Terpenoids are an important and diverse group of plant secondary metabolites. Up to 10 % of recently fixed carbon may be dedicated to de novo terpenoid biosynthesis (Peñuelas & Staudt, 2010). Terpenoids are structurally derived from isoprene units. All terpenoids are synthesised from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). The terpenoids of wine grapes have long been known to contribute substantially to the varietal typicity and sensory quality of wines (Rapp & Mandery, 1986; Wüst, 2018). Monoterpenes and terpenoid degradation products, such as carotenoid-derived C ₁₃-norisoprenoids, are powerful odorants that shape the sensory quality of grapes and wine, particularly in aromatic grape varieties such as Gewürztraminer, Muscat varieties, and Riesling.

The majority of terpenoids in grapes are present in their glycosylated form (Günata et al., 1985), linked to either glucose alone or glucose and a second sugar moiety (Mateo & Jiménez, 2000). Glycosylation increases the water solubility of terpenes, making the storage of large amounts of terpenoids possible without risking toxicity. The release of glycosylated compounds may occur in response to external stimuli in plant tissues, but also post-harvest during grape processing, fermentation, and wine storage.

Terpenoids are thought to play a key role in plant communication and are associated with general stress and defence responses (Gershenzon & Dudareva, 2007). Plants have been demonstrated to increase the emissions of volatile organic compounds (VOCs) under abiotic stress (drought, salinity, heat, and high light) and biotic stress in a straightforward way, leading Holopainen and Gershenzon (2010) to generalise that “stress brings VOC release”. Similarly, de novo terpenoid biosynthesis is upregulated by environmental stress (Boncan et al., 2020). This response is strongly mediated by jasmonic acid signalling in grapes (D'Onofrio et al., 2018; Wang et al., 2022) and other crops (Wüst, 2018).

During the ongoing process of climate change, drought stress is arguably the single most important factor threatening viticultural yields in many traditionally dry-farmed regions of Europe, where viticulturists see drought stress as one of the major risks to their economic sustainability (Loose & Pabst, 2019). Apart from a perceived or actual loss of yield, it has also been reported that growers fear decreased typicity and sensory quality of wines due to drought stress (Loose & Kiefer, 2020).

Several recent publications have investigated the effects of drought stress on the accumulation of aroma compounds in wine grapes (Palai et al., 2023; Savoi et al., 2020; Savoi et al., 2016; Wang et al., 2019; Kovalenko et al., 2021). While the general conclusion of these studies is that drought stress increased the accumulation of individual monoterpenes, its effects could often not be discriminated from microclimatic effects induced by reduced vigour due to drought stress. In this paper, we combined a set of experimental data gathered on the Vitis vinifera L. cultivar Riesling over four years and in two different climate zones. Our objectives were to confirm the increase of aroma compounds associated with better wine quality under drought stress in Riesling grapes and wines and to separate the direct effect of drought stress on the accumulation of terpenoids in grapes from its indirect effect, which increases aroma compound biosynthesis via increased light interception by the grapes due to lower vigour. To do so, we maximised light interception by the grapevines using total leaf removal in and around the bunch zone on vines with different water status to exclude light exposure of the bunches as a confounding factor for the drought stress effect. We compared these treatments with vines without leaf removal to assess drought and leaf-removal effects and their interaction.

Experiment 1a: The 2022 growing season in the Rheingau region was characterised by severe drought stress, particularly in shallow soils such as the one in the experimental vineyard, accompanied by extreme temperatures during the growing season. August was the most extreme month in this regard, with an average temperature of 23.9 °C (+ 5.1 °C compared to 2021) and only 3.7 mm of precipitation (Figure 1).

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Drought stress in the experimental vineyard increased progressively from Ψ _PD = ‒0.26 MPa at bloom to ‒0.67 MPa at veraison in the unirrigated treatment (‒0.17 and ‒0.34 MPa in the irrigated treatment) and was only alleviated by substantial rainfall in the first half of September, close to harvest (Figures S1 and S2). The difference in Ψ _PD between irrigation treatments resulted in a reduction of leaf area (27 % lower total leaf area on 28.07.2022; Figure S3) and a substantial decrease in berry size from > 1.5 g in the irrigated treatment blocks to < 1 g in the unirrigated treatment blocks (Table 1), but berry temperatures were not affected by irrigation (Figure S4).

Additionally, some leaf shedding was observed during the ripening phase (Figure S3). δ ¹³C was 2.25 ‰ lower in berry extracts from irrigated vines. The effects of drought stress on berry extract aroma compound concentration in 2022 showed some interactions with the leaf removal treatments, with strong irrigation effects: irrigation reduced aroma compounds only where partial leaf removal was conducted, but not where full leaf removal was conducted (Table 1, Figure 2, Table S7).

