How Rootstocks Impact the Scion Vigour and Vine Performance of Vitis vinifera L. cv. Tempranillo

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Introduction
Vitis vinifera scions are commonly grafted onto various rootstocks of other Vitis species (i.e., Vitis berlandieri, Vitis rupestris, and Vitis riparia) to infuence scion vigour and provide resistance to biotic and abiotic stresses (Marín et al. 2021).
As evapotranspiration and the water needs of vines are predicted to increase as a result of climate change [1], much research has focused on the identifcation of new adaptation mechanisms to save water and improve the water use efciency of crops without negatively afecting the quality of crops [2,3]. Te responses of diferent grapevine rootstocks to water stress conditions have been widely investigated to obtain a better understanding of the mechanisms involved in drought responses [4] and the diferential responses that grafted plants attain due to rootstock behaviour [5][6][7][8][9]. Tis knowledge could enable specifc rootstocks to be used to increase crop quality in areas that are susceptible to drought as an efcient strategy to avoid permanent drought damage to vineyards. However, in addition to drought stress, other factors should be also considered when choosing the best rootstock for a specifc soil. Soil characteristics and cultural practices also represent important controlling factors in the development of viticulture. Tus, depending on the wine growers' interests and the soil characteristics, a variety of requirements need to be solved in order to obtain the desired vine and grape qualities for specifc sites. It is widely known that certain rootstocks produce the best results in soils with specifc characteristics. Tus, several studies have studied the efects of various rootstocks under diferent conditions on fruit quality, nutrient uptake, plant growth, root development, cold tolerance, water stress adaptation, and resistance to diferent diseases (reviewed in [10]).
Te ideal rootstock will increase reproductive growth or yield, without leading to an excessive increase in vegetative growth. However, the efects of rootstocks on berry composition are generally considered to be an indirect result of the impact of the rootstock on vegetative and reproductive growth, for example, by altering the uptake of water or nutrients [11,12]. Indeed, water and nutrient uptake were identifed as two key processes that difered between ownrooted and grafted plants [12][13][14]. Tus, rootstocks diferently modulate water supply and nutrient uptake and have an impact on the performance of grafted vines.
On the one hand, water supply to the grapevine may be modulated by root growth dynamics. Diferences in root quantity, distribution, and/or the apparent efciency of water uptake and transport would promote better grapevinewater relations [15,16]. Indeed, compared to droughtsensitive rootstocks, drought-tolerant rootstocks formed more new roots in the soil profle during a dry and hot season which increased the uptake of water by the grapevines [13]. Root hydraulic conductance has been employed to describe changes in water uptake from the root-soil interface to the apoplast in the leaves [17]. Higher hydraulic conductance is observed in more drought-tolerant rootstocks, which exhibit improved development of xylem and lower vessel embolization; these properties can confer higher conductance [13]. According to Tramontini et al. [18], the hydraulic system is not only infuenced by genetics but can also be afected by the soil type, which can signifcantly impact the development of xylem tissue, and thereby afect hydraulic conductance in the whole plant.
On the other hand, an efcient root system is advantageous for nutrient uptake by grapevines and allows the vine to better exploit the nutrient resources available in the soil. Indeed, it is well known that the rootstock confers vigour to the scion, and higher vigour is related to a higher nutrient uptake capacity [19]. However, some rootstocks allow excessive uptake of a number of nutrients that can be damaging to grapevines, including sodium, chloride, and boron [20]. More recent reports showed that rootstocks modifed the mineral composition of the scion petioles and blades [7-9, 14, 19]. Gautier et al. [14] reported that the genetic background of a rootstock can modify the concentrations of phosphorus, magnesium, and sulfur in scion petioles. Moreover, several authors [12,19] presented updated information on how diferent rootstocks infuence the absorption of nutrients and the composition and sensory properties of the wine. Indeed, other researchers used different rootstocks to achieve lower pH and higher titratable acidity (TA) in grape juice by reducing berry potassium concentrations [21]. Overall, this strategy may serve as a tool or criterion to establish guidelines for fertilization by considering the rootstock employed, and thus, may help to increase the efciency of fertilizers use, reduce costs, and avoid environmental contamination [19]. However, given the complex interactions between the rootstock, cultivar, and environment, local long-term studies are necessary before a specifc type of rootstock can be recommended for a specifc edaphoclimatic condition (Marín et al. 2021). Tis implies that the results obtained for a particular cultivarrootstock combination in a specifc environment cannot be widely extrapolated to other situations [22,23]. In this context, these specifcities may explain the contradictory results obtained in previous studies. Moreover, although data from pot experiments and controlled conditions are highly valuable for the comparison of genotypes, such data must be considered with caution before extrapolation to the feld [24].
To address these issues, the present feld study conducted in a 30-year-old vineyard aimed to assess the efects of four well-established rootstocks, 41B Millardet et de Grasset (41B), 161-49 Couderc (161-49C), 110 Richter (110-R), and 1103 Paulsen (1103-P), on the vine performance and fruit composition of the scion cultivar Tempranillo, one of the most widely cultivated black grapes in Spain.1103-P and 110-R have previously been classifed as more vigorous rootstocks and 41B and 161-49C as low-vigour rootstocks [5,25]. Moreover, by evaluating the vines across three years, we aimed to account for the efects of seasonal climatic variability on the performance of the four scion-rootstock combinations. Finally, we demonstrate the practical application of the nitrogen balance index (NBI), which has been proven to be a useful tool for monitoring nutrient absorption capacity, to rapidly obtain, accurate, objective nondestructive information, and in almost real-time.

