This study was conducted during the growing seasons of 2009 and 2010 in two 18-year-old vineyards located in the Goumenissa region of north Greece (40°52' N, 22°29' S). The vineyards were planted with the red winegrape varieties Cabernet-Sauvignon (CS) and Xinomavro (XM), both Vitis vinifera L. cultivars, and grafted onto the 1103 Paulsen rootstock. Both vineyards had the same vine spacing (2.2 m and 1.3 m between and within row respectively) and a north to south row orientation. The vines were spur-pruned to 12 buds per vine and trained to a bilateral Royat system with three fixed trellising wires. Soil had a clay loam and a sandy clay loam texture in the CS and XM vineyard respectively, with an average effective rooting depth of between 60 to 90 cm for both vineyards. Climatic conditions were generally similar for both growth seasons in terms of average temperature and total precipitation for the April to September period; the average temperature was 21.2 and 21.8 ^oC for 2009 and 2010 respectively, whereas precipitation for the same period was 226.2 mm for 2009 and 227.0 mm for 2010.

In each vineyard, three blocks were delineated, each containing a 2×3 grid of 6 plots with 6 vines per plot. The plots were separated with four guard vines along the row and with a guard row from each side. The combination of two irrigation levels [Irrigation (I): 70 % of crop evapotranspiration (ET_c) and no irrigation (NI)] and three rates of ammonium nitrate [0 (N₀), 60 (N₆₀) and 120 (N₁₂₀) kg/ha N] was randomly assigned to each block. A randomised complete block design was applied with six treatments (2 irrigation levels × 3 N fertiliser rates × 3 blocks) resulting in 18 plots per vineyard. Only the mean of each plot was used in the statistical analysis of the results.

Drip irrigation started at berry set (growth stage E-L 27, according to the modified Eichhorn-Lorenz system) in both seasons and was continued at weekly intervals, according to the estimates obtained from the potential evapotranspiration measured by an automated weather station located in the XM vineyard. Ammonium nitrate (34-0-0) was applied to the soil surface at budburst in both years of the study.

Carbon isotope composition (δ¹³C) and total nitrogen content were measured in the bulk dry matter (BDM) of leaf blades, sampled at four time points (BS, BC, VE and MT, corresponding to the growth stages of berry set, bunch closure, veraison and maturity respectively) during each growing season. The leaf tissues were dried at 65 °C and ground to fine powder with a rotary mill and a 0.20 mm mesh. From the final sample preparation, 2.8±0.1 mg, enclosed in SC0009 8×3 mm (SerCon Ltd, Gateway, Crewe, UK) capsules, was passed to an automated combustion elemental analyzer interfaced with a continuous-flow isotope ratio mass spectrometer (IRMS; PDZ Europa, Cheshire, UK).

For δ¹³C in cane BDM, twelve canes from each plot were randomly selected at dormancy. From the middle of each cane, a section containing a latent bud and the internode immediately below the bud was used for δ¹³C determination following the same procedure as for leaf blades.

At technological maturity (late September of each year for both varieties), all clusters from each plot were collected, then transferred to the laboratory and a sample of 200 berries from all parts of each cluster was randomly selected and hand-pressed for must extraction. After immediate must clarification (0.2 NTU), 5 μL of bulk must (BJ) was directly injected to the IRMS for must δ¹³C determination.

In all cases, the PDB standard was used and δ¹³C was calculated according to Equation 1.

Stem water potential (Ψ_s) was measured at the BS, BC, VE, and MT stages, using a pressure chamber, according to Choné et al. (2001). In each measurement set, four leaves per plot from the inner part of the canopy were enclosed in plastic bags and covered with aluminum foil for at least 90 min before measurement to allow equilibration of Ψ_s. The measurements were taken at solar noon (between 12:30 and 13:00) under clear sky conditions.

The same days when sampling for δ¹³C and for Ψ_s measurements in each growth stage, net assimilation rate (A_n), stomatal conductance (g_s), intercellular CO₂ concentration (C_i) were determined in four mature sunlit leaves, adjacent to Ψ_s leaves, in each plot with a portable gas exchange system LCi (ADC BioScientific Ltd, Hoddesdon, UK). The measurements were performed within the same time window of Ψ_s measurement. Intrinsic water-use efficiency (WUE_i) was estimated as the A_n/g_s ratio. The mean of the four measurements of each plot was used in the statistical analysis.

