Introduction

Wine varietal aroma relates to a large range of compounds found in grape berries as precursors that are released during winemaking. Among those, glycosylated aroma precursors are possibly the most extensively studied group in Vitis vinifera and other Vitis sp. (González-Barreiro et al., 2015; Lee et al., 2016; Ghaste et al., 2015). Meanwhile, despite their significance in terms of wine aroma, volatile sulfur compounds such as thiols and their precursors have received less attention. Varietal thiols, such as 3-mercaptohexan-1-ol (3MH) and 4-mercapto-4-methylpentan-2-one (4MMP), are known for their contribution to the characteristic bouquet of Sauvignon blanc wines and other V. vinifera varieties (Rauhut and Kiene, 2019; Capone et al., 2015; Piano et al., 2015). Their presence has also been reported in wines from other Vitis varieties, including the interspecific hybrid Cayuga grown mainly in the State of New York for wine production (Musumeci et al., 2015).

Thiol precursors are present as non-volatile and odourless cysteinylated, dipeptide and glutathionylated precursors in berries (e.g., cysteine-3MH, cysteine-4MMP, glutathione-3MH, glutathione-3MH-aldehyde, glutathione-4MMP, cysteinyl-glycine-3MH and γ-glutamyl-cysteine-3MH) and are further released into volatile and odorous compounds by certain yeast strains (Saccharomyces sp.) during alcoholic fermentation (Bonnaffoux et al., 2017; Santiago and Gardner, 2015). In addition, C₆-compounds, such as hexenal and hexenol, can also contribute to the formation of 3MH during alcoholic fermentation (Harsch et al., 2013, Araujo et al., 2017; Thibon et al., 2016). Thibon et al. (2016) identified S-3-(hexanal)-glutathione as an additional precursor of 3MH.

The concentration of thiol precursors has been investigated in red and white Vitis vinifera varieties, such as Cabernet-Sauvignon, Sauvignon blanc, petit Manseng, Riesling and others (Roland et al., 2011; Fracassetti et al., 2018; Roman et al., 2017), as well as in red and white fungus-resistant interspecific Vitis varieties, such as Leon Millot (red), Baco noir (red), Villard blanc (white), Villaris (white) and Floreal (white), among others (Dournes et al., 2020; Nicolini et al., 2020). Glutathione-3MH is the main thiol precursor reported in interspecific hybrid grapes thus far, with levels ranging from 4.4 to 1141 μg/kg berry fresh weight (FW), whereas cysteine-3MH is second, with concentrations ranging from 0.3 to 136 μg/kg berry FW (Nicolini et al., 2020). The concentration of thiol precursors increases in berry with maturation, but the final concentration is influenced by grapevine variety, soil composition, climate and cultural practices (Capone et al., 2011; Dufourcq, 2018; Roland et al., 2010b).

In grapevine, thiol precursor biosynthesis occurs in berries and has been shown to be closely associated with the biosynthesis of amino acids and glutathione (Chen et al., 2019), which is further related to the nitrogen status of grapevine (Choné et al., 2006; Helwi et al., 2016). In the vineyard, nitrogen and sulfur supplementation may enhance thiol precursors in berries. For instance, soil nitrogen supply has been shown to enhance the synthesis of cysteinylated precursors and glutathione in grapes; in some cases, it also promoted vegetative growth and/or plant susceptibility to pathogens (Choné et al., 2006; Helwi et al., 2016). In trials on white Vitis vinifera varieties, foliar nitrogen supplementation (in the form of urea, 10 to 20 kg/Ha) along with sulfur (5 to 10 kg/ha) during veraison increased thiol concentration in wines by up to five times, depending on the variety and growing conditions, with limited impact on grapevine vigour (Buica et al., 2016; Dufourcq et al., 2010; Dufourcq et al., 2009). Foliar supply has several benefits compared to soil fertilisation: (i) a faster assimilation of the urea is achieved through the leaf cuticle due to its small molecular weight, non-ionic nature and high solubility, (ii) a smaller amount of nitrogen is required, because losses are limited, and (iii) it has a lower impact on vine vigour (Witte et al., 2002). The impact of nitrogen and sulfur on the concentration of thiol precursors of interspecific hybrid varieties has been little investigated thus far. Many interspecific hybrids, especially those adapted to a northern climate, show a higher vigour and noticeable biochemical differences with V. vinifera varieties, including, among others, a tendency to accumulate higher levels of nitrogen in berries (Pedneault and Provost, 2016).

