3.1. Effect altitude on physicochemical behaviour of grapes and wine

3.1.1. Effect of air temperature

Temperature decreases with altitude creating a more temperate climate due to adiabatic cooling of the air (Gutiérrez-Gamboa et al., 2021). ʻChasselasʼ grapes cultivated on steep terraced slopes at altitudes of between 375 and 575 m were monitored by Rienth et al. (2020) in the AOC-Lavaux region in Switzerland. For all three of the consecutive study years, altitude was the main driver of precocity and was consistently associated with the date of budburst and flowering. The temperature decreases with altitude, usually in the range of 0.65 to 1.0 °C for 100 m gain in elevation, were associated with plots at higher elevations showing a delay in budburst and flowering. Because the vineyards at higher altitude ripen later during the cooler periods of the year, they have the highest potential for producing high-quality grapes (with higher acidity and lower alcohol levels) under future warmer temperatures, leading to maximum terroir expression.

Cabré and Nuñez (2020) used indices related to air temperature to evaluate the impacts of future climate on Argentinean winegrowing regions, including the Cool Night Index (CNI) and the average growing season temperature (GST) index. CNI is considered as the mean minimum night temperature in the later maturity stages of the ripening period; it gives a measure of ripening potential, indicating the suitability of a winegrowing region notably in relation to the secondary metabolites (polyphenols, aromas) in grapes and wines. GST is used to determine a cultivar's suitability in a given site and its projected changes could be useful for identifying the best appropriate grapevine varieties for current and future locations. These authors showed that CNI and GST are projected to increase (more than 6 °C for CNI and between 4 and 7 °C for GST) mainly in a distant future in the most pessimistic emission scenario for greenhouse gases (RCP8.5). They also reported that more and new suitable areas may be available for cultivating cool climate varieties, while less suitable areas may be available for warm and hot climate varieties to maintain their current quality.

Major biochemical changes occur during grape ripening, in particular the accumulation of total soluble solids (TSS), increased pH and decreased acidity (Almanza-Merchán et al., 2012). Total titratable acidity (TTA) decreases in grapes with increasing temperatures. Meneghelli et al. (2018) showed that a vineyard at 500 m a.s.l. and with higher mean air temperature exhibited lower TTA and higher pH and TSS/TTA ratios in ‘Niágara Rosada' and ‘Isabel' cultivars when compared to the same values obtained for a vineyard at 650 m altitude. However, in the same study, relatively high values of TTA were obtained at 250 m altitude and this was attributed to the excess of leaf nitrogen that increases vine vigour. Vigorous vines increase shading in the bunch area, which decreases the temperature of the grapes, extends the vegetative growth period and delays fruit maturity.

In a study conducted on ‘Syrahʼ grapes, higher temperatures at the lower altitude regions (350 m a.s.l.) led to a degradation in malic acid and an increase in the concentrations of glucose and fructose compared to the vineyards at higher altitude (1100 m a.s.l.) (Barreto de Oliveira et al., 2019). Regina et al. (2010) reported that ʻPinot Noir' grapes from a high-site vineyard (1150 m a.s.l.) characterised by lower temperatures showed higher concentrations of malic acid compared to the same variety cultivated at 873 m a.s.l. (8.04 g/L vs 4.45 g/L in 2008). This suggested that low temperatures are more favorable for producing sparkling wines of high organoleptic quality.

3.1.2. Choice of grape variety

The altitude effect should be evaluated separately for each grape variety. Malinovski et al. (2016) showed that the effects of high-altitude regions in Água Doce in Brazil on the viticultural performance of six Italian native grapevines differ from one cultivar to another. ʻAncellotta', ʻGarganega' and ʻFiano' varieties showed satisfactory levels of TSS (i.e., above 18 °Brix), for the production of quality wines. In contrast, the TSS values of ʻLambrusco', ‘Nero d'Avola' and ‘Negroamaro' at harvest were below 18 °Brix, thus likely compromising the quality of the wines to be produced from these three cultivars.

In the same study, ‘Nero d’Avola’ and ‘Negroamaro’ cultivars originating from Sicily and Apulia respectively in Southern Italy, had difficulties in developing their berry components, like sugars and acidity, during ripening; this may be due to the annual air temperature being lower than in their region of origin. Meanwhile, the ‘Ancellotta’ cultivar, originating from Central Italy with similar thermal conditions to Água Doce, stood out for its good qualitative indices and high adaptability to the local area.