Site Characteristics and Plant Material.
Te trial was carried out over three growing seasons (2018-2020) in a 30year-old "Tempranillo" (Vitis vinifera L.) vineyard located in Aldeanueva de Ebro, La Rioja (Spain). Randomized soil sampling was performed in the frst year at 0-30 and 30-60 cm depth for soil characterization purposes. In each of the four experimental units, three single samples were taken using a stainless steel drill to make a pooled sample representative of each depth. In the laboratory, the soil samples were desegregated, air-dried to constant mass, sieved (2 mm), and stored until chemical analysis. Te soil was classifed as a Haplocalcids typical [26] which corresponds to loam soil with the following average characteristics: clay 23.1%, silt 45.2%, sand 31.7% (USDA classifcation), organic matter 0.94%, and pH of 8.3 (additional information on the soil profle is given in Supplementary Table 1). Te climate is between warm and temperate, with hot and dry summers and mean annual rainfall of about 500 mm·year −1 . Te drought period usually lasts from May to September, but its length is highly variable from year to year. Meteorological data were provided by an automatic meteorological station 2 Australian Journal of Grape and Wine Research belonging to the AgroClimatic Information Service of La Rioja (SIAR) located 1.4 km from the experimental site. Te grapevines (Vitis vinifera cv. Tempranillo) were grafted onto seven diferent rootstocks, and the vine spacing was 2.7 m × 1.2 m. Two vigorous (1103-P and 110-R) and two less vigorous (41B and 161-49C) rootstocks were selected for the current study. Rootstocks 1103-P and 110-R (both Vitis berlandieri x Vitis rupestris parentage) are commonly characterized as high vigour and drought-resistant rootstocks [5,25], rootstock 41B (Vitis berlandieri × Vitis vinifera parentage) is characterized as having moderate vigour and medium tolerance to drought, and rootstock 161-49C (Vitis berlandieri × Vitis riparia parentage) is characterized as having low vigour and drought intolerant [5]. Te experimental design was a randomized complete block divided into four experimental units (n � 4). Each experimental unit consisted of 48 vines per rootstock-scion combination, distributed within one row. Bufer vines with the same rootstock (R99) were distributed between rows.
Te training system was a vertical shoot positioning trellis with movable wires, and the vines were spur-pruned on a bilateral royat cordon system, leaving an average of 10 to 12 buds per plant. Shoots were trimmed twice a year, between bloom and veraison, at a height of about 1.0 m. All grapevines were rain-fed until veraison; thereafter, the irrigation dosage was adjusted using a drip system up to 30% of the reference evapotranspiration (ET 0 ) as calculated from the Penman-Monteith relationship and adjusted using a grape crop coefcient (Kc) and evaporation from a Class A pan [27]. Tus, irrigation began on diferent dates in each year depending on the weather conditions: irrigation began in July 2018, June 2019, and August 2020.

Grapevine Water Status and Gas Exchange.
One of the four experimental units was selected for water status and gas exchange measurements. In the selected experimental unit, with 48 vines per rootstock, six (n � 6) well-established plants along the row (one per post) were selected to monitor vine water status. Predawn and midday leaf water potential (Ψ PD and Ψ MD , respectively) were measured ones using a Scholander pressure chamber (Soilmoisture Equipment Corp., Santa Barbara, CA, USA) at two phenological stages: (i) fowering and (ii) veraison. On the same day, stomatal conductance (g s ), transpiration rate (E), and net photosynthesis (A N ) were measured on six mature, healthy, sun-exposed leaves from six diferent plants per rootstock (n � 6) using a portable open gas exchange system (Li-6400; Li-Cor Inc., Lincoln, NE, USA) with a CO 2 concentration of 400 mmol CO 2 mol −1 air in the cuvette. Measurements were taken at midmorning, from 10:00 am to 12:00 pm, on sunny days.
Intrinsic water use efciency (WUE), obtained from instantaneous measurements, was calculated as the ratio between A N and g s .

Whole-Plant Hydraulic Conductance Per Unit Leaf Area.
Whole-plant hydraulic conductance per unit leaf area (K plant ) was estimated on the basis of Ohm's law analogy for the soil-plant-atmosphere continuum as described by Nardini and Salleo [28]as follows: where E max , Ψ leaf , and Ψ soil are the maximum diurnal transpiration rate, leaf water potential, and soil water potential, respectively. Ψ PD was taken as a proxy for Ψ soil and Ψ MD was taken as Ψ leaf [29].

Measurements of Vine Vigour and Chlorophyll Content
Using Optical Sensors. DUALEX (Force-A, Paris, France) is a hand-held device for measuring the chlorophyll and polyphenols contents of leaves. Te chlorophyll content is estimated through the leaf transmittance ratio of two wavelengths in the red and infrared bands of the spectrum, and the favonol content is measured by a chlorophyll fuorescence screening method at 375 nm [30]. Te DUALEX calculates the nitrogen balance index (NBI) as the ratio between the content of chlorophyll and favonoids [31,32]. Tis index introduces the favonoid content as a stress factor and indicates possible nutritional defciencies in the plant. In each of the four experimental units per rootstock, 15 measurements were taken using this hand-held sensor, at two phenological stages: fowering and veraison.
Vine vigour was assessed using a Crop-Circle ACS-430 (Holland Scientifc, Inc., Lincoln, NE, USA), an active light sensor, independent of natural light conditions, that emits radiation and measures refectance in three wavelengths: 670, 730, and 780 nm (NIR). In addition to the refectance values, the device generates NDVI values as an estimate of vegetation cover [33].
Using this equipment, continuous measurements (10 measurements per second) of each replicate were carried out at both fowering and veraison.