The objective of the current study was to screen the thiol precursor content of seven white hybrid Vitis varieties grown in Eastern Canada for wine production, and evaluate the impact of foliar urea and sulfur sprays starting at mid-veraison on grapevine physiology and thiol precursor in fully mature berries. In addition, amino acids and glutathione were analysed, and their relationship with berry thiol precursor concentration was studied.

Materials and methods

Results and discussion

1. Thiol precursors in the berries

Seven white interspecific hybrid varieties grown in the cold climate of Quebec (Canada) were analysed for the first objective of this study. The varieties studied included selections from different breeders (e.g., French hybrids, such as Seyval blanc and Vidal and American hybrids, such as Saint-Pepin and Frontenac blanc) that have different genetic backgrounds and are locally known to produce wines with very different aroma profiles.

Total soluble solids, pH, monosaccharide and organic acid concentrations of the juices from the sampled varieties are shown in the Supplementary Material (Table S1). No significant differences were found between the varieties for those parameters, except for Frontenac blanc, which showed a higher citric and malic acid content than the other varieties. Frontenac blanc is known to be a highly acidic variety compared to other white hybrids grown in Eastern Canada (Slegers et al., 2017).

A total of eight thiol precursors were investigated in the sampled varieties. Among those, only the four precursors of 3MH (Cys-3MH, G-3MH, CysGly-3MH and γ-GluCys-3MH) were detected in all varieties and, for a given precursor, no significant differences were found from one variety to another (Figure 1; means and standard deviations are available in Table S2 of Supplementary Material). The absence of 4MMP precursors from all samples is likely attributable to the varieties' genotypes. Indeed, 4MMP precursors have been found in only two out of 38 recently analysed hybrid varieties (Nicolini et al., 2020), suggesting that the genes responsible for the biosynthesis of these compounds have a lower occurrence in hybrid Vitis varieties than in Vitis vinifera.

Figure 1. Concentration (μg/kg berry FW) of cysteine-3MH (A, Cys-3MH), glutathione-3MH (B, G-3MH), cysteinyl-glycine-3MH (C, CysGly-3MH) and γ-glutamyl-cysteine-3MH (D, γ -GluCys-3MH) in the hybrid Vitis varieties Frontenac blanc (FB; n = 5), Saint-Pepin (SP; n = 5), Vidal (V; n = 6), Seyval blanc (SB; n = 4), Louise Swenson (LS; n = 1), Adalmiina (Al; n = 1) and L’Acadie blanc (Ac; n = 1) harvested in the province of Quebec, Canada, during the 2016 season. The bar represents the mean concentration and the error bar the standard deviation.

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The concentrations of the 3MH precursors showed large variations in the biological replicates of Frontenac blanc, Vidal, Seyval blanc and Saint-Pepin. Such results were expected due to the completely randomised plan selected for the sampling in order to carry out a broad survey of 3MH precursors in hybrid varieties. However, the high variability also suggests that the accumulation of these compounds is likely highly affected by bioclimatic conditions, cultural practices and/or berry ripeness; for instance, the concentration of G-3MH, which was the main thiol precursor found in the studied varieties, varied from 21 to 395 μg/kg berry FW in Frontenac blanc, from 91 to 605 μg/kg berry FW in Saint-Pepin and from 141 to 711 μg/kg berry FW in Vidal (Figure 1B). The other 3MH precursors (Cys-3MH, CysGly-3MH and γ-GluCys-3MH) were found in quantities equal to or lower than 7 μg/kg berry FW and, except for CysGly-3MH, were detected in all varieties. However, additional analyses would be necessary to validate the presence/absence or the variability of 3MH precursors concentration in L'Acadie blanc, Aldamiina and Louise Swenson berries, as only one replicate could be analysed for those varieties (Figure 1A, 1C, and 1D).