In each of the two experimental vineyards, a randomised complete block design, replicated in three blocks and repeatedly measured over the two years, was used to evaluate the effects of two irrigation levels and three fertilisation rates on vine properties. To estimate the fixed and random effects and their size, a linear mixed model with the restricted maximum likelihood method for the estimation of covariance parameters was used. For leaf data that were collected repeatedly within each growing season, the growth stage at each sampling was incorporated as an additional factor into the mixed model. Vineyard, year, irrigation, fertilisation and growth stage were considered as fixed effects, whereas blocking as random effects. Since each vineyard was planted with a different variety, the “vineyard” effects in our experimental design represent the combined effects of vineyard location (soil, for example) and of variety. The least significant difference test was used to detect differences between the means of the fixed effects at p < 0.05. The relationships between the measured variables were evaluated by linear correlation and regression analysis. Data analysis was conducted using Statistical Analysis System software, version 9.3 (SAS Institute, Cary, NC).

Irrigated vines of both vineyards had higher values of stem water potential (Table 1). Ψ_s decreased from the BS to MT stage, and it decreased more steeply between BC and VE (Table 1). The greater differences in Ψ_s between the irrigation treatments were observed at the VE and MT stages [Table 1; data previously published in Taskos et al. (2015)]. No difference in Ψ_s was observed among N treatments. Both fertilised treatments in CS and the N₁₂₀ treatment in XM had higher concentrations of leaf N (Table 1). Leaf N concentration declined progressively from BS to MT stage (Table 1). Irrigation had no effect on leaf N content (Table 1).

Irrigation had significant effects on gas exchange of single leaves in the XM vineyard only, where the irrigated vines had higher rates of stomatal conductance and net assimilation, but lower WUE_i (Table 1). However, leaves of both cultivars presented higher net assimilation and stomatal conductance rates at BS and BC compared to VE and MT samplings (Table 1). Contrary to irrigation, the influence of N addition on gas exchange was small (Table 1). However, significant relationships were observed between leaf N content and A_n, g_s, WUE_i, C_i (Figure 1) in both vineyards: with increasing leaf N, net assimilation and stomatal conductance rate increased, whereas intrinsic water use efficiency decreased (Figure 1). The relationship between A_n and leaf N was stronger in the CS vineyard than in the XM vineyard, while the opposite was observed for the C_i values (Figure 1). These relationships were better described by quadratic regression models, except for C_i in the XM vineyard.

In both vineyards, water application and N fertilisation affected leaf carbon isotope composition, but in different directions; leaf δ¹³C values were lower in the irrigated vines compared to the non-irrigated vines, but were higher in the CS fertilised treatments and in the N₁₂₀ XM vines compared to the non-fertilised vines (Table 1). The N₁₂₀ CS and XM vines had consistently higher leaf δ¹³C than N₀ vines, irrespective of growth stage (Figures 2B, D). On the contrary, the irrigated vines had lower leaf δ¹³C only after the BC and VE stages (Figures 2A, C) in the CS and XM vineyards respectively, because of a significant interaction between irrigation and the growth stage (F = 9.3, p < 0.0001). Leaf δ¹³C values also varied with growth stage, mainly between the BS and BC stages, when δ¹³C values decreased in both vineyards (Table 1), except for non-irrigated XM vines (Figure 2C). The size of the effect of N fertilisation and irrigation on leaf δ¹³C depended on growth stage (Figure 3A, B): N application accounted for a higher proportion of leaf δ¹³C variance at the BS and the BC stages, whereas irrigation induced higher variance of leaf δ¹³C at the VE (only CS) and MT stages.

As in the case of the leaf carbon isotope ratio, different responses of cane δ¹³C to irrigation and N fertilisation were observed in both vineyards: the values increased with N supply but decreased with irrigation (Table 1). Berry must δ¹³C was lower in the irrigated vines of both varieties (Table 1). However, due to significant interaction between irrigation and N fertilisation (F = 8.98, p = 0.0018), irrigated and not-irrigated treatments did not differ in their must δ¹³C values under the higher nitrogen rate (Figure 4).

In both vineyards and irrespective of irrigation and fertilisation treatment, bulk berry must at maturity was more enriched with ¹³C compared to leaves and canes (Table 1). The size of the effect of irrigation on must and cane δ¹³C was higher compared to that of fertilisation in both vineyards (Figures 3C, D), and the mixed model accounted for a higher variance of δ¹³C in the must and canes than in leaves (Figures 3C, D). The leaf δ¹³C variance explained by the leaf N content was higher at the BS and BC stages, whereas Ψ_s explained most of leaf δ¹³C variance at the VE and MT stages (Figures 5A, B). Similar responses were observed for cane δ¹³C variance (Figures 5E, F). Only Ψ_s accounted significantly for must δ¹³C variance, at all stages in XM (Figure 5D) and after the BC stage in CS (Figure 5C).