Leaf Chemical Analysis.
Tirty complete, healthy leaves were sampled per experimental unit in each rootstock, at a rate of one leaf per plant on fruit-bearing shoots of average vigour from visually representative vines along the row. Leaves were collected opposite to the frst bunch at fowering and opposite to the second bunch at veraison [34]. Both sides of the trellis were alternatively considered.
Leaf blades and petioles were separated, washed three times with tap water, rinsed with distilled water, oven-dried (Dry-big, J.P. Selecta, Barcelona, Spain) at 70°C for 48 hours, ground in an ultracentrifugal mill (ZM1, Retsch, Haan, Germany), and passed through a 0.5 mm mesh.
To determine total N (N-organic + N-NH 4 + ) in leaf blades and petioles, 0.20 g of ground samples were subjected to dry combustion analysis (Leco CNS, St. Joseph, MI, USA) using the Dumas method [35]. For the remaining nutrients,phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), and boron (B)-0.20 g samples were subjected to wet digestion with H 2 SO 4 (95%) and H 2 O 2 (30%) [36] and analysed by inductively coupled plasma-optical emission spectrometry (Optima 3000DV, PerkinElmer, Norwalk, CT, Each year, the evolution of grape maturity was evaluated by random berry sampling. In each of the four experimental units, 500 berries per rootstock were sampled within the entire row to analyse the evolution of technological and phenolic maturity. Technological maturity was analysed by determining the total soluble solids (TSS) by refractometry, pH, total acidity, malic acid, potassium content, and colour intensity (CI) according to EEC methods [37], and tartaric acid, according to the Rebelein method [38] in the juice of berries crushed using a blender. Te evolution of phenolic maturity was assessed by extracting phenolic compounds from the grapes; briefy, 200 berries were weighed, extracted with 50 mL of 1% HCl twice in a mixer without breaking the seeds, and the paste was heated with shaking up to 40°C, another fraction of 1% HCl was added, and the paste was heated and shaken again up to 60°C. Te paste was cooled to 10°C in an ice bath, fltered through a cloth and the extract volume was measured. After dilution of the extracts, total phenolics were determined as total polyphenol index (TPI) by spectrophotometric absorbance at 280 nm. TPI was determined by the spectrometer Helios Omega (Termofsher Scientifc). Total anthocyanins were determined by decolouring using sulfur dioxide [39].

Statistical Analysis.
Pearson's correlations were calculated with Graphpad Prism software (GraphPad Software, Inc., La Jolla, CA, USA). Te diferences between means were assessed by both one and two-way analysis of variance (ANOVA) using SPSS 22.0 (IBM Corp., Armonk, NY, USA); if the diferences were signifcant, multiple comparisons were performed using Duncan's post hoc test (P < 0.05) in SPSS 22.0.
Principal component analysis (PCA) was performed using the FactoMinR package with RStudio software, version 1.1.463, to visualize the grouping of rootstocks based on macronutrients and micronutrients at fowering and veraison for the petiole and leaf blade separately.

Climate and Soil Water Status.
Te mean annual rainfall was 554, 373, and 482 mm in 2018, 2019, and 2020, respectively ( Figure 1). Spring precipitation (from April to June) varied greatly from year to year. Precipitation was below average in spring 2019 (124 mm), and above average in 2018 and 2020 (240 mm in 2018 and 188 mm in 2020). Moreover, the distribution of precipitation varied between years. While rainfall mainly occurred in early spring and was scarce in summer in 2018 and 2020, accumulated precipitation was lower during spring but continued to accumulate during summer in 2019 ( Figure 1). Indeed, the dynamics of plant water status refected here by predawn water potential (Supplementary Table 2), which is assumed to represent the mean soil water potential next to the roots, varied according to the annual rainfall conditions. Tus, the 2018 vintage showed the highest Ψ PD values at fowering and veraison.

Leaf Gas Exchange and Whole Plant Hydraulic
Conductance. Table 1 shows the gas exchange parameters. At veraison, vines grafted on 1103-P maintained a signifcantly higher leaf photosynthesis rate (An) than those grafted on 41B in every year. Rootstocks 110-R and 161-49C generally, behaved similarly and presented intermediate An values, with some variation each year. In contrast to the above, stomatal conductance (g s ) difered between the highvigour rootstocks (1103-P and 110-R) and low-to-moderate vigour rootstocks (41B and 161-49C) at both phenological stages, though no marked diferences in g s were obtained between rootstocks at fowering in 2019 and 2020. 110-Rigorous In addition, K plant was also substantially lower for the 41B and 161-49 rootstocks than 1103-P and 110-R rootstocks at both stages, however, some diferences were observed for this parameter in each year. Tus, in general, the fne stomatal regulation observed in Tempranillo plants grafted on the two least vigorous rootstocks resulted in higher WUE at the leaf level.
Te year had a signifcant efect on all parameters measured. Terefore, a signifcant interaction between the rootstock and the year was observed at both phenological stages except for WUE at veraison. In 2018, which was the wettest year in spring, gas exchange values difered between rootstocks depending on their vigour, with the high-vigour rootstocks always showing higher values. Tis efect was less pronounced in 2019 and even less so in 2020, which coincided with lower gas exchange values in these years and therefore less pronounced vegetative development.