Bolivian viticulture is mostly carried out at altitudes of between 1660 and 2360 m a.s.l, and ʻTannatʼ grapes as well as Mediterranean cultivars (e.g., ʻGrenacheʼ, ʻCarignanʼ, ʻTempranilloʼ and ʻSangioveseʼ) have been shown to adapt well to the subtropical conditions of the Central Valley of Tarija, Bolivia. However, other cultivars (e.g., ʻCabernet-Sauvignonʼ, ʻMerlotʼ, ʻPinot Noirʼ, ʻChardonnayʼ, ʻSyrahʼ and ʻMalbecʼ) are not well-adapted due to their need for very cold winter conditions. Therefore, understanding the differences in chilling needs could help grapevine breeders and growers adapt to the expected climate change (Gutiérrez-Gamboa et al., 2021).

3.2 Altitude effect on phenolic and aroma compounds in grapes and wine

3.2.1. Effect of air temperature

High altitude and the related lower air temperature have an influence on the ripening and polyphenolic composition of grapes (Xing et al., 2016). Coklar (2017) found that under the effect of low temperatures in high elevation vineyards, the total monomeric anthocyanin content in ʻEkşikaraʼ whole berries and skins was significantly higher than that at lower altitude. The author also found that tannin levels in grape seed and skin for the high site were significantly higher than those at the low site. Moreover, higher total phenolic content in whole berries, skins and seeds were obtained for the high site.

The high-altitude regions and their associated climatic conditions seem to be favourable for the production of certain types of phenolic compounds, whereas the climatic conditions in low-altitude regions appear to be favourable for the production of other types. Higher levels of anthocyanins monoglucosides and trihydroxylated flavonols in skins of ʻCabernet-Sauvignonʼ, ʻCarmenereʼ, ʻSyrahʼ and ʻMerlotʼ were generated in the western regions in China (up to 1214 m a.s.l.) characterised by large day- and night-time temperature differences. However, the grapes from the lower altitude eastern regions near the sea characterised by higher daily minimum temperatures and small day- and night-time temperature differences, had a higher proportion of acylated anthocyanins and dihydroxylated flavonols (Liang et al., 2014). Furthermore, Coklar (2017) reported that with decreasing altitude, the relative amount of malvidin-3-O-glucoside in both skin and whole berry of ʻEkşikaraʼ grapes increased, whereas that of petunidin-3-O-glucoside, cyanidin-3-O-glucoside, peonidin-3-O-glucoside and acylated anthocyanins in whole berry decreased. This study, however, did not decouple the effect of temperature from UV radiation.

In another study performed by Barreto de Oliveira et al. (2019), the difference in altitude had a significant impact on ʻSyrahʼ grapes. The concentrations of total phenols (1,440 mg/ kg fresh fruit), non-flavonoids (200 mg/kg), flavonoids (1,240 mg/kg) and total anthocyanins (890 mg/ kg) at the site at 1100 m a.s.l. were significantly greater than those at the site at 350 m altitude (450, 130, 320 and 350 mg/ kg fresh fruit respectively). This result could be explained by the fact that the high-altitude region is characterised by large day- and night-time differences in temperature and by the fact that the maximum temperatures during the productive cycle stayed below 30 °C, thus favouring the accumulation and preservation of phenolic compounds during the grape ripening period. The grape skins at the high site contained higher levels of total condensed tannins, including monomeric, oligomeric, and polymeric 3-flavanol, whereas the grape seeds contained higher levels of the same compounds at the low site. On the other hand, the concentrations of trans-resveratrol in the first and the second year of the study were 5.71 and 8.17 mg/Kg fresh fruit respectively at the low site and 4.11 and 4.72 mg/Kg fresh fruit respectively at the high site. The synthesis of stilbenes is induced by environmental stresses; therefore, the maximum daily temperatures that exceeded 30 °C during berry growth and ripening in the low-altitude area may have stressed the vines, resulting in higher resveratrol levels.

