mv-3-glu). The anthocyanin content was calculated as follows:

In this formula, A is the absorbance, MW (463.3 g mol^-1) is the molecular weight of Mv-3-glu, ε (28,000 M^-1 cm^-1) is the molar absorptivity of Mv-3-glu, and C is the concentration of the grape extract in mg mL^-1. The anthocyanin content was expressed as mg of Mv-3-glu equivalents (Mv-3-glu E) per 100 g fresh weight. All extracts were assayed in triplicate and the results were expressed as means ± standard errors.

Statistical analysis

Results are expressed as means and standard errors calculated over all replicates. All the data were subjected to an ANOVA using the SPSS 23.0 software package, and Tukey's test was used to establish the significance of differences between means at a p<0.05 level. Student’s t-test is also used in this work. The data were analysed using irrigation and crop load as the main factors, and including the irrigation × crop load, irrigation × year, and crop load × year interactions.

Figure 2. Differences between irrigation treatments (NIr: no irrigation; Ir: irrigation at 100% ETc) and shoot thinning treatments (LT: low shoot-thinning; HT: high shoot-thinning) on 'Tempranillo' berry weight (A, B, C, D, E, F) and total phenolics (G, H, I, J, K, L) as a function of days after anthesis (DAA) in 2014 (first row), 2015 (second row), and 2016 (third row). I: Fast growth phase. II: Lag phase. III: Ripening phase. Vertical bars indicate standard errors (n=150 for berry weight and n=18 for total phenolics). Statistical significances are based on Student's t-test: ***, p<0.001; **, p<0.01; *, p<0.05; n.s., p>0.05.

Figure 3. Differences between irrigation treatments (NIr: no irrigation; Ir: irrigation 100% ETc) and shoot thinning treatments (LT: low shoot-thinning; HT: high shoot-thinning) on 'Tempranillo' proanthocyanidins (A, B, C, D, E, F) and anthocyanins (G, H, I, J, K, L) as a function of days after anthesis (DAA) in 2014 (first row), 2015 (second row), and 2016 (third row). I: Fast growth phase. II: Lag phase. III: Ripening phase. Vertical bars indicate standard errors (n=18). Statistical significances are based on Student's t-test: ***, p<0.001; **, p<0.01; *, p<0.05; n.s., p>0.05.

DiscussionTop

Effects of cultural practices on berry development

In our study, berry growth followed a double sigmoid pattern in concordance with results described in varieties such as Cabernet Sauvignon (Basile et al., 2011), Shiraz (Ollé et al., 2011), and Tempranillo (Girona et al., 2009; Valdés et al., 2009; Intrigliolo & Castel, 2011).

Irrigation generally led to greater berry weights, in accordance with most other published data (Esteban et al., 2001; Girona et al., 2009; Valdés et al., 2009; Intrigliolo & Castel, 2011). However, we found a reverse pattern in the 2014 season. A possible explanation for this discrepancy could be that 2014 was characterised by a heavy rainfall (26.34 mm) during stage II. This important rainfall could mask the effect of irrigation practice due to the high water accumulation in soil.

Concerning thinning practices, our findings were that high shoot-thinning produced greater berry weight, in agreement with previous published data (Reynolds et al., 1994a,b; Intrigliolo & Castel, 2011). Similar results have been found with cluster thinning treatments (Diago et al., 2010).

Our results also showed that the effects of the different combinations of irrigation and thinning treatments on berry weight varied according to the year. In 2015 and 2016, the Ir-HT treatment led to the greatest weights during ripening. While this is in concordance with the results of Intrigliolo & Castel (2011), Keller et al. (2008) found no effect of the interaction between irrigation and crop load treatments on berry weight in any year studied. On the other hand, although Valdés et al. (2009) reported irrigation effect on berry weight, they did not detect neither significative effects of crop level nor interaction between irrigation and crop load on berry weight.

Effects of cultural practices on total phenols during grape development

In the present study, we found a sharp decrease of total phenols at Stage I but no changes at Stage II, as has been reported by other workers during berry development (Crippen & Morrison, 1986). Also, we observed total phenolics amounts declined at the beginning of ripening, followed by a stabilization around harvest, in concordance with other studies (Romero et al., 2010).

Irrigation contributed to lower amounts of total phenols in the grape berries, in agreement with other studies on the same cultivar (Esteban et al., 2001; Intrigliolo & Castel, 2008; Valdés et al., 2009; Garrido et al., 2014, 2016). Some authors claim that the total phenols increase is a simple consequence of reduction in berry size (Kennedy et al., 2000; Roby et al., 2004). However, we have to discard this possibility because our results indicated an increase in phenolics irrespective of berry weight, in agreement with some other studies (Garrido et al., 2014). It could be checked at harvest in 2014 when grapes under NIr treatments reached higher total phenols concentration but also higher weight.

The present results showed that HT treatments contributed to greater amounts of total phenols at harvest. This is in concordance with studies employing cluster thinning (Valdés et al., 2009; Diago et al., 2010; Gamero et al., 2014; Garrido et al., 2016) or defoliation practices (Garrido et al., 2014). There are various possible explanations for the greater phenolics content at harvest caused by thinning. For instance, thinning could promote greater nutrient availability due to an improved source-sink ratio (Pastore et al., 2013). The high accumulation of total phenolics at harvest could be explained as a response of the high temperatures registered during this period (Garrido et al., 2014).