Efects of the Rootstocks on Vine
Growth. Early grapevine vegetative growth, i.e., the ground-based normalized diference vegetation index (NDVI), was evaluated at two distinct grapevine phenological stages: fowering (May or June) and veraison (August) for the four grapevine rootstocks. Te NDVI values discriminated the vigour of the plants, with high-vigour rootstocks (1103-P and 110-R) obtaining higher NDVI values than the rootstocks that are commonly considered to have low-to-moderate vigour (41B and 161-49C) ( Figure 2 and Supplementary Table 3). Te same pattern occurred over the three consecutive years of fowering (P < 0.01). However, at veraison, diferent NDVI responses were obtained within the Tempranillo plants grafted on the four rootstocks, which may be a consequence of a noticeable overall decline in NDVI throughout the season or saturation of the NDVI values, which occur when crops show high physiological potential (Figure 2 and Supplementary Table 3).
Te vine vigour parameters at pruning were consistent with the NDVI values. Vines grafted on the rootstock 1103-P, and to a lesser extent on 110-R, had higher pruning mass (PM) than vines grafted on the 41B and 161-49C rootstocks. However, this diference was only signifcant for the 1103-P rootstock ( Table 2) as 110-R and 161-49C rootstocks exhibited intermediate values during both years. Indeed, the main shoot mass (MSM) was also higher for vines grafted on the 1103-P rootstock (Table 2), although the number of main shoots per vine (NMS) was lower for this rootstock and the number of basal buds was similar for all rootstocks (Table 2). Furthermore, the Ravaz index, i.e., the ratio of yield and pruning mass, was higher for vines on the 41B than 1103-P, 110-R, or 161-49C. Te diferent vine balances observed in this study indicate that each of the rootstocks led to diferent crop load ratios, which was lower than the optimum range for 1103-P (low yield and larger vine), and higher than the expected range for 41B (more fruit and smaller vine).

Berry Yield and Fruit Composition.
At harvest, the grape yield was higher on the rootstocks 1103-P and 110-R than 161-49C and 41B (Table 3). For vines on the 1103-P rootstock, this diference was mainly due to the higher cluster mass, and not the number of clusters per vine (Table 3). However, the high yield of vines on 110-R was due to a higher number of clusters compared to vines on lessvigorous rootstocks. However, the grape yield was higher in 2018 and 2020 than in 2019. Te grape yield, number of clusters per vine, mean cluster mass, and 100-berry mass were higher in 2018 and 2020 than in 2019, which may be directly related to the lower precipitation recorded during t late spring of 2019 ( Figure 1). Even when taking into account the higher irrigation dosage applied at the beginning of June (before fowering) in 2019 in an attempt to compensate for increased water demand, higher predawn leaf water potential values were obtained at fowering in 2019 than in 2020 (Supplementary Table 2) Te marked diferences in grape yield and in pruning mass (Tables 2 and 3), which was up to two-fold higher for the 1103-P rootstock than 41B in 2019 and 2020, with intermediate values for 110-R and 161-49C rootstocks-led to high and low Ravaz indexes for the 41B and 1103-P rootstocks, respectively, although the Ravaz index for 1103-P was similar to 110-R and 161-49C (Table 2).
Moreover, must composition at harvest was also infuenced by the rootstock, as can be seen in Table 4. Vines grafted on the 41B rootstock produced the lowest TSS content, total acidity, malic acid, and yeast-assimilable nitrogen (YAN). Contrary, must from vines grafted on 1103-P showed the highest pH, total acidity, tartaric acid, and malic acid contents. Vines grafted on 161-49C produced the berries with the highest total polyphenol index (TPI). Indeed, higher TPI values were observed for the vines on this rootstock in all three years of the study. In general, higher potassium concentrations and consequently higher pH values, which are related parameters [40][41][42], diferentiated the more vigorous rootstocks from the less vigorous rootstocks. Berry composition also varied according to the year of the study, with the year having a higher seasonal efect on fruit composition that was independent of the rootstock (Table 4). TSS and the anthocyanin content were higher and total acidity, tartaric acid, malic acid, and YAN were lower in 2019 compared to 2018 and 2020, can be directly related to the lower precipitation recorded at fowering and late summer of 2019, which resulted in lower yields. At fowering, the frst principal component (PC1), which accounted for 51.7% and 47.2% of the variability in the petiole and the leaf blade, respectively, was positive for all nutrients (Figures 3(a) and 3(b)). Te rootstocks were grouped according to their vigour. 1103-P and 110-R were higher in the analysed mineral compounds (Figure 3), whereas 161-49C and 41B had the lowest concentrations of those nutrients.