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Vice versa, drought stress ( i.e., the unirrigated treatment) increased the concentration of free monoterpenes, bound monoterpenes, and bound C ₁₃-norisoprenoids by 88 %, 35 %, and 29 % when only partial leaf removal was conducted but had the same levels as the irrigated treatment under full leaf removal. In comparison, only free monoterpenes increased (20 %) in the unirrigated vines compared to the irrigated vines when full leaf removal was conducted (Figure 2). Leaf removal effects in the unirrigated vines were much weaker in 2022 than in Experiment 1b (same site; seasons 2021, 2023, 2024; Figure 3) and were mostly restricted to glycosylated norisoprenoids (Table 1). Full leaf removal of irrigated vines raised free and bound terpenoid compounds to similar levels as did withholding irrigation with only partial leaf removal (Table 1) but did not increase overall monoterpene concentration above the levels of partial leaf removal in the unirrigated vines. In contrast, the content ( i.e., amount per berry) of glycosylated monoterpenes and norisoprenoids was higher in berries with low δ ¹³C ( i.e., from the irrigation treatment; Table S2).

Grapes for winemaking were sourced from the irrigated and unirrigated treatments with only partial leaf removal. The effect of drought stress on the wines was comparable to its effect observed in the grapes, except that a larger fraction of norisoprenoids and monoterpenes was present in their free form in wine (Table S3) and the concentration of bound monoterpenes and norisoprenoids was substantially lower than in berry extracts (Table 1). The concentration of free and bound monoterpenes and free and bound norisoprenoids was 48, 16, 56, and 37 % higher, respectively, in wines from unirrigated grapes compared to wines from the irrigated treatment, while the ratio of monoterpenes to norisoprenoids was unaffected by irrigation (Table S3). Similar to Rossmann et al. (1996), δ ¹³C was about 2 ‰ higher in grapes than in ethanol of the corresponding wines, while the difference between irrigated and control treatments was comparable (~2 ‰).

Experiment 1b: The years 2021, 2023, and 2024 were all characterised by above-average precipitation (+42 to +122 mm compared to the annual average for 1991–2020). Hence, the irrigation system was not put into operation in these seasons, and the vineyard remained dry-farmed. In all years, however, long dry periods around the time of veraison led to drought stress during the ripening phase (Average minimum Ψ _PD = ‒0.74, ‒0.81, and ‒0.47 MPa for 2021, 2023, and 2024, respectively; Figure S1). A high spatial variability of drought stress was observed in the experimental vineyard, with block 1 showing higher juice δ ¹³C, more negative water potentials and lower berry weights compared to blocks 2 and 3 (Table S4, Figure S1). Although these spatial differences were relatively small compared to the irrigation-induced differences in other experiments presented here ( e.g., the coefficient of variance of berry weight across the vineyard was 8 % without irrigation in 2021 and 23 % with irrigation in 2022), the concentration of practically all aroma compounds was significantly higher in berries with high δ ¹³C values, while the content of aroma compounds per berry was little affected (Tables S4 and S5). The effect of drought stress was more pronounced on free and bound monoterpenoids than on bound norisoprenoids (Table S4). A PCA of the data set showed that terpenoids and δ ¹³C values were tightly associated with the first PC, on which leaf removal treatments were also separated (Figure 3). Vintage differences were modelled on PC2 and were associated with berry weight and TSS accumulation.

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The effect of leaf removal observed in Experiment 1b (years 2021, 2023, and 2024) was stronger than in Experiments 1a and 2 (year 2022). The effect was particularly pronounced in the 2021 growing season, which was characterised by low temperatures and low global radiation during berry development (Figure 1). Leaf removal increased monoterpenes and C ₁₃-norisoprenoids to a similar extent (38 % and 29 %, respectively), with free monoterpenes being the most strongly affected (+67 %, Table S4).

Although no irrigation was applied in the 2021 and 2023 growing seasons, samples were taken from the previously irrigated and unirrigated blocks to test for drought stress-induced carry-over effects. Although carry-over effects on yield were detected (mean yield decrease –25.1 %, p = 0.0057; data not shown), there were no such effects on aroma composition.

An optimisation procedure (Figure 4) was used to uncover crucial time points for drought stress influence on the biosynthesis of aroma compound groups and other drought-sensitive berry traits measured at harvest.

