Volatile compounds were analysed by the procedure proposed by Ortega et al. (2001) with slight modifications. A volume of 2.7 mL of the samples was transferred to a 10 mL screw-capped centrifuge tube that contained 4.05 g of ammonium sulphate and the following compounds were added; 6.3 mL of Milli-Q water and 20 μL of a standard internal solution consisting of 2-butanol, 4-methyl-2-pentanol and 2-octanol from Sigma Aldrich (Darmstadt, Germany) at 140 μg m.L^-1 each, in absolute ethanol from LiChrosolv-Merck (Darmstadt, Germany), and 0.25 mL of dichloromethane. The tube was shaken mechanically for 120 min and was later centrifuged at 2,900 g for 15 min. The dichloromethane phase was recovered with a 0.5 mL syringe, transferred to the autosampler and analysed. The chromatographic analysis was carried out in an HP-6890, equipped with a ZB-Wax plus column (60 m × 0.25 mm × 0.25 µm) from Phenomenex. The column temperature, initially set at 40 °C and maintained at this temperature for 5 min, was then raised to 102 °C at a rate of 4 °C.min^-1 to 112 °C at a rate of 2 °C.min^-1, to 125 °C at a rate of 3 °C.min^-1 and this temperature was maintained for 5 min and then raised to 160 °C at a rate of 3 °C.min^-1; to 200 °C at a rate of 6 °C min^-1 and was then kept at this temperature for 30 min. The carrier gas was helium, which was fluxed at a rate of 3 mL.min^-1. The injection was done in split mode 1:20 (injection volume 2 μL) with a flame ionisation detector (FID). In addition, Kovats retention indices (KI) were calculated for the GC peaks corresponding to identifying substance by the interpolation of the retention time of normal alkane (C8–C20) by Fluka Buchs (Schwiez, Switzerland), analysed under the same chromatographic condition. The calculated KI were compared with those reported in the literature for the same stationary phase.

The statistical analysis used to examine the dataset was a one-way Analysis of Variance (ANOVA). When the ANOVA revealed significant differences (p < 0.05), a post hoc mean separation was performed using either the Duncan test (for normally distributed data) or the Kruskal–Wallis procedure from the “Statgraphics Centurion XVI” software package (version 16.0.07) (Statgraphics Technologies, The Plains, VA, USA).

In general, the vineyard water status became more negative until mid-season, when sustained deficit irrigation and rainfall led to an increase (Figure 1). In both seasons, the most negative _stem values reached - 1.5 to - 1.3 MPa, indicating severe water stress (Poni et al., 2018). It should be noted that ESP showed, on most dates, less negative values than WSP (Figure 1). In addition, on some dates, the WSP showed higher vine water stress than the VSP. Nonetheless, it should be noted that the _stem reflects the water status at midday, and that this varies in response to the trellis leaning throughout the day (Santesteban et al., 2019; Buesa et al., 2020). Therefore, it is to be expected that the WSP will recover its water status in the afternoon compared to the VSP and, above all, to the ESP.

Figure 1. Stem water potential (_stem) evolution in ‘Bobal’ grapevines from the vertical shoot positioned (VSP), East shoot positioned (ESP), and West shoot positioned (WSP) treatments in 2016 and 2017 in Requena (Valencia), Spain (n = 16). Vertical bars indicate the standard error. Different letters denote significant differences among treatments at p < 0.05.

Regarding vegetative growth, both pruning mass and total leaf area per vine (LA) were significantly affected by the treatments (Table 1). The ESP tended to increase the pruning mass compared to WSP, with VSP showing intermediate values between the two. However, differences among treatments were statistically significant only in 2016. A similar pattern showed LA due to the highest leaf area of main and secondary shoots. WSP treatment showed in both seasons significantly lower leaf area in secondary shoots (Table 1). The differences found among treatments in vegetative growth were in good agreement with its water status evolution (Figure 1). Nevertheless, Buesa and co-workers (Buesa et al., 2020), reported that leaning trellises towards the West increased LA by 13 % and yield by 12 % compared to traditional VSP. In the present trial, however, there were no overall significant effects of the trellis leaning on yield components (Table 1). The only exception was in 2017 when the number of clusters per vine was significantly decreased by 14 % in the ESP compared to the other treatments. However, neither cluster mass nor berry mass was affected, despite the slight differences in vine water status (Figure 1).