In both vineyards must and cane δ¹³C values were positively correlated with the δ¹³C values in leaves at the MT stage (r = 0.69 & 0.61 respectively, p < 0.001 in CS; r = 0.50 p < 00.1 & 0.74 p < 0.001 respectively in XM).

The increased leaf N content in the CS fertilised treatments and in the XM N₁₂₀ treatment (Table 1) is consistent with previous studies (Keller et al., 2001) and indicates a modification of vine N status. The progressive decline of leaf N from the BS stage towards the MT stage (Table 1), can be explained by N translocation from the aging basal leaves to younger actively growing organs (Keller et al., 2001). Regarding water status, according to van Leeuwen et al. (2016), irrigated vines were under weak water deficit after VE, while the non-irrigated vines were under moderate to severe water deficit after veraison [data previously published in Taskos et al. (2015)]. The decline of Ψ_s values between BC and VE (Table 1) indicates a corresponding depletion of available water reserves in the soil of both vineyards.

In grapevines, increasing soil water deficit causes a chain of gas exchange responses: stomata closure and a subsequent reduction in g_s and of CO₂ available in the chloroplasts, as observed in the non-irrigated XM vines (Table 1). According to their g_s values, XM vines (Table 1) were under mild water stress after the VE stage (Cifre et al., 2005). According to Chaves et al. (2010), some varieties maintain higher g_s during the day for similar predawn water potential under field conditions. Thus, despite the alteration of vine water status, the smaller effects of irrigation on CS gas exchange (Table 1) could be attributed to varietal differences. However, the two varieties were not replicated in our experimental design. Consequently, the “variety” effects may as well represent in fact “location” effects.

Table

titre du tableau
	Cabernet-Sauvignon	Xinomavro
	Leaf	Juice	Cane	Leaf	Juice	Cane
Effect	N	δ13C	Ψs	A	gs	WUEi	Ci	δ13C	δ13C	N	δ13C	Ψs	A	gs	WUEi	Ci	δ13C	δ13C
Year
2009	2.14a	-26.87a	-0.95a	19.7a	1.07a	24.0b	256a	-25.61	-26.95	2.07	-27.49	-0.85a	17.9a	0.74a	32.8	247	-24.87	-27.12
2010	1.90b	-27.07b	-1.00b	17.6b	0.57b	38.5a	238b	-25.57	-26.93	2.00	-27.62	-0.98b	14.5b	0.50b	37.4	247	-25.34	-27.24
Stage
BS	2.65a	-26.39a	-0.76a	21.4a	0.83b	31.7b	243b	nd	nd	2.59a	-27.32a	-0.62a	17.6b	0.77b	24.7b	265a	nd	nd
BC	2.13b	-27.00b	-0.78a	20.6a	1.25a	21.1c	258a	nd	nd	2.04b	-27.64b	-0.74b	19.3a	0.93a	25.8b	252b	nd	nd
VE	1.78c	-27.33c	-1.16b	17.5b	0.77b	32.4b	245b	nd	nd	1.82c	-27.74b	-1.12c	13.6c	0.42c	42.3a	237c	nd	nd
MT	1.53d	-27.17bc	-1.20b	15.0c	0.44c	39.8a	244b	nd	nd	1.69c	-27.53ab	-1.18c	14.3c	0.35c	47.6a	234c	nd	nd
Irrigation
NI	2.03	-26.77a	-1.04b	18.4	0.78	33.0	245	-24.90a	-26.61a	2.06	-27.37a	-0.99b	15.6b	0.52b	38.8a	241b	-24.51a	-26.87a
I	2.01	-27.18b	-0.91a	18.9	0.87	29.5	249	-26.28b	-27.27b	2.01	-27.74b	-0.84a	16.9a	0.72a	31.4b	253a	-25.70b	-27.49b
Nitrogen
N0	1.86c	-27.28b	-0.96	18.6ab	0.85	30.0	249	-25.93b	-27.27b	1.94b	-27.78b	-0.88	16.0	0.57	36.8	246	-25.22	-27.33b
N60	1.99b	-26.91a	-1.00	17.6b	0.72	34.8	245	-25.24a	-26.83a	1.99b	-27.66b	-0.92	15.8	0.64	33.8	250	-25.19	-27.33b
N120	2.22a	-26.72a	-0.96	19.7a	0.88	29.0	248	-25.59ab	-26.72a	2.17a	-27.22a	-0.94	16.9	0.64	34.7	245	-24.91	-26.88a
Tissue	nd	-26.97*	nd	nd	nd	nd	nd	-25.59**	-26.94*	nd	-27.55*	nd	nd	nd	nd	nd	-25.10**	-27.18*