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Positive correlations were observed between modelled mean Ψ _PD in the post-flowering phase and berry weight, as well as TSS in the post-veraison period (Figure 4). All other parameters were negatively correlated with Ψ ( i.e., increasing under increasing drought stress) at certain phenological stages: δ ¹³C was most strongly correlated with water deficit roughly two weeks before veraison (‒22 dav, window size 20 d, R _cv = 0.55 for undefoliated vines, Figures 4 and S6) and during shoot development. Bound norisoprenoids were also most strongly (R = ‒0.86 with LR) correlated with water deficit shortly before veraison, irrespective of leaf removal. Correlations of bound and free monoterpenes with Ψ _PD showed different minima, depending on the leaf removal treatment. Without leaf removal, the strongest correlations of free monoterpenes were observed for a window roughly spanning the entire vegetative development phase (centre = ‒39 dav, size = 75 d). Bound monoterpenes were most strongly associated with pre-veraison water deficits. When leaf removal was conducted, free monoterpenes were uncorrelated with water status (Figures 4 and S6). The strongest correlations with bound monoterpenes were observed shortly before Veraison, with large window sizes of 45 and 60 days (R = ‒0.69 and ‒0.62) for LR and no/partial LR, respectively. The correlation of Ψ _PD during the pre-veraison period and harvest TSS showed a pattern inverse to that with berry weight.

Experiment 2: A strong effect of drought stress on monoterpene and norisoprenoid concentration was observed in Experiment 2 (Table 2), with free monoterpene concentration increasing by 30 % and bound norisoprenoids by 40 %.

This experiment was carried out in a hot and dry summer (2022), in which irrigation was completely withheld from flowering to veraison in the DS (drought stress) treatment, leading to strong water deficits (Ψ _MD up to ‒1.5 MPa) before irrigation was reinitiated (Figure S5). The water deficit strongly affected berry size (‒40 % berry weight), with variance similar to the experiment carried out at the Rüdesheim site in the same year (CV 19 % and 23 %, respectively; Table 2). Besides berry size, δ ¹³C was also strongly affected by the pre-veraison water deficit (Table 2). This was surprising, as δ ¹³C is normally associated with water deficit during the ripening phase, but it was in line with the results of Experiment 1. There was a significant positive relationship between δ ¹³C and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN), vitispirane, and total bound norisoprenoids, as well as some free and glycosylated monoterpenes, and a negative relationship with three monoterpenes (free geraniol, nerol, and bound nerol oxide) in berry extracts. The leaf removal effect was relatively weak, and mostly limited to glycosylated monoterpenes, similar to the experiment carried out at the Rüdesheim site in the same year. Another similarity between the two experiments was that the effects of leaf removal were small when carried out in the drought stress treatment, compared to the fully irrigated treatment. Late leaf removal (LLR) had a larger effect on aroma compound concentration than early leaf removal (ELR, Table 2). Likely due to the strong effect of the drought treatment on berry size, there were no effects of irrigation on free monoterpene and bound norisoprenoid content per berry (Table S6), while the bound monoterpene content was elevated in berries from fully irrigated vines.

To dissect drought- and sun exposure-induced effects on the monoterpenoid and C ₁₃-norisoprenoid content of Riesling grapes, we collected data from three experiments combining irrigation and leaf removal treatments and δ ¹³C measurements under different climatic conditions and drought stress levels.

By combining drought stress experiments with leaf removal treatments, we showed that drought stress and increased bunch sun exposure via leaf removal both increase the concentration of monoterpenes and C ₁₃-norisoprenoids in Riesling grapes. The indirect effect of drought stress on aroma compound concentration – a reduction of canopy density – was reduced by leaf removal, and similarly, the effects of leaf removal were reduced in drought-stressed vines. Correlations between glycosylated aroma compounds and simulated drought stress were particularly strong ~14 days before veraison. Wines made from the grapes of an irrigation trial showed that the effects measured in the berries can be expected in wines to a similar extent. Drought stress, whether as a terroir factor or as part of a quality-oriented management and irrigation strategy, can therefore be considered a positive factor for wine quality through an indirect, microclimatic effect as well as a direct effect on aroma compound biosynthesis. Leaf removal can support quality-oriented management strategies, but its effect appears to decrease with increasing drought stress.

We thank the students Carl Baumberger, Alena Mosbacher, Gianna Kozlovic, and Nicola Sorrenti, who did much of the fieldwork for the project in 2021-2024. The laboratory team of the HGU Department of Viticulture is acknowledged for their assistance in the project. The wineries "Hessische Staatsweingüter Kloster Eberbach" and "Weingut Peter Jakob Kühn" are acknowledged for their valuable collaboration in the field experiments. The team at WSU Prosser is thanked for their assistance in conducting the irrigation trial in Washington and processing the samples from the experiments conducted there. The authors also thank Christian Borsch and Silvia Rudloff of JLU Giessen for their support in δ ¹³C analysis.