VSP; vertical shoot positioned, ESP; East shoot positioned and WSP; West shoot positioned.

2. Effect of the leaning canopy on the physicochemical properties of musts

Differences in the basic physicochemical parameters of ‘Bobal’ grapes among treatments were found (Table 2). For instance, in 2017, the TSS of musts at harvest showed a significant increase in VSP compared to the other treatments. Moreover, in both vintages, the TTA tended to be the highest in WSP, with the grapes harvested in ESP decreasing TTA. However, no significant differences were found in pH among treatments (Table 2).

Different letters indicate significant differences between treatments at 95 % p < 005 (*), ns (not significant) based on Duncan’s multiple range test. Titratable total acidity expressed as g/L of tartaric acid.

Regarding grape colour intensity and phenolic characteristics, in both seasons, musts from WSP vines showed significantly the highest colour intensity (Table 3). This fact could be related to the trend observed for anthocyanins with higher values in WSP and lower values in ESP compared to VSP. Significant differences were also observed for TPI, the content of polyphenols and tannins. In general, higher values were observed in WSP and lower values in ESP, regarding control (VSP). These results agree with previous authors who reported that, across training systems, increasing cluster sun exposure was related to higher anthocyanin and polyphenol concentrations in grapes (Wolf et al., 2003). In this sense, the WSP had a less dense canopy due to a significant reduction in secondary shoot growth (Table 1). Moreover, WSP clusters are also more protected from exposure to direct sunlight during the afternoon than ESP and VSP. Consequently, ESP clusters may increase the microclimate temperature during the warmest and driest hours of the day to the extent of diminishing berry colour and phenolics (Bergqvist et al., 2001; Oliveira and Nieddu, 2016). Furthermore, mild water stress can also have a positive effect by increasing the synthesis of berry anthocyanin and polyphenols (Castellarin et al., 2007; Bucchetti et al., 2011; Berhe, 2022; Martínez-Vidaurre et al., 2024). This highlights the relevance that other potential factors may have on the water status of the vines such as soil type, rootstock used, crop load, soil management and irrigation strategies, etc., which may influence the effects on grape composition of canopy management practices (Ashley, 2004; Uriarte et al., 2016).

Different letters indicate significant differences between treatments at p < 005 (*), p < 001 (**), p < 0001 (***) and ns (not significant). TPI: Total Polyphenol Index.

3. Effect of the leaning canopy on the physicochemical properties and phenolic composition of wines

In wines, a general trend was observed regarding canopy leaning according to the basic oenological parameters of wines. Thus, lower pH and higher TTA were observed in WSP, showing significant differences among treatments in 2016 (Table 4). Nevertheless, no differences in volatile acidity were found among treatments in any vintage. In 2017, the alcoholic strength was significantly higher in VSP wines compared to ESP and WSP.

Different letters indicate significant differences between treatments at p < 005 (*) and p < 0001 (***). TTA (Titratable Total Acidity expressed as g/L of tartaric acid and volatile acidity in g/L of acetic acid). ns (not significant).

Regarding phenolic parameters and colour characteristics, a similar trend was observed in musts, accordingly higher colour intensity and anthocyanins were obtained in the case of WSP with significant differences in both vintages (Table 5). Moreover, a significantly higher content in TPI, total phenols, total flavonoids, total tannins, and catechins was observed for WSP compared to both VSP and ESP in both vintages. These results can be related to a greater amount of radiation in the morning when the photosynthetic capacity of the vines is higher, and to the fact that the bunches are protected from the afternoon sun when the air temperature is higher in the WSP system (Buesa et al., 2020; Intrigliolo et al., 2017). Indeed, when vines lean towards the West, an early interception of light in the morning increases the rate of foliar photosynthesis (Intrigliolo et al., 2017) that can lead to a greater accumulation of polyphenols in grapes (Vitrac et al., 2000). Moreover, the more severe vine water stress experienced by WSP (Figure 1), can accelerate grape ripening and polyphenols biosynthesis (Castellarin et al., 2007; Intrigliolo and Lakso, 2011; Lovisolo et al., 2010). On the contrary, the ESP showed no increase in the phenolic parameters of wines compared to VSP, with the exception in 2017 of total phenols, total flavonoids, and catechins (Table 5). This may be explained by the slight increases observed in vine water status in ESP (Figure 1) and its effects on the phenolic compounds in this cultivar (Salón et al., 2005). However, no decreases in the phenolic parameters compared to VSP were found. Therefore, increasing solar radiation exposure of the cluster may have had counteracting effects, positively influencing flavanol synthesis (Friedel et al., 2016; Wolf et al., 2003) while provoking a depletion by overheating. Differences in canopy temperature throughout the day among treatments showed that WSP reduced canopy temperature by 3–4 degrees compared to VSP and WSP (Supplementary Figure 2). These differences in canopy temperature cannot be directly translated to differences in grape temperature, but they show that these are temperatures that could lead to the degradation of phenolic compounds (Mori et al., 2007). In this regard, Torres and co-authors reported that higher light interception affected flavonoid accumulation, both because of its synthesis but also its degradation (Torres et al., 2020).