ʻCabernet-Sauvignonʼ wines made from grapes grown at different altitudes (774, 960, 1350 and 1160-1415 m a.s.l.) were studied by Falcão et al. (2007) in Santa Catarina State, Brazil. Altitude did not affect the concentrations of α- and β-ionone and β –damascenone in the produced wines. However, in both winter and summer, the highest levels of 2-methoxy-3-isobutylpyrazine (MIBP) were found in wines of the highest site (1415 m a.s.l.), where the temperature was lower in comparison to the other studied sites. Consequently, these wines were correlated with a “bell pepper” aroma, whereas wines from the lowest altitude (774 m a.s.l.) were correlated with a “red fruits” aroma. Grape MIBP content is closely linked to viticulture parameters, like the growing temperature (Allen et al., 1994); in a previous study, lower temperatures during the period preceding véraison had a greater impact on the MIBP content than after the grapes had matured (Lacey et al., 1991).

However, in some winegrowing regions and depending on the geographic location of the vineyards, it is possible for a higher altitude cultivation site to have warmer conditions that affect the sensory attributes of the produced wines; for instance, in a study performed by Alessandrini et al. (2017), the high site (380 m a.s.l.) was warmer than the low one (200 m.a.s.l.) due to its higher heat accumulation degrees favouring the accumulation and preservation of the aroma compounds and enhanced the elegance and the floral aroma of the produced wine. The levels of volatiles were most likely temperature-dependent rather than altitude-dependent. The minimum air temperatures at the low site were approximately 2 ˚C lower than those at the high site and were thus considered to be the main limiting factor for the biosynthesis of the aroma compounds at the lower altitude.

Rotundone is described by Ferreira (2012) as being an important wine aroma-impacting compound, and the anecdotal evidence that rotundone was more common in ʻShirazʼ wines from cool climate locations was corroborated in an Australian red wine survey (Black et al., 2015). Geffroy et al. (2016) evaluated the variability of its concentrations in ʻGamay Nʼ wines in four French wine-growing areas. The results showed that Auvergne - the coolest vineyard over the whole wine-growing season and ripening period - had the highest rotundone concentrations and the most intense peppery notes. Peppery aroma scores and the concentration of rotundone in wine were shown to be significantly correlated. However, the effects of temperature and vine water status were not totally differentiated in this study: the vineyard with the highest rotundone content was also the wettest during the véraison-harvest period.

In cooler vintages and vineyards, higher levels of rotundone are expected to accumulate in ʻVespolinaʼ grapes (Caputi et al., 2011). The highest levels of rotundone were obtained in ʻShirazʼ berries from vines exposed to cooler temperatures, showing a within-vineyard variation and that the topography of the vineyard, particularly the aspect, was the most important factor in the formation of rotundone (Scarlett et al., 2014). In another study, rotundone was typically present at the top and in shaded areas of bunches of ʻShirazʼ grapes, which correlates with lower grape surface temperatures, and its concentration was negatively affected by fruit temperatures above 25 °C (Zhang et al., 2015a). Furthermore, a study on fifteen vintages of ʻShirazʼ wine produced from the same vineyard block at the same winery showed that wines from cooler seasons tend to contain higher levels of rotundone (Zhang et al., 2015b). Water abundance was another influential factor in this study, since a higher amount of rotundone was detected in wetter vintages.

3.2.2. Effect of UV radiation

The increase in UV radiation noted at higher altitudes promotes the synthesis of skin anthocyanins; this can explain why wines produced from high-altitude regions have a higher colour intensity (Jin et al., 2017). At higher altitudes, the increase in UV-B radiation can reach 8 % per decade leading to the enhancement of colour, flavonol and tannin synthesis in red grapes on the one hand (van Leeuwen and Darriet, 2016), and to higher concentrations of 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) - which can give the wine an intense and sometimes unpleasant smell of hydrocarbons - on the other.

UV‐B up-regulates the biosynthesis of anthocyanin in grapevines, counteracting the negative effects of increasing atmospheric carbon dioxide and increased temperature on anthocyanin content in berries (Gutiérrez-Gamboa et al., 2021). Exposure to UV-B has been found to slow down berry growth and increase the biosynthesis of anthocyanins and flavonols (Martínez-Lüscher et al., 2016). It also leads to an increase in levels of both total bound glycosidic secondary metabolites and phenolics (Keller and Torres-Martinez, 2004; Lafontaine et al., 2005), and in the synthesis of anthocyanins in grape skins (Berli et al., 2008; Carbonell-Bejerano et al., 2014).