Vidal and Saint-Pepin showed the highest concentration of G-3MH, with 50 % of the replicates showing a concentration higher than or close to the overall average value of 240 μg/kg berry FW (Figure 1B). During winemaking, the conversion rate of 3MH precursors to 3MH volatile thiol is very low and ranges from 0.1 to 12 % (Robinson et al., 2014). This process is affected by oenological conditions, such as must turbidity, temperature and yeast strain used for winemaking. However, it ultimately depends on yeast assimilable nitrogen (Subileau et al., 2008), with ammonium ions and amino acids as the main sources in grape must. Since the olfactory perception threshold of 3MH is 60 ng/L in wine, the concentration of G-3MH found in the studied varieties could significantly impact the resulting wine aroma. Furthermore, in Vitis vinifera varieties, freezing whole grape berries has been shown to increase the concentration of G-3MH in berries (Chen et al., 2019; Capone et al., 2011). Such a phenomenon has not yet been demonstrated in interspecific hybrids; therefore, an analysis of thiol precursors in fresh berries would be necessary to fully account for the thiol potential of these varieties in table wine production. However, most of the studied varieties, especially Vidal, are used for ice wine production in Quebec. Our results suggest that thiols contribute to the aroma of these speciality wines, and that their concentration can increase given the selection of proper yeast strain and fermentation conditions (Santiago and Gardner, 2015).

2. Amino acids in the berries

Thiol precursors contain an amino acid or a peptide moiety. Despite not being fully understood, the accumulation of thiol precursors has been suggested to relate to amino acid and glutathione biosynthesis in berries (Chen et al., 2019). Glutathione (L- γ-glutamyl-L-cysteinyl-glycine and GSH) is a tripeptide that results from the condensation of glutamic acid, cysteine and glycine: Glu and Cys are first condensed to form γ-glutamylcysteine through the action of γ-glutamylcysteine synthetase, and then Gly is added via GSH synthetase activity to form GSH (Kritzinger et al., 2012). In berries, GSH may combine with hexenal via the activity of GSH S-transferases (VvGST3 and VvGST4), resulting in the production of the thiol precursor G-3MH (Kobayashi et al., 2010). Other thiol precursors (CysGly-3MH and Cys-3MH) derive from G-3MH through the subsequent action of γ-glutamyl transferase (CysGly-3MH), and carboxypeptidase (Cys-3MH) (Dubourdieu and Tominaga, 2009).

In the present study, 21 amino acids and the GSH content of seven hybrid varieties were quantified and their potential relationship with 3MH precursors was evaluated. The total amino acid contents (including GSH) of the varieties analysed were similar (Table 1), but large ranges were observed for biological replicates; for instance, the total amino acid + GSH content of Frontenac blanc ranged from 196 to 2167 mg/kg berry FW. Such variation is likely attributable to the different climatic conditions, soil composition (e.g., organic matter content and pH) and/or vine management practices (including fertilisation) that occurred in the commercial vineyards where the berries were sampled (Ortega‐Heras et al., 2014). However, large variations in nitrogen compound accumulation (e.g., primary amino nitrogen and yeast assimilable nitrogen) are frequent in cold-hardy and disease-resistant grape varieties, such as interspecific hybrids (Pedneault and Provost, 2016).

Arg, Ala, Gln and Pro were the main amino acids found in all analysed varieties, accounting for 61 (Louise Swenson) to 82 % (Adalmiina) of the analysed amino acids (Table 1). Thirteen of the 22 amino acids showed significantly different concentrations from one variety to another, with Frontenac blanc generally showing the highest concentration for most amino acids. Arg was the main amino acid in L'Acadie blanc (42 %), Frontenac blanc (33 %) and Louise Swenson (26 %), whereas Gln was the main amino acid in Adalmiina (33 %) and Seyval blanc (26 %). Ala was predominant in Saint-Pepin (27 %). Other amino acids, as well as GSH, were found in lower concentrations. In contrast with the other varieties studied, the Vidal amino acid profile showed a significant proportion of Pro (31 %). This is noteworthy because, as a secondary amino acid, Pro cannot be assimilated by yeast. Furthermore, Vidal generally accumulates a lower concentration of primary amino nitrogen than other hybrid varieties (Pedneault and Provost, 2016; Slegers et al., 2017). Thus, sometimes, in Vidal juice it is necessary for the supplementation of primary amino nitrogen to ferment correctly.