Leaf and
At fowering, the second principal component (PC2) explained 16.8% and 21.8% of the variability in the petiole and leaf blade, respectively. Within the petiole, PC2 segregated the 110-R and 161-49C rootstocks from the two other rootstocks. PC2 was mainly explained by Mg and Na which are opposite to each other, indicating lower concentrations of these elements in the vines on the 110-R and 161-49C rootstocks. However, in the leaf blade, 110-R appeared to be displaced independently from the other three rootstocks. PC2 in the leaf blade is explained by K and Mg.
Some diferences in the PCA of the petiole and leaf blade were also obtained at veraison (Figure 4). In the leaf blade, PC1 clearly segregated 1103-P and 161-49C by a gap of around fve units of distance, whereas 110-R and 41B grouped together between 1103-P and 161-49C. Tereby, the PCA showed that 1103-P, on the positive side of PC1, led to higher Zn, P, Na, and Cu concentrations at veraison, whereas 161-49C, led to a lower concentration of these nutrients. Overall, these results suggest that Tempranillo vines grafted onto the 1103-P rootstock have higher nutrient concentrations at veraison while vines grafted on 161-49C have lower nutrient concentrations.
Within the leaf petiole, there was a clear segregation between 161-49C and the other three rootstocks, which was explained by PC2. 1103-P and 41B were positively correlated with the Mg, Cu, Na, and Zn concentrations, while 110-R was positively correlated with K and negatively correlated with Mg.

Dependence of Optical Indices on Individual Leaf Macro and Micronutrient
Compositions. Te dependence of the optical indices on leaf blade macro-and microelement concentrations was analysed for all of the Tempranillo plants grafted on the four rootstocks over three years period (2018-2020) ( Table 5). Pearson's correlation tests confrmed a strong negative correlation between the abundance of chlorophyll and the leaf N concentration, with chlorophyll more strongly correlated to N than the favonol content or NBI. Moreover, signifcant correlations were also observed between chlorophyll and other nutrients such as P, K, Ca, Fe, and Mn (P < 0.01).
Signifcant correlations were also observed between favonols and the N, P, and K concentrations (Table 5). Te NBI is the ratio of chlorophyll to favonols, thus signifcant negative correlations (P < 0.01) were also observed between this index and the N and K concentrations (Table 5).

Infuence of Rootstock and Vintage on Scion Leaf Composition Determined by a Noninvasive Chlorophyll Fluorescence Sensor and Nitrogen Status.
As signifcant correlations were observed between the leaf composition determined by using either the noninvasive chlorophyll fuorescence sensor or chemical mineral determination (Table 5), we assessed the efects of the rootstock and season on optical indices and leaf N ( Table 6) to determine if these optical indices could be used to discriminate the capacity of the four rootstocks to concentrate nutrients within the leaf blade. In general, both .40 a * , * * , and * * * indicate signifcant diferences at the 0.05, 0.01, and 0.001 levels of probability, respectively. In each column and for each factor or interaction, diferent letters indicate signifcant diferences according to Duncan's multiple range test at the 95% confdence level. Regarding the year efect, for each factor, diferent letters denote statistically signifcant differences between years based on the Student's t-test(P < 0.05). NBB: number of shoots arising from basal buds/vine, NMS: number of main shoots/vine, MSM: main shoot mass (g), PM: pruning mass (kg/vine), and Ravaz Index: yield/pruning mass.
rootstock and year signifcantly infuenced the leaf parameters determined using both the noninvasive sensor and by directly measuring N at the leaf level.
Te 2018 vintage had the highest spring precipitation and 2019 had the highest summer precipitation (Figure 1). Consequently, a higher chlorophyll index and lower favonol index were obtained fowering in 2018, resulting in a higher NBI, whereas a higher NBI value was obtained at veraison in 2019. In accordance with the negative correlation observed between NBI and leaf N (Table 5), leaf N at veraison was lower in 2019 than in 2018 and 2020.
As described above, NDVI values ( Figure 2) distinguished the rootstocks into two groups based on vigour, with 1103-P and 110-R being the most vigorous (with higher NDVI) and 161-49C and 41B the least vigorous (with lower NDVI). At veraison, the chlorophyll index and NBI were higher for Tempranillo leaves on 1103-P and 110-R than 161-49C and 41B. However, this pattern was not observed at fowering, as the more vigorous rootstocks led to higher leaf N than the less vigorous rootstocks, while later the opposite trend was observed at veraison. Tus, at veraison, Tempranillo leaves on 1103-P and 110-R had lower N values, suggesting a dilution efect on scion leaf composition.
Looking at the NBI values over diferent years, it was only possible to distinguish between the rootstocks with the highest or the lowest vigour in 2019, which was the year with the highest NBI, suggesting that NBI is not strictly related to the vigour conferred by the rootstocks to the scion.

Discussion
Te role of root systems in scion performance is a subject of intense interest to vine-growers. Variations in genetic pedigree, are assumed to alter the ability of grapevine roots to explore deeper and more humid soil layers [15] and tolerate several biotic and abiotic stresses [43].

How Rootstocks Diferently Infuence Scion Vigour.
We studied 30-year-old Tempranillo scions grafted onto four feld-grown rootstocks over three consecutive years. Previous reports indicated that diferent rootstocks may confer low, moderate, or high vigour to the scion Galet 1988, [44,45]. Te 1103-P and 110-R rootstocks conferred higher vigour overall than the 141-49C and 41B rootstocks (Table 2). Te low vigour imparted by 141-49C and 41B has previously been reported by Romero et al. [7]. Moreover, the two more vigorous rootstocks (1103P and 110-R) led to higher NDVI values than the two lower vigour rootstocks (Figure 2 and Supplementary Table 3). NDVI is frequently used in agricultural applications to estimate various croprelated parameters such as biomass [46]and leaf area index (LAI) [47], and for crop management [48,49] and mapping vigour zones [50,51]. Tis study confrms the potential of NDVI to evaluate vine vegetative development, as previously reported by Acevedo-Opazo et al. [49]. Tus, NDVI has the potential as a reliable index to estimate vigour and also to estimate pruning wood mass (PM). It is worth noting that two distinct grapevine phenological stages were selected for   data acquisition in this study: fowering (May or June) and veraison (August). PW correlated better with NDVI values collected at fowering than with NDVI values collected at veraison, in agreement with a previous study [52]. Te higher correlation between PW and NDVI at fowering in our trial (Supplementary Table 4) was probably related to the more even distribution of the vine canopy by midseason, which leads to saturation of the NDVI by the end of the season [53]. Viña et al. [54] also reported that NDVI measurements become less sensitive for estimating biomass as vegetative growth increases. Consequently, the relationship between early-season NDVI and PW may provide grape growers with a useful tool for yield estimation, as the higher the NDVI, the greater the PW, and therefore the greater the vigour. Higher vigour correlates with increases in other agronomic parameters, including grape yield (Supplementary Table 4), although this fnding needs to be confrmed in future studies.