In Bolivia and Argentina, some wines produced from vineyards located higher than 1500 m a.s.l. were found to have higher total antioxidant capacity and phenolic content (including resveratrol) than wines produced from vineyards located at a lower altitude (Osorio-Macías et al., 2018). UV-B radiation activates the phenylpropanoid biosynthesis pathway, resulting in phenolic-based resistance against Botrytis cinerea that causes grey mould or botrytis bunch rot in grapes (Elmer and Reglinski, 2006; Gutiérrez-Gamboa et al., 2021). Plant responses to heat stress at high altitudes may be regulated by abscisic acid and salicylic acid (Larkindale and Huang, 2005). Abscisic acid plays a role in the response of grape leaf tissues to UV-B radiation by augmenting the synthesis of UV-absorbing compounds, antioxidant enzymes and membrane sterols (Gutiérrez-Gamboa et al., 2021). Salicylic acid might be synthesised by the grapes as a defense response to stress conditions, such as UV radiation that increases with increasing altitude; Jin et al. (2017) found that its amount increased dramatically with altitude in the wines produced from ʻMerlotʼ and ʻCabernet-Sauvignonʼ.

The phenolic content of ʻCarignanʼ grapes grown in two different locations, referred to as early and late ripeness parcels corresponding to a higher (370 m a.s.l.) and lower (305 m a.s.l.) altitude respectively, was studied by Edo-Roca et al. (2013). The total leaf area (TLA) in the early (warm) parcel (3.4 m²/vine) was smaller than the TLA in the late (moderate) parcel (4.8 m²/vine). As a result, the grapes in the warm parcel were more exposed to solar radiation and synthesised larger amounts of anthocyanins. Furthermore, flavonol compounds can serve as natural sunscreen for grapes; their synthesis is induced by light and their levels are positively related to radiation. Karaoğlan et al. (2015) showed that the highest amount of quercetin-3-O-glycoside (5.16 mg/L) was obtained for the ʻMuscat of Bornovaʼ wine from the site at the highest altitude and with the highest solar radiation (587,465 W/m²). Furthermore, the amounts of quercetin-3-O-glycoside found in 2.22 and 3.88 mg/L of lower altitude wines correlated with solar radiation values of 542,875 and 553,782 W/m² respectively).

Plants accumulate large quantities of various types of compatible solutes and important osmoprotectants (e.g., proteins, proline and carbohydrates) in response to diverse stresses, caused by, for instance, low temperature and UV radiation. These osmoprotectants protect plants from stressing conditions by adjusting cellular osmosis, protecting membrane integrity and contributing to the detoxification of reactive oxygen species and the stabilisation of proteins/enzymes activities (Hayat et al., 2012). Indeed, Berli et al. (2013) showed that the application of a high solar UV-B treatment under field conditions to ʻMalbecʼ grapevine leaves in a high-altitude vineyard (1450 m a.s.l.) in Argentina augmented the levels of photoprotective pigments and proline, thereby increasing the antioxidant capacity of leaves.

3.2.3. The combined effect of soil and vine water status

In many studies, the effect of soil and vine water status cannot be clearly distinguished from the altitude factor. Vines with limited access to water in a vineyard cultivated with ʻCarignanʼ grapes at a higher altitude and characterised by a shallow stony soil produced smaller berries with higher anthocyanin content compared to another vineyard at a lower altitude with a deeper soil (Edo-Roca et al., 2013).