Table 1. Amino acid and glutathione content (mg/kg berry; fresh weight) in berry juices from seven white interspecific hybrid grape varieties (Frontenac blanc, n = 5; Saint-Pepin, n = 5; Seyval blanc, n = 4; Vidal, n = 6, Adalmiina, n = 1; L'Acadie blanc, n = 1; and Louise Swenson, n=1) which were harvested in the province of Quebec, Canada, during the 2016 season.

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AA 2	Frontenac blanc			Seyval blanc			Saint- Pepin			Vidal			Adalmiina	L'Acadie blanc	Louise Swenson	p- value
Ala	185	±	102	177	±	105	235	±	143	66.5	±	31	147	43.4	116	0.0727
Arg	474	±	273 b1	90.1	±	109 a	175	±	137 ab	130	±	111 a	257	258	221	0.0114
Asn	13.2	±	6.1	11.9	±	9.9	10.5	±	6.7	4.88	±	2.14	6.62	8.92	5.45	0.1823
Asp	32.0	±	13	15.1	±	9.8	20.0	±	10.4	16.9	±	3.7	26.0	22.2	48.9	0.0601
Cys	0.26	±	0.11 b	0.19	±	0.08 ab	0.17	±	0.05 ab	0.12	±	0.02 a	0.12	0.14	0.24	0.0415
GABA	53.5	±	19	23.6	±	11	34.6	±	14.5	27.5	±	6.6	34.4	30.0	47.5	0.0144
Gln	263	±	167	221	±	239	228	±	171	73.2	±	61	359	103	125	0.2468
Glu	53.5	±	29 b	20.1	±	14 a	19.0	±	11.3 a	17.0	±	5.4 a	40.6	21.3	59.1	0.0089
Gly	4.09	±	2.1 b	2.58	±	1.9 ab	2.61	±	1.2 ab	1.24	±	0.51 a	1.24	1.72	1	0.0451
His	20.2	±	10.5	6.08	±	4.6	12.8	±	6.1	13.9	±	7.0	8.91	6.76	17.3	0.0833
Ile	11.1	±	5.0 b	5.73	±	4.3 ab	2.52	±	0.80 a	11.1	±	5.5 b	1.36	1.57	7.1	0.0141
Leu	28.0	±	14 b	8.04	±	6.2 a	8.47	±	4.9 a	12.8	±	3.6 a	6.94	3.22	16.8	0.0041
Lys	5.07	±	2.2 b	1.32	±	0.38 a	1.31	±	0.61 a	1.41	±	0.76 a	1.62	1.87	2.62	0.0003
Met	5.18	±	3.0 b	1.21	±	0.83 a	1.29	±	0.94 a	2.55	±	1.36 ab	0.95	0.38	2.09	0.0109
Phe	16.4	±	8.3 ab	6.28	±	3.52 a	8.88	±	4.2 a	19.3	±	4.8 b	2.89	2.93	4.99	0.0061
Pro	135	±	76	166	±	200	42.8	±	24.9	214	±	169	135	57.0	50.8	0.2462
Ser	48.5	±	27	25.9	±	23.2	34.8	±	21	17.6	±	7.4	20.8	18.3	26.3	0.1201
Thr	43.1	±	25	23.3	±	19	21.8	±	13	17.5	±	8.1	17.0	15.3	29.8	0.1152
Trp	8.21	±	2.9 b	2.56	±	0.55 a	3.83	±	1.09 a	4.78	±	1.95 a	6.77	5.15	6.09	0.0023
Tyr	6.78	±	4.1 b	1.17	±	0.49 a	3.64	±	2.89 ab	4.85	±	1.18 ab	3.61	0.56	11.6	0.0344
Val	23.7	±	11.2	11.6	±	8.1	11.3	±	5.67	12.9	±	3.6	7.98	4.04	16.0	0.0556
GSH	7.81	±	5.2 a	18.92	±	11.4 b	5.44	±	3.44 a	16.6	±	8.8 ab	7.37	7.24	29.3	0.0299
TOTAL	1438	±	767	839.45	±	769.53	883	±	564	686	±	346	1093	612	845