Rootstocks Infuence Water Uptake and Leaf Gas
Exchange. Water stress induces complex physiological regulation in grapevines at both the root and shoot levels (especially leaves). Terefore, the interrelationship between scions and rootstocks is difcult to predict. Indeed, the signifcant scion X rootstock interactions indicate that the scion cultivar must be taken into account during the selection and classifcation of rootstocks Ferlito et al. 2020, [14]. Tis study demonstrates that diferent rootstocks confer diferent levels of vigour to Tempranillo cv. Compared to 1103-P and 110-R, the 161-49C and 41B rootstocks conferred lower vigour and led to smaller vines, potentially to reduce transpiration and, hence, decreased water requirements due to the development of smaller canopies [55]. In this study, the scions grafted onto the two more vigorous rootstocks (1103-P and 110-R) exhibited higher photosynthesis and stomatal conductance rates, compared to the same scion grafted onto the two least-vigorous rootstocks (161-49C and 41B) ( Table 1). Similar results were reported by Alsina et al. [15], Romero et al. [7], and Lovisolo et al. [5], who attributed the improvements in root water uptake and transport capacity to the presence of Vitis rupestris in the genotypic background of 1103-P and 110-R compared to less vigorous rootstocks, such as 41B or 161-49C produced by crossing with Vitis riparia. Te efects of the rootstocks on leaf photosynthesis and stomatal conductance were associated with the K plant values, which were higher for 1103-P and 110-R than 161-49C and 41B, at both fowering and veraison. Similarly, Gambetta et al. [56] described higher fne root hydraulic conductance, even under well-watered conditions, in 1103-P and 110-R rootstocks, compared to other less vigorous rootstocks, such as 420A and 101-14. For the more vigorous rootstocks, increased hydraulic conductance of the fne roots correlated with a higher leaf area and higher transpiration rates in the scion. Collectively, this data suggests that diferent   rootstock-scion combinations may explain the varied neariso/anisohydric behaviours of certain cultivars reported in diferent studies [57,58]. Indeed, several factors contribute to the drought response of the rootstock, including root anatomy, growth patterns, and chemical and physical signals related to stomatal regulation. Smart et al. [22] found that diferences in the proportion of roots in the diferent soil layers, rather than diferences in the ability of rootstocks to develop roots at depth, conferred high-vigour rootstocks with improved access to water and minerals from the deeper soil profle. Tus, it is likely that a combination of all of these factors contribute to the drought response when a rootstock is subjected to drought conditions. Water use efciency (WUE) based on instantaneous gasexchange data indicated the rootstocks led to diferent water performances ( Table 1). Evaluation of the scion WUE identifed 161-49C and 41B as the most water-efcient rootstocks and 1103-P and 110-R as less water-efcient rootstocks.
Teoretically, a larger size of the root system maintains favourable plant water status, while a smaller size of the root system leads to a lower water transport capacity [15]. Terefore, chemical signals, transported to the leaves in the transpiration stream, may reduce stomatal conductance and/or growth, and thus increase water-use efciency (WUE).