Soil and water availability affect the branch points of the biosynthetic pathway of flavonoids in grape berries, which are regulated by a number of enzymes (Li et al., 2011): Flavonoid 3'-hydroxylase (F3'H) and flavonoid 3', 5'-hydroxylase (F3'5'H) convert dihydrokamepferol into dihydroquercetin and dihydromyricetin, in addition to their derivatives, respectively. Cyanidin-derived anthocyanins and delphinidin-derived anthocyanins are synthesised from dihydroquercetin in the F3'H branch pathway and dihydromyricetin in the F3'5'H branch pathway respectively in the downstream pathways of both branches. Li et al. (2011) found that the regional ʻCabernet-Sauvignonʼ wines from vines at an altitude of 1900-3500 m a.s.l. in China contained the greatest levels of both quercetin derivatives and cyanidin-derived anthocyanins, while the regional wines from vines at an altitude of 214 m a.s.l. contained the second highest level of myricetin derivatives and the highest levels of delphinidin-derived anthocyanins. The authors related this result to different factors, such as soil and water availability, which might have promoted the directional flow of carbon into the myricetin synthetic branch or to the quercetin synthetic branch. The region at the highest altitude is characterised by a brown sandstone soil and a specific water retention curve (the relationship between soil water content and soil water pressure head). This could incite the flow of more carbon to the F3'H branch pathway, leading to higher concentrations of quercetin derivatives and cyanidin-derived anthocyanins in the grapes and in the resulting wines. The authors did not exclude possible interference of other factors, like the cool-warm climate existing in the higher altitudes and the warm climate existing in the lower altitudes.

A study performed on wines from ʻMuscat of Bornovaʼ grapes grown in Turkey found that phenolic levels were the highest in wines of the Halilbeyli sub-region (115 m a.s.l.), followed by wines of the Menderes (90 m a.s.l.) and Kemaliye (245 m a.s.l.) sub-regions (Karaoğlan et al., 2015). In particular, the concentrations of trans-caftaric and trans-coutaric acids in the produced wines from the Kemaliye sub-region (39 and 5.8 mg/L respectively) were lower than those of the Menderes sub-region (55 and 23.6 mg/L respectively) and the Halilbeyli sub-region (80 and 25.3 mg/L respectively). The lowest values obtained at Kemaliye may be related to the soil composition of this sub-region, which is characterised by having the highest percentage of lime, which affects water retention and drainage.

3.2.4. The effect of other parameters

Many studies have shown that other factors, such as humidity, vintage and grapevine variety, are closely associated with temperature, UV radiation and vine water status. The lower humidity of the high cultivation sites (at approximately 2900 m a.s.l.) in southwest China induced an increase in the production of cyaniding-type anthocyanins and quercetin-type flavonols from the F3'H branch of the flavonoid biosynthetic pathway in ʻCabernet-Sauvignonʼ, when compared with sites at lower altitudes (2300 and 2150 m a.s.l.) (Xing et al., 2016). Another study on the effect of altitude on carotenoids of ʻTouriga Francaʼ and ʻTouriga Nacionalʼ grape varieties was conducted by Oliveira et al. (2004) in the Douro Valley. The altitudes ranged from 85 and 145 to 180 m a.s.l. for ʻTouriga Francaʼ and from 90 and 155 to 210 m a.s.l. for ʻTouriga Nacionalʼ. High-elevation terraces with higher humidity levels promoted the accumulation of carotenoids, which degrade during the grape ripening period to form odour-active C13-norisoprenoids.

Mateus et al. (2001) showed that at the lower cultivation locations (100-150 m a.s.l.), humidity levels of 95-100 % during the night were not favourable for the biosynthesis of total anthocyanidin monoglucosides in the grape skins of ʻTouriga Nacionalʼ and ʻTouriga Francesaʼ compared to the higher sites (250-350 m a.s.l.). Nevertheless, higher concentrations of catechin monomers and low-molecular procyanidin oligomers in the grape skins of both varieties, as well as of total extractable proanthocyanidins in the skins of both cultivars and the seeds of ʻTouriga Nacionalʼ, and of low-molecular procyanidin oligomers in the ʻTouriga Nacionalʼ wine, were obtained at the lower altitudes. None of these studies, however, separated the effect of humidity from temperature.

The latitude effect was more pronounced than the altitude effect for ʻMalbecʼ wines produced by different regions in Argentina. The vineyards at latitudes of 31-33° produced the most desired sensory attributes in wine (e.g., floral, sweetness, cooked fruit and raisin) in contrast to regions outside these latitudes which exhibited sourness, bitterness and a strong herbal aroma (Goldner and Zamora, 2007). The levels and the composition of phenolics in grapes vary as a result of vine vigour and water inputs. The variability in microclimatic conditions of the grapevines may be caused by vine vigour heterogeneity (Asproudi et al., 2016). When compared to less vigorous vines, high vigour vines usually have lower fruit exposure and a greater fruit MIBP content (Mendez-Costabel et al., 2014); as a result of increasing water input from rainfall and irrigation, MIBP synthesis can increase (Mendez-Costabel et al., 2014).