In our work, the greatest amount of total phenols at harvest was reached under the combination NIr-HT, in agreement with previous studies (Valdés et al., 2009; Intrigliolo & Castel, 2011). We also found that the year had a significant effect on total phenolics concentrations, in agreement with previous studies (Gamero et al., 2014). No statistically significant interaction was observed between the two practices (irrigation and thinning) in the total phenolics content, as also reported by other studies (Ortega et al., 2007; Valdés et al., 2009; Gamero et al., 2014).

Effects of cultural practices on proanthocyanidins during grape development

We observed a progressive decline in proanthocyanidin concentrations towards a nearly constant level at the end of ripening, as previously described in the same cultivar (Niculcea et al., 2013).

Our results also demonstrated that irrigation treatments reduced proanthocyanidin concentration in whole berry from the beginning of the irrigation onwards, but not at harvest in 2016 (Fig. 3C). González & Ferrer (2008) stated that the lower tannin content under greater water availability might be a consequence of dilution because of the greater berry size. Niculcea et al. (2013) detected no significant differences in proanthocyanidin concentrations in the whole grape berry under different irrigation treatments. In our study, the HT treatment led to greater proanthocyanidin accumulation at harvest, it coinciding with previous studies applying cluster thinning (González & Ferrer, 2008) or defoliation (Risco, 2012).

To the best of our knowledge, this has been the first study to analyse the influence of combined NIr and HT treatments on proanthocyanidin concentrations during grape berry development. The results show that NIr-HT treatments contributed to favouring these concentrations at harvest. In agreement with the authors cited above, this could be due to the additive effect of these treatments leading to a greater proanthocyanidin concentration.

Effects of cultural practices on anthocyanins during grape development

The anthocyanin accumulation throughout ripening followed by a stabilization or decline around harvest that was found in our study is in concordance with previous studies (Bindon et al., 2013; Niculcea et al., 2013). Some authors have explained the decline in anthocyanin concentration near harvest as an effect of environmental and viticultural conditions, since high berry temperatures and sunlight exposure could inhibit anthocyanin biosynthesis (Tarara et al., 2008).

Our results indicated that the Ir treatment reduced the anthocyanin concentration and thus berry quality, in agreement with previous studies (Intrigliolo & Castel, 2008, 2011; Valdés et al., 2009). Surprisingly, the anthocyanin concentrations in 2016 presented a reverse pattern at harvest. This could be attribute to water stress since, once water deficit surpasses a certain threshold, anthocyanin synthesis may be delayed or hindered by a decrease in photosynthesis and carbon limitation (Girona et al., 2009). We also found that the NIr treatment affected anthocyanin concentrations positively, in concordance with previous studies (Roby et al., 2004; Girona et al., 2009).

In concordance with previous published data (Ortega et al., 2007; Intrigliolo & Castel, 2011), we found that the HT treatments increase anthocyanin levels. This finding is also coherent with other studies concerning cluster thinning (González & Ferrer, 2008; Valdés et al., 2009; Santesteban et al., 2011; Gamero et al., 2014) or defoliation (Freese, 1988) on anthocyanin concentrations during berry ripening. The effect was associated with greater exposure of the clusters to direct sunlight and to higher berry temperatures because of the reduced vegetation cover (Ginestar et al., 1998; Bergqvist et al., 2001).

With regard to the combination of treatments, some authors have reported that the coupling of non-irrigation and low crop load increases the quantities of anthocyanins (Valdés et al., 2009; Intrigliolo & Castel, 2011). In the present study, the NIr treatments favoured anthocyanin accumulation at harvest in 2014 and 2015, but we found no significant differences between the NIr-HT and NIrLT treatments. On the other hand, we found that the year had a significant effect on anthocyanin concentrations, in agreement with Gamero et al. (2014). We detected no statistically significant irrigation × thinning interactions affecting anthocyanin content, again in concordance with the reports of other workers (Ortega et al., 2007; Valdés et al., 2009; Gamero et al., 2014).

In summary, although seasonal variability difficulties the study of phenolics content under natural conditions, we found that hot and dry years favoured high concentrations of these compounds. Generally, a supply of water reduced the concentrations of proanthocyanidins, anthocyanins, and total phenols during berry development. The stress induced in non-irrigated vines could stimulate the biosynthesis of phenolic compounds. In the highly shoot-thinned vines, anthocyanin synthesis was markedly stimulated throughout ripening, probably because of a higher level of exposure to sunlight. However, the only observable effect of high thinning on proanthocyanidin and total phenols concentrations was at harvest, probably due to the extreme temperatures at that time. Although the non-irrigation and high thinning treatments favoured the accumulation of phenolic compounds, the effect of their combined treatment was non significative. The effect of the year on berry weight and on phenolic compounds was checked at harvest. In particular, the quality of the berries varied depending on the specific weather conditions of each year. A significant effect of irrigation × year was observed on berry size and phenolic content, but thinning × year interaction only was detected on proanthocyanidin and anthocyanin concentration. Therefore, as irrigation practice is an important factor influencing to phenolic content of berry grape it could be studied in future works through different intensities of water deficit in order to improve the grape quality and, consequently, the wine resulting.

ReferenesTop