Rootstocks Infuence Mineral Nutrition.
Vines grafted on more vigorous rootstocks, such as 1103-P or 110-R, which are both classifed as rootstocks with high drought tolerance [5,15], maintained higher root water uptake during the growing season, probably due to diferences in root quantity, distribution, and/or apparent efciency of water uptake and transport (Marin et al. 2021). Consequently, these rootstocks were able to exploit soil water resources more efciently, and this increased transport capacity was refected by higher leaf blade and petiole nutrient contents. Indeed, leaf and petiole mineral compositions were signifcantly afected by both the rootstock and the vine phenological stage (Figures 3 and 4). Te concentrations of each element follow diferent trends throughout the season and, despite the fact that the evolution of the nutrient concentrations was similar in the blade and petiole, the concentrations of the elements are generally signifcantly diferent between the leaf blade and petiole at each phenological stage [59,60]. Indeed, the nutrient concentrations at each phenological stage, specifcally fowering and veraison, difer so signifcantly that specifc references for nutritional diagnosis have been proposed for each element at each phenological stage [61].
In general, rootstocks exhibit diferent root architectures, root cation exchange capacities, and root exudates and, in turn, these factors infuence the leaf nutrient concentrations [62]. Tereby, it may be possible to select the rootstocks that most efciently capture and translocate mineral elements in the soil, which would allow the use of fertilizers to be reduced [63]. Tus, we performed principal component analysis (PCA) to classify the infuence of the rootstocks on mineral composition. In general, the PCA diferentiated the most invigorous rootstocks (1103-P and 110-R) from the least vigorous rootstocks (41B and 161-49C), which suggests that diferent rootstocks, essentially their root water absorption capacity, signifcantly infuence mineral nutrition in the scion. Indeed, recent studies by Gautier et al. [64] and Gautier et al. [14] confrmed the existence of a signifcant relationship between the genetic background of a rootstock and its ability to modify concentrations of phosphorus, magnesium, and sulfur in the petioles of the scion. In these studies, rootstocks with a Vitis riparia genetic background (i.e., 161-49C) conferred lower petiole P concentration compared with other rootstocks with V. rupestris or V. berlandieri genetic backgrounds (i.e., 1103-P and 110-R), which increased petiole P concentration. Accordingly, in the present study, 1103-P, which centered on all axes in the PCA, led to the best position with respect to macro and micronutrients, i.e., led to higher N, P, K, Ca, Mg, Na, Cu, Fe, B, and Mn concentrations in the petioles and leaf blades compared to the other rootstocks. 161-49C and 41B, with the lowest mineral concentrations, were in the most opposite position to the positive axes, while 110-R was more closely related to 1103-P (Figures 3 and 4). Moreover, the petiole, 1103-P was dominated by N, Cu, and Zn while 110-R was dominated by B. Within the leaf blade, the concentrations of N, P, Ca, Mn, and Cu were explained by PC,1 and the concentrations of K and Mg were explained by PC2. In this case, 1103-P was dominated by the concentrations of Ca and Mn, N, Cu, P, K, and Zn while 110-R was dominated by the concentration of B.
Te rootstock 1103-P stands out due to its high capacity to absorb Mg [65]. High levels of potassium in the soil solution can limit the absorption of magnesium, and thus reduce the availability of magnesium to the plant [66]. Tus, in highpotassium soils, selecting a "magnesium-absorbing" rootstock, such as 1103-P, may represent a simple strategy to avoid a defciency of this nutrient. In this regard, the genetic diversity within Vitis ssp. Can provide new functional abilities to match specifc scion/rootstock/site combinations. Furthermore, we also highlight the inefciency, or maybe inability, of 161-49C to absorb and translocate Na. Tus, this rootstock may be a good candidate for high-salinity soils, although we cannot ignore the fact that salt tolerance can also be conferred by Cl uptake, which we did not analyse in this study. Rootstocks are considered as one important way to improve the salt tolerance of grapevines [67,68], which represents another example of how specifc scion/rootstock/site combinations may contribute to better vineyard management.

Nondestructive Diagnostic Testing of Mineral Nutrition of
Grapevine Based on DUALEX ® Measurements and its Applicability in Detecting Leaf N Content in Diferent Grapevine Rootstocks. We also assessed the ability of optical sensors to characterize the infuence of the rootstock on scion leaf composition and plant vegetation indices over three years (Table 5). Our results indicate the universality of these indices, at least for the Tempranillo cultivar.
Overall, Pearson's correlation tests confrmed a strong association between the chlorophyll abundance and N content within the leaf, with the chlorophyll (Chl) index better related to N than favonols (Flav) or the nitrogen balance index (NBI).
In general, chlorophyll meter leaf Clips are the most precise optical technique for assessing N levels [69]. In that study, signifcant correlations were also observed between Chl and the N content (P < 0.01) in wheat [69]. Cartelat et al. [31] proposed the NBI, which is the ratio of Chl to epidermal Flav leaf content, for the evaluation of nitrogen nutrition in wheat in the context of precision agriculture. Te NBI index is proposed to be more sensitive to phenology and therefore refects N availability better than either of the other two indicators (Chl and Flav) individually [69][70][71] because leaf Chl and Flav contents on a surface area basis are both dependent on the age of the leaf and light exposure during growth, especially during the frst part of the season [72,73]. During the second part of the season, Chl tends to decrease while Flav remains constantly high [72]. However, in our study, the Chl index was the most robust optical index for N estimation in the leaf scion as a diagnostic method. Tis divergence from previous results might be either due to the study of varied crops with diferent performances or because the turnover between Chl and Flav had not yet been established at the time the measurements were taken.
Less signifcant correlations were also observed between the optical indices and nutrients other than N, such as P, K, Ca, Mg, Fe, and Mn (Table 5). Similarly to N, the concentrations of these nutrients tended to decrease throughout the season and showed signifcant negative correlations with the determined optical indices (Table 5). On the other hand, nutrients that increase in concentration [59] positively correlated with the three optical indices (NBI, Chl, and Flav) ( Table 5). Tis is because many essential elements are involved in photosynthetic processes and are therefore related to the chlorophyll and favonoid contents of leaves, including elements from leaf structures such as Mg or N, those involved in chlorophyll synthesis or elements that play a role in maintaining the structure of chloroplasts, such as Mg, Mn, and Fe.
Tus, this study showed the potential of optical indices to predict leaf N content independently of the phenological stage in four rootstocks. However, the potential of DUALEX to discriminate between rootstocks was not clear (Table 6). Although a clear positive correlation between leaf Chl and vine vigour had already been reported (Sampaio 2007 and Blank et al. 2018), in this study, the more vigorous rootstocks did not always result in a higher N status in the scion. Indeed, positive correlations between vine vigour and the N concentration were only found at fowering in 2019 and 2020, but not at fowering in 2018 or veraison in any year (Table 6).
Cartelat et al. [31] reported that Chl values to increase with N concentration in wheat, irrespective of the growth stage, cultivar, or year. However, in grapevines, the relationship between the optical indices and leaf N content varied depending on the year and phonological stage, thus these indices did not adequately discriminate between the rootstocks.