Vintage is one of the main factors to influence the concentrations of proanthocyanidins in grapes, in addition to the effect of air temperature, which varies between altitudes. Of the monomeric and the small oligomeric compounds in ʻSyrahʼ seeds, Barreto de Oliveira et al. (2019) found higher levels of gallocatechin, epigallocatechin and B1, B2, B3 and B4 dimers at a higher altitude (1100 m a.s.l.) in the first year of their study, and high concentrations of catechin and B2 dimers esterified with gallic acid at a lower altitude (350 m a.s.l.) in the second year. Vintage was observed to have a higher effect than altitude for ʻChardonnayʼ and ʻPinot Noirʼ cultivated in Minas Gerais, Brazil (Regina et al., 2010); a greater number of sunny days in the growing season contributed to a greater accumulation of anthocyanins and phenolic compounds in the grapes.

The highest content of total phenols and flavonoids in ʻMoscatelʼ grape stems sampled form three different regions in northern Portugal at 120, 670 and 730 m a.s.l. were found in the lowest altitude region over two consecutive vintages (Gouvinhas et al., 2020) ; nevertheless, they increased significantly in the second year of the study, which was characterised by an atypical summer with a 3-day heat wave (temperatures above 40°C) near the beginning of the harvest. Under these stressful conditions, which scalded the grapes, the plants produced secondary metabolites as a defense mechanism. The obtained lower levels of total phenols and flavonoids in the high-altitude regions may be attributed to the absence of significant water or thermal stresses in these regions. However, higher biological capacities were induced by high precipitations and the climate, which has an Atlantic influence in the low-altitude regions.

The amount of polyphenols in ‘Ancellotta', ‘Lambrusco', ʻNegroamaro', ‘Nero d'Avola', ʻFiano' and ‘Garganega' cultivated at 1300 m a.s.l. was greater in the year which had a higher number of rainy days (Malinovski et al., 2016). This result was correlated with the fact that fungal diseases are common in areas with high rainfall, inducing plants to produce phenolic compounds as a stress response. The intensity of this response was found to vary depending on the grape varieties, showing that different cultivars respond differently even when subject to the same stressful climatic conditions.

The antimicrobial activity in ʻMoscatelʼ grape stems was shown by the multivariate analysis to be lower in the high-altitude regions (730 m a.s.l.) than in the lower altitude regions (120 m a.s.l.) (Gouvinhas et al., 2020). This antimicrobial activity seemed to be more affected by the genetic characteristics of the grape stem varieties than by the climate conditions and altitude of the growing sites. Jiang et al. (2013) concluded that the magnitude of the effect of environmental factors on the volatile compounds in Cabernet-Sauvignon and Merlot wines produced from four wine-growing regions in China (at 214, 450–600, 1036 and 1100 m a.s.l.) may be related to the cultivar.

Different studies have shown that the effects of temperature and sunlight cannot be isolated from the effect of vine water status. In high-altitude areas in China (2282, 2435 and 2608m a.s.l.), due a decrease in temperature and an increase in rainfall and sunlight hours, the quercetin, trans-resveratrol and tannin content in ʻCabernet-Sauvignonʼ wines increased with increasing altitude (Jin et al., 2017); however, no significant effect of altitude on the same parameters was found in ʻMerlotʼ wines. Overall, altitude followed by sunlight hours mostly affected the phenolic characteristics and antioxidant activity of tested red wines. Vine water status is a key determinant of terroir expression, which depends on climatic conditions, such as rainfall and evapotranspiration (van Leeuwen et al., 2020). In a study conducted on ʻGrenacheʼ berries (Edo-Roca et al., 2013), the effects of vine water status and temperature were not completely differentiated: levels of anthocyanins in grapes declined in the last ripening control in the warmest year of the study, in which dryness and high temperatures occurred during a 3-day heat wave (temperatures reaching an unusual 40 °C) in the period before harvest.