Different letters indicate significant differences between treatments at p < 005 (*), p < 001 (**), p < 0001 (***) and ns (not significant). TPI (Total Polyphenol Index).

The PVPP Index increased in both vintages for WSP, showing significant differences from VSP and ESP only in 2017 (Table 5). A higher combination of anthocyanins and flavanols in WSP treatment can influence positively the wine colour stability (Escribano-Bailón et al., 2001). In this sense, the DMACH Index was significantly lower in WSP treatment compared to VSP and ESP, which may be caused either by precipitation of some of the proanthocyanidin molecules or by increasing their degree of polymerisation (Zanchi et al., 2007). Wines showed no differences in the Gelatine Index, which is commonly used to evaluate astringency. Regarding the formation of polysaccharides–tannin–protein complexes, assessed through the Ethanol Index, a higher value was found in WSP wines (no significant differences within each vintage), indicating a higher content of tannins dissolved but in turn higher formation complexes likely due to the higher content of polyphenols observed (Table 5).

4. Effect of the leaning canopy on the aromatic composition of wines

A total of thirty-eight volatile compounds were quantified to determine the effect of the leaning canopy on the aromatic composition of wines (Table 6). The total concentration of each compound is shown individually and grouped in five representative families according to their chemical structure (aldehydes, esters, alcohols, acids, and lactones).

Different letters indicate significant differences between treatments at p < 005 (*), p < 001 (**), p < 0001 (***) and ns (not significant).

Regarding aldehydes, the different treatments did not influence their overall concentration. In 2017, VSP wines did show a higher concentration of acetaldehyde (Table 6). This compound can provide pleasant or unpleasant notes to wines depending on their concentration. Thus, low concentrations provide positive notes such as sweet fruits, but high concentrations can lead to undesirable aromas such as baked apple, walnuts, butter or glaze (Zea et al., 2015). Concerning esters, in both vintages, WSP wines consistently showed significantly higher concentrations than VSP and ESP. Particularly, isobutyl acetate, ethyl isovalerate, ethyl hexanoate, ethyl lactate, hexyl acetate, diethyl succinate, and 2- phenyl-ethyl acetate obtained significant differences (Table 6). These compounds are related to fruity aromas and pleasant notes, being a high incidence in the aromatic profile of wines (Ferreira et al., 2000). Therefore, WSP wines could lead to aromatically fresher wines with a more marked youthful character. This can be explained by the increased sunlight exposure of the clusters in WSP due to the lower secondary shoots development (Belancic et al., 1997; Zhang et al., 2017).

The concentration of higher alcohols in WSP was significantly higher than in VSP in 2016 and significantly higher than ESP in both seasons (Table 6). This pattern showed differences mainly between WSP and ESP treatments and could indicate that the canopy leaning towards the West is most suitable to improve the level of higher alcohol in wines. In Tempranillo wines, Talaverano et al. (2017) reported higher levels of total alcohol in response to more negative vine water potentials, as is the case of WSP in comparison to VSP and ESP (Figure 1).

In relation to fatty acids (aromatic precursors of fruity esters), a different tendency was found depending on the vintage. In 2016, a higher content of butyric acid and hexanoic acid was found in ESP and WSP compared to the control (Table 6). Octanoic acid tended to be reduced in WSP for both vintages. Nevertheless, in 2017, isobutyric acid, butyric acid and hexanoic acid reached significantly higher levels in WSP than in control wines (VSP). Regarding lactones, as occurred in higher alcohols and fatty acids, a different pattern was observed between vintages showing significant overall differences between WSP and ESP only in 2016. Pantolactone was the only compound that consistently increased in both seasons by WSP compared to VSP and ESP (Table 6).