Ν_{0 =} unfertilised; N_{60 =} 60 kg /ha N; N₁₂₀ = 120 kg/ha N; NI = non-irrigated; I = irrigated at 70 % of ETc; BS = berry set; BC = bunch closure; VE = veraison; MT = maturity.Total nitrogen (N, %); Carbon isotope ratio (δ¹³C, ‰); Stem water potential (Ψ_s, MPa); Net Assimilation rate (A, μmol CO₂ m^-2 s^-1); Stomatal conductance (g_s, mol m^-2 s^-1 H₂O) ; Intrinsic water use efficiency (WUE_i, μmol CO₂ /mol H₂O); Intercellular CO₂ concentration_{(Ci = mmol /mol^- CO2)Data for leaf N, Ψs have previously been published in Taskos et al., (2015); nd = no dataMeans within factors and vineyards followed by different lowercase letters are significantly different at the 0.05 probability level according to LSD. For Tissue, means within vineyards followed by different numbers of asterisks are significantly different at the 0.05 probability level according to LSD}

Table

Growth stage: BS = berry set; BC = bunch closure; VE = veraison; MT = maturityData are plot means at each growth stage for two years.

Since leaf δ¹³C was measured in leaf BDM, leaf δ¹³C values at each growth stage integrated the isotope effects during the preceding period of leaf development, when the carbon forming the leaf structural carbohydrates was fixed (Brugnoli and Farquhar, 2000). In addition, part of leaf δ¹³C can be determined by the carbon isotope composition of non-structural carbohydrates like starch and primary photosynthates close to the time of leaf sampling. Therefore, whatever the underlying mechanisms of leaf δ¹³C modulation by nitrogen and water availability, and whatever the extent to which leaf δ¹³C was determined by these two factors, leaf δ¹³C at a specific growth stage will integrate the opposite effects of irrigation and fertilisation during leaf development on leaf structural and non-structural carbohydrates up to that specific stage. Considering that any significant water deficit before the BS stage was unlikely under the experimental conditions, the stronger effect of N fertilisation at the BS and BC growing stages (as shown in Figures 3A, B) represents a higher contribution of nitrogen availability to leaf δ¹³C variation, but only in the context of minimal influence of water availability. The opposite was observed at the VE and MT stages when water status effects on leaf δ¹³C modulation prevailed (Figures 3A, B; Figures 5A, C). That means that a water deficit at the early stages of leaf development could have equally impacted leaf δ¹³C at the BS and BC stages. Furthermore, carbohydrate allocation in grapevine leaves continues well past the time needed for completing lamina expansion (Poni et al., 1994), and the amount of structural carbohydrates does not change much after the completion of leaf development. Thus, the carbon isotope composition of the non-structural fraction of leaf carbon may have increasingly contributed to leaf δ¹³C close to the later grapevine stages. Consequently, the increased differences in leaf δ¹³C between the irrigation treatments (Figure 2) at the VE and MT stages are probably the result of the more significant irrigation effects on the non-structural fraction of leaf carbon.

Table

Growth stage: BS = berry set; BC = bunch closure; VE = veraison; MT = maturity.Asterisks denote significant effects at the 0.05 probability level.

Carbon isotope composition of leaf soluble sugars provides a reliable estimation of C_i /C_a variation induced by environmental stresses, such as drought or salinity (Brugnoli and Farquhar, 2000). In water deficit conditions, diffusional limitations reduce the C_i /C_a ratio leading to an enrichment of primary photosynthates with ¹³C (Farquhar et al., 1989). This is why the carbon isotope composition of the purified hexose fraction of berry sugars at maturity provides a consistent and integral measure of vine water status during berry ripening (van Leeuwen et al., 2001; Gaudillère et al., 2002; Des Gachons et al., 2005; Zufferey et al., 2018). In addition, δ¹³C of bulk berry must at harvest correlates strongly with δ¹³C of purified berry sugars (Gaudillère et al., 2002), and thus also provides an efficient measure of vine water status during the berry maturation period (Gómez-Alonso and García-Romero, 2010; Herrero-Langreo et al., 2013). Therefore, the higher δ¹³C values for the bulk must of the non-irrigated vines (Table 1) are consistent with the higher water deficit they were subjected to during the VE and MT stages. The irrigation treatment means (Table 1) fall within the range of δ¹³C values as reviewed by Santesteban et al. (2015) for berry must, indicating moderate to severe water deficit for the non-irrigated grapevines.