¹ Data represent mean values ± standard deviations of 4 to 5 biological samples, except for the Adalmiina, L'Acadie blanc and Louis Swenson varieties, for which n = 1 biological sample; samples were analysed in duplicate. For a given variable, values followed by different lower-case letters within a row are significantly different from each other according to Tukey's HSD test at p ≤ 0.05. ² Abbreviations: Amino acid, AA; L-alanine, Ala; L-arginine, Arg; L-asparagine, Asn; L- aspartic acid, Asp; L-cysteine, Cys; γ-aminobutyric acid, GABA; L-glutamic acid, Gln; L-glutamine, Glu; L-glycine, Gly; glutathione, GSH; L-histidine, His; L-isoleucine, Ile; L-leucine, Leu; L-lysine, Lys; L-methionine, Met; L-phenylalanine, Phe; L-proline, Pro; L-serine, Ser; L-threonine, Thr; L-tryptophan, Trp; L-tyrosine, Tyr; L-valine, Val.

The presence of an amino moiety in thiol precursors suggests that the biosynthesis of those precursors may relate to the biosynthesis of certain amino acids, such as Cys and GSH. In the current study, the potential relationships between the accumulation of thiol precursors (Cys-3MH and G-3MH) and the amino profile of berries were investigated using correlation analysis. The results show moderate (0.56 ≤ r ≤ 0.68, P ≤ 0.01) to strong (r ≥ 0.70, P ≤ 0.001) positive correlations between variables (Table 2). The strongest correlations occurred between amino acids, rather than between amino acids and thiol precursors. Despite the high concentrations of both GSH and thiol precursors found in certain varieties, such as Seyval blanc and Vidal (19 mg/L and 17 mg/L for GSH respectively), no significant correlation was found between those variables.

In contrast with Chen et al. (2019), no significant correlation was found between thiol precursors and the amino acids Glu, Cys and Gly, which, as constituents of GSH, may be involved in the metabolism of G-3MH and Cys-3MH. Conversely, a significant correlation was found between G-3MH and Pro (r = 0.57, P ≤ 0.01). As Pro is biosynthesised from Glu, and Glu is necessary for GSH biosynthesis, Pro is likely a competitor of G-3MH from a metabolic point of view; for this reason, this correlation was unexpected. Furthermore, in contrast with our results, Chen et al. (2019) found a negative relationship between G-3MH content of Sauvignon blanc berries and their content in Pro and Glu, among other amino acids. Such differences between the results suggest that interspecific hybrid Vitis varieties accumulate thiols differently than V. vinifera.

Table 2. Correlation matrix of thiols precursors (Cys-3MH; G-3MH) and amino compounds (amino acids, glutathione) content in berry juices from seven white interspecific hybrid grape varieties (Frontenac blanc, n = 5; Saint-Pepin, n = 5; Seyval blanc, n = 4; Vidal, n = 6, Adalmiina, n = 1; L'Acadie blanc, n = 1; and Louise Swenson, n = 1) harvested in the province of Quebec, Canada, during the 2016 season.