In general, the content of esters and higher alcohols seems to be favoured by WSP, producing a higher aromatic intensity of wines, as reported by other authors in response to the effect of the increase in vegetation height (Moreno et al., 2015b). These results could be related to the fact that WSP grapes, compared to VSP and ESP ones, intercepted a greater amount of radiation early in the morning and from the lower in the afternoon, when the air temperature and VPD are higher (Supplementary Figure 2). This may be due to, on the one hand, the increase in sunlight exposure of the clusters (Belancic et al., 1997; Zhang et al., 2017) and, on the other hand, a reduction in the degradation of aromatic precursors by heat (Marais et al., 2001), both processes leading to a higher aromatic concentration in wines.

5. Integrating the effects of canopy leaning on physiology, agronomic and oenological responses

Canopy architecture significantly affected vine water status, and thus vegetative vigour, mainly due to the increase and reduction in leaf area of secondary shoots of ESP and WSP, respectively, with respect to the VSP control (Table 1). This, together with the different radiation loads intercepted by the canopy due to its leaning, likely increased the irradiance and temperature of the clusters throughout the morning and afternoon in WSP and ESP, respectively. This generated microclimate conditions that in turn increased and reduced phenolic and aromatic compounds in WSP and VSP, respectively, with respect to the VSP. Note the importance of the timing of radiation interception, as it is in the afternoon, when VPD is higher, that the greatest interception seems to be negative for acidity and phenolic compounds and aromatic composition (ESP), whereas the opposite is true in the morning (WSP). Nevertheless, our two seasons results also highlight the complex relationships between meteorological conditions and field management practices as other authors reported in other cultivars (Buesa et al., 2021a; Talaverano et al., 2017; Vilanova et al., 2019). For instance, the smaller differences in the total leaf area of the vines among treatments in 2017, when canopies were additionally denser compared to 2016, seem to dampen the effects of canopy leaning on cluster microclimate and thus on grape and wine composition. Consequently, canopy leaning should be adjusted to the specific environmental conditions of the vintage, as the angle of leaning and the timing can be modulated according to the grape ripening process. As an example, in the face of heat waves during grape ripening, leaning the trellis to the West might minimize the detrimental effects on grape composition.

Leaning canopy treatments affected canopy radiation load, causing significant differences in vine water status, and resulting in differences in vegetative growth. Thus, the WSP treatment increased vine water stress (_stem) resulting in a 15 % lower both pruning mass and total leaf area compared to the VSP and ESP treatments. However, no significant differences were observed in the yield components. Musts from the WSP showed a tendency to increase TTA compared to VSP, while ESP tended to reduce it. This is attributable to the temperature reduction in the cluster microclimate in the WSP compared to ESP and VSP. Furthermore, grapes from WSP treatment also exhibited heightened colour intensity, anthocyanins and TPI, likely due to its more negative potentials inducing the synthesis of phenolic compounds. Consequently, wines from the WSP treatment displayed lower pH and higher TTA, as well as higher colour intensity, anthocyanins, and polyphenolic composition than the other treatments. Aromatic analysis revealed that WSP wines had higher concentrations of esters and higher alcohols than the other treatments, which could be related to fruity aromas and a higher aromatic intensity of wines. These findings demonstrate that canopy leaning towards the West can be a useful technique for adapting must composition and wine quality to climate change through increasing radiation interception in the morning and decreasing cluster heating in the afternoon. These results encourage further research into the potential of canopy management practices to regulate radiation load and its timing under different cultivars and environmental conditions. It also provides insights into the physiological basis of its effects on vine water status and cluster microclimate.

This work was supported by the Spanish Ministry of Science and Innovation through the Spanish Research Agency (MCIN/AEI) and the European Union Next GenerationEU/PTR [grant numbers PID2021-123305OB-C31]. R. Ferrer-Gallego acknowledges the funding of MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTR [RYC2021-031638-I] and [CNS2023-144458]. The authors express their gratitude to Felipe Sanz, Antonio Yeves, Diego Pérez, Giulio Caccavello, Maria Clara Merli and Javier Castel for their help in the fieldwork. Thanks are also due to ‘Cajamar’ and ‘Lucio Gil de Fagoaga’ for facilitating the experimental field.

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