Rootstock Performance and Grape Quality Parameters.
Compared to rootstocks commonly considered to have low-to-moderate vigour (161-49C and 41B), the higher soil water uptake by the high-vigour rootstocks (1103-P and 110-R), probably explained by its deeper root proliferation during the hottest and driest part of the season [15], was refected in a higher pruning mass (Table 2) and higher yield response (Table 3). Tus, the signifcant efects of the rootstock on the scion yield provide evidence that selection and classifcation of rootstocks based on conferred vigour may help to control the productivity of Tempranillo cv. Rives [74] found that both, the inherent vigour of the scion (own vigour) and that conferred by the rootstock were contributing factors to yield performance. Furthermore, detailed studies of crop development, including the assessment of shoot fruitfulness, fower number, and fruit set, are required to further elucidate the diferent scion responses.
In this study, the bunch mass, but not the number of bunches, mainly contributed to the yield variability between the rootstocks, with 1103-P and 110-R being the most productive and 41B and 161-49C being the least productive. Traditionally, high vine vigour and yields are associated with grapes and wines of low-quality [7,[75][76][77]. Herein, the grape yield was higher on the rootstock 1103-P than on the low-moderate vigour rootstocks. Te higher vine vigour and grape yield of the 1103-P rootstocks usually corresponds with low-quality grape parameters such as a higher pH, higher malic acid, higher potassium, and lower polyphenol content. Te higher potassium uptake and malate levels observed for this rootstock may require the addition of higher amounts of tartaric acid during winemaking to adjust the pH [78]. For all rootstocks, YAN was present at acceptable concentrations for successful fermentation (i.e., >150 mg/L) [79]. Interestingly, of the low vigour rootstocks (161-49C and 41B), rootstock 41B strongly reduced vegetative development of the scion in comparison with 161-49C (Table 2), while the yield was similarly afected by both of this rootstock (Table 3). Terefore, we observed higher Ravaz index values for Tempranillo vines grafted onto the 41B rootstock than for 161-49C rootstock ( Table 2). Te Ravaz index (yield/pruning mass), often referred to as the vine balance, ranges from fve to ten for the balanced vine in warm climates, whereas from 3 to 6 may be more appropriate for cool climates [80]. Tus, we assume that the 41B rootstock contributes to excessive crop yield (more fruit and smaller vines), and, therefore contributes to the unbalanced development of the vine and thus negatively afects the grape quality. In contrast, compared to 41B, Tempranillo vines grafted onto 161-49C had optimal Ravaz index ratios and may be considered well balanced, with higher fruit quality, indicated by higher TSS content, total acidity, and IPT.
Overall, these results indicate the potential for the selection of an appropriate rootstock to modulate fruit composition, with the rootstock signifcantly afecting TSS (Brix), pH, TA, malate, potassium, YAN, anthocyanin, and TPI.

Conclusions
Te current study aimed to investigate the infuence of four diferent rootstocks on the performance of 30-year-old Tempranillo cv. vines. Te varied efects of these well-established30-year rootstocks in the feld were consistent throughout the three years of the study, although interactive efects between year and rootstock existed for most parameters. Overall, the less vigorous rootstocks (41B and 161-49C), as determined by NDVI, conferred drought adaptability traits and infuenced the capacity for water-saving, thereby increasing WUE. In contrast, the more vigorous rootstocks (1103-P and 110-R) increased water transport capacity, which was related to higher nutrient uptake efciency.
Moreover, yield increases were generally associated with increased cluster mass, likely due to increased water uptake in vines grafted to a particular rootstock. Tus, this study provides evidence that appropriate selection and classifcation of rootstocks based on their conferred vigour may help to improve productivity. Indeed, Tempranillo vines grafted onto the rootstock 161-49C may be considered well balanced, as they had an optimal Ravaz index ratio, which favours better fruit quality such as a higher TSS content, total acidity, and TPI.
Finally, correlations were observed between the leaf optical indices (Chl index, Flav index, and NBI) and the concentrations of nutrients such as N, P, K, Ca, Mg, Fe, and Mn, independently of the phenological stage, in all four rootstocks. However, these indices did not clearly discriminate between the rootstocks.110-R.
Overall, this study demonstrates that the appropriate selection of rootstocks is crucial for grape growers seeking to improve vine performance and wine quality.

Data Availability
Te data analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.

Supplementary Materials
Supplementary Table 1: properties of the soil of the vineyard used in the experiment, Supplementary Table 2: mean values of predawn and midday leaf water potential (leaf, MPa) measured at fowering and veraison onto each rootstock and year, and their interactions, Supplementary Table 3: mean values of NDVI at fowering and veraison onto each rootstock and year, and their interactions, and Supplementary Table 4: correlation between NDVI values at fowering and veraison and several vegetative developments and productive parameters (NMS: number of main shoots·vine-1, PW: pruning weight Kg·vine-1, yield: Kg·vine-1, clusters: number·vine-1, cluster weight: g·clusters-1, and 100-berry weight, g) in Tempranillo scions grafted onto four selected rootstocks at three seasons for NDVI values (n = 12) and two seasons for vegetative growth parameters (n = 8). (Supplementary Materials)