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Compounds1	Cys-3MH	Glut-3MH	Ala	Arg	Asn	Asp	Cys	GABA	Gln	Glu	Gly	His	Ile	Leu	Lys	Met	Phe	Pro	Ser	Thr	Trp	Tyr	Val	GSH
Cys-3MH	12
Glut-3MH	0.52	1
Ala	0.37	0.37	1
Arg	0.32	0.10	0.62	1
Asn	0.15	0.16	0.86	0.68	1
Asp	0.38	0.23	0.71	0.92	0.74	1
Cys	0.10	-0.09	0.69	0.82	0.78	0.78	1
GABA	0.14	-0.04	0.60	0.87	0.66	0.81	0.80	1
Gln	0.21	0.16	0.89	0.70	0.96	0.75	0.76	0.64	1
Glu	0.29	0.03	0.60	0.97	0.70	0.90	0.89	0.85	0.72	1
Gly	0.00	-0.03	0.65	0.68	0.87	0.67	0.76	0.75	0.75	0.71	1
His	0.33	0.39	0.54	0.90	0.54	0.85	0.64	0.76	0.58	0.83	0.53	1
Ile	0.07	0.11	0.05	0.58	0.28	0.51	0.39	0.46	0.28	0.59	0.29	0.70	1
Leu	0.29	0.04	0.44	0.94	0.56	0.89	0.76	0.82	0.58	0.96	0.59	0.88	0.74	1
Lys	0.13	-0.12	0.36	0.88	0.57	0.76	0.76	0.84	0.49	0.89	0.78	0.73	0.55	0.87	1
Met	0.24	0.00	0.33	0.86	0.44	0.79	0.67	0.72	0.48	0.88	0.51	0.84	0.80	0.96	0.82	1.00
Phe	0.14	0.31	0.13	0.62	0.21	0.59	0.32	0.53	0.26	0.58	0.20	0.82	0.86	0.73	0.51	0.77	1
Pro	0.16	0.57	0.12	0.28	0.32	0.34	0.17	0.16	0.24	0.29	0.21	0.44	0.56	0.34	0.21	0.26	0.50	1
Ser	0.38	0.25	0.85	0.88	0.90	0.92	0.82	0.82	0.90	0.87	0.82	0.79	0.41	0.79	0.74	0.68	0.45	0.31	1
Thr	0.26	0.15	0.72	0.92	0.87	0.91	0.88	0.86	0.85	0.94	0.82	0.82	0.59	0.88	0.83	0.79	0.56	0.41	0.95	1
Trp	0.05	-0.18	0.27	0.81	0.35	0.67	0.67	0.76	0.40	0.81	0.48	0.77	0.66	0.85	0.80	0.88	0.69	0.01	0.57	0.68	1
Tyr	0.14	0.11	0.31	0.76	0.27	0.69	0.52	0.60	0.38	0.74	0.31	0.81	0.57	0.79	0.60	0.80	0.76	0.16	0.53	0.59	0.79	1
Val	0.30	0.15	0.59	0.93	0.72	0.92	0.81	0.84	0.72	0.95	0.70	0.88	0.73	0.97	0.84	0.92	0.71	0.43	0.89	0.95	0.77	0.73	1
GSH	-0.16	0.09	-0.02	-0.15	0.1	-0.01	0.24	-0.13	0.1	0.02	-0.07	-0.11	0.23	0.01	-0.2	0.02	0.19	0.39	-0.05	0.05	-0.1	0	0.09	1

¹Amino compounds: L-alanine, Ala; L-arginine, Arg; L-asparagine, Asn; L- aspartic acid, Asp; L-cysteine, Cys; γ-aminobutyric acid, GABA; L-glutamic acid, Gln; L-glutamine, Glu; L-glycine, Gly; L-histidine, His; L-isoleucine, Ile; L-leucine, Leu; L-lysine, Lys; L-methionine, Met; L-phenylalanine, Phe; L-proline, Pro; L-serine, Ser; L-threonine, Thr; L-tryptophan, Trp; L-tyrosine, Tyr; L-valine, Val; glutathione, GSH. Thiol precursors: cysteine-3MH, Cys-3MH; glutathione-3MH, G-3MH.

² Values in bold are significant at P ≤ 0.001 and values in italics at P ≤ 0.01.

Interest in the amino acid content of grape juice with regards to thiols is also driven by the relationship between the presence of specific amino acids and the release of free thiols during winemaking. The amino acid content of berry juice and the relative content of amino acids to each other's have been found to impact the consumption of thiol precursors by yeast and the consequent release of free thiol in wine (Alegre et al., 2017). For instance, previous studies on Vitis vinifera sp. Sauvignon blanc juice have shown moderate relationships (r = 0.33 to 0.43) between the concentration of amino acids Glu, GABA and Gln and the release of free 3MH in wine (Pinu et al., 2014; Chen et al., 2019). In model solutions, a high proportion of Asp, Thr, Phe and Ser were related to a higher release of 3MH during fermentation, whereas His, Pro and Met were associated with a lower release of free thiols (Alegre et al., 2017).