Although well-established knowledge exists on the effects of water deficit on the carbon isotope composition of berry must, data for any corresponding effects on vine nitrogen status are scarce and inconsistent. Gaudillère et al. (2002) did not observe any significant influence of nitrogen status, or any interaction between nitrogen and water status on the δ¹³C of berry purified sugar in a study in which variation in leaf nitrogen was indirectly induced by different soil tillage treatments; however, as the authors pointed out, the leaf nitrogen range of their data was narrower than that in our study. In agreement with these findings, and under the conditions of our study, the variation in must δ¹³C at harvest was not explained by nitrogen regime at any of the four growth stages (Figures 5C, D). However, in a one year study, Brillante et al. (2020) found a strong positive relationship between leaf blade N concentration at anthesis and berry must δ¹³C at maturity, but only when the data from two different vineyards were pooled; the authors attributed the absence of a similar relationship within each vineyard to the narrower leaf N variation in comparison to the pooled data. On the other hand, we observed a significant interaction between irrigation and N fertilisation (Figure 4), which shows that the water supply effects on bulk juice δ¹³C depended on the fertiliser rate in both vineyards: while N rate increased, bulk juice δ¹³C values of the unirrigated and the irrigated treatments tended to converge. Whatever the underlying mechanisms, we expect these effects to interfere with interpretations of berry must carbon isotope composition in terms of water availability only in cases of high nitrogen variation and availability. Nevertheless, it should be noted that by veraison most of the added N will have been allocated to or incorporated into various vine organs. Thus, in order to better study the effect of N supply on must δ¹³C, a late (e.g., at VE) N treatment would be necessary.

Relatively few data exist on cane BDM δ¹³C, and these are related to grapevine genotypic variation in cane δ¹³C as a proxy of WUE (Virgona et al., 2003) and the effect of water deficit (Glenn et al., 2010). The contrasting effects of irrigation and N fertilisation on cane δ¹³C resemble their effects on leaf δ¹³C (Table 1), suggesting that leaf and cane δ¹³C were modulated in a similar manner within both organs. However, water availability contributed more to cane δ¹³C than N fertilisation, because irrigation accounted for more than double the variance of cane δ¹³C than that accounted for by N fertilisation (Figures 3C, D). In addition, the influence of water availability, as indicated by stem water potential, was evident for the BS stage in XM and from BC in CS (Figures 5A, B) when water conditions were not limiting (Table 1). This probably reflects the high sensitivity of shoot growth to water availability (Keller, 2005). Despite its regulation by water availability, cane δ¹³C cannot be considered an indicator of vine water status since stem potential accounted for at best 35 % of its variance (Figures 5E, F); nor can it be considered as an indicator of WUE_i on a seasonal basis, since the average WUE_i of the four samplings and cane δ¹³C were only positively correlated in the second year of the experiment and in the CS vineyard (R² = 0.48, p =0.0008, n = 18, data not shown). Thus, the interpretations of cane δ¹³C in terms of single leaf WUE_i may not be correct.

Canes are lignified shoots that develop and mature during the preceding growing season. Although green shoots can photosynthesise if they contain chlorophyll, their own carbon assimilation activity contributes to their carbon pool much less than the carbon they receive from leaves and other vine parts. However, metabolic processes in various plant organs or during translocation of metabolites from one organ to another can cause carbon isotope fractionations (Cernusak et al., 2013). Therefore, the carbon isotope abundance of canes was modulated by the isotope composition of translocated carbon and any associated fractionations. Additionally, carbohydrate composition is known to vary in response to N and water supply (Holzapfel et al., 2010), thus nitrogen and water status effects may be mediated by cane compositional changes. However, based on our data we cannot make inferences regarding the relative contribution of these effects and how water and nitrogen availability impacted them. Despite this limitation, the stronger positive relationship between leaf and cane δ¹³C from the later samplings (r = 0.61, p < 0.001 in CS and r = 0.74 p < 0.001 in XM at MT) probably represents the more intense allocation of leaf-originated carbon that was fixed during the maturation period (Holzapfel et al., 2010).