The potential of releasing free thiol from the studied hybrid varieties was evaluated using principal component analysis (PCA) and the relative proportion (as a percentage) of amino compounds (amino acids and GSH). The result primarily showed the compositional differences between the Seyval blanc and Saint-Pepin varieties (Quadrat I), Frontenac blanc (Quadrat II) and Vidal (Quadrats III), as can mainly be seen in PC 1 (39.5 % of variance explained; Figure 2). The variables graph shows that amino acids that readily release thiols (e.g., Asp, Thr and Ser) are mostly located in Quadrat II of the PCA, while amino acids associated with a lower rate of thiol release (e.g., Pro, His and Met) are in Quadrat III. Quadrat II is associated with Frontenac blanc, suggesting that thiol release could be facilitated in this variety. In contrast, amino acids associated with lower thiol release rates are mostly found in Vidal (Quadrat III), which also showed a higher range of G-3MH. Based on results from Chen et al. (2019), a high Pro content may negatively impact the release of thiols during fermentation. However, it is unclear whether the positive impact of thiol-promoting amino acids would bypass the negative impact of Pro, His and Met during the fermentation for this variety. On the other hand, some varieties containing high levels of thiol precursors (e.g., Saint-Pepin and Seyval blanc; Quadrat I) showed low amounts of amino acids associated with low-thiol conversion rates (Quadrat III). These results suggest that adjusting the amino profile by adding thiol-promoting amino acids can contribute to higher conversion rates in certain varieties, including Vidal, Saint-Pepin and Seyval blanc. However, the impact of the amino profile on the rate of conversion of thiol precursors into free thiols during winemaking using interspecific hybrid varieties remains to be studied

Figure 2. Principal component analysis showing (A) the distribution of 20 samples from 4 white interspecific hybrid varieties (Frontenac blanc, n = 5, purple diamond; Saint-Pepin, n = 5, blue triangle; Seyval blanc, n = 4, brown square; and Vidal, n = 6, green circle) from the province of Quebec, Canada, during the 2016 season, and (B) the loadings plot based on concentrations of amino acids and glutathione (refer to Table 1 for abbreviations).

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Conclusions

Thiol precursors, amino acids and glutathione were characterised for the first time in 23 grape samples from seven white hybrid varieties (Adalmiina, Frontenac blanc, L'Acadie blanc, Louise Swenson, Saint Pepin, Seyval blanc and Vidal) grown in the province of Quebec, Canada. Precursors of 3-mercaptohexan-1-ol (3MH) were the only thiol precursors identified in the hybrid varieties analysed, glutathione-3MH being the main abundant thiol precursor. The varieties Vidal, Seyval blanc and Saint-Pepin showed the highest concentrations of glutathione-3MH. Correlation analyses on amino acids, glutathione and thiol precursors (glutathione-3MH and cysteine-3MH) in grape berry juices showed little correlation between amino acids and thiol precursors in berries, except for a positive correlation between glutathione-3MH and proline; this contrasts with previous findings for V. vinifera, suggesting that thiol/amino acid metabolisms differ in interspecific hybrids when compared to V. vinifera.

Based on literature data, principal component analysis was used to evaluate the potential of amino acid profile to promote thiol precursors conversion during winemaking. The results showed that certain varieties containing high levels of thiols precursors (e.g., Saint-Pepin and Seyval blanc) have low amounts of amino acids typically associated with low-thiol conversion rates (e.g., proline, histidine and methionine), whereas others such as Vidal have high levels of proline. Although amino nutrition is usually considered as a whole in winemaking (e.g., yeast assimilable nitrogen), these results suggest that supplementing juice with specific amino acids could be a strategy to increase thiol conversion in interspecific hybrid varieties.

In the second part of the study, the effect of urea and sulfur foliar supply on vine physiology, berry maturity and thiol precursor production was investigated on Vidal. To our knowledge, this was the first essay to be conducted on Vidal grapes grown in Quebec. No significant differences were found between treatments (combinations of urea and sulfur) on yield components and thiol precursor concentrations in grape berry juice. However, despite berries being at a similar ripening stage, cysteine-3MH accumulation was significantly different between each experimental site, suggesting that other factors, such as cultural practices, soil and climate, also affect thiol precursors concentration in berries.

These findings open avenues for further investigations on the biogenesis of thiol precursors in berries and on the improvement of wine quality in interspecific hybrid wine production.

Acknowledgements

The authors would like to thank MAPAQ and AAFC for financing this study. The authors acknowledge Hugo Bonnaffoux (IFV, France) and Dr Florine Cavelier (CNRS-IBMM, France) for providing certain analytical standards for grape characterisation. We wish to thank Dr Caroline Provost (CRAM, Quebec, Canada) and the Conseil des vins du Québec for the grape donation. We would also like to thank Véronique Richard (INAF, Quebec, Canada) for her help with the sugar and organic acid analyses.

Conflict of interest

The authors declare no conflict of interest.

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