Phytohormones and Mineral Nutrient Changes in Young Plants of Grapevine Genotypes at Different Growth Stages

Abstract

Climate change is considered a threat for viticulture by altering phenology, yield, and key physiological processes. The plant responses depend on the genotype characteristics and the microclimate of crop area. In this research, “Castellana Negra”, “Negramoll”, and “Tintilla” were cultivated for 102 days, and physiological variables were assessed under natural conditions. Results indicated similar trends in growth between “Negramoll” and “Tintilla”, while ”Castellana Negra” grew slowly and possessed fewer leaves compared to the other genotypes. Stomatal conductance was constant among the genotypes, excepting “Negramoll”, which demonstrated lower values at d 76 compared to “Castellana Negra” and “Tintilla”, coinciding with the elevated leaf temperature. Regarding the hormonal changes, “Castellana Negra” accumulated the highest concentration of salicylic acid (SA) compared to “Negramoll” and “Tintilla”, which showed similar content. Furthermore, an antagonistic change between SA and jasmonic acid (JA) was observed in all genotypes, as well as between abscisic acid (ABA) and JA at the beginning and end of the trial. The variations in micronutrients did not show a clear tendency between cultivars. Therefore, to thoroughly elucidate the role of phytohormones and other physiological factors in the growth and development of these genotypes under varying environmental conditions, long-term experiments could be conducted.

1. Introduction

Climate change and global warming are currently considered a significant concern in numerous grapevine-growing regions worldwide, impacting phenology, growth, yield, and physiological responses, including leaf gas exchange and photosynthetic pigments in grapevine [1,2]. For instance, temperatures above 35 °C can disturb the photosynthetic processes in plants. In several regions, midday air temperatures during summer often exceed 40 °C, which can have detrimental effects on grapevines, especially during berry ripening. This, in turn, leads to reduced yield and economic profitability in vineyards [3,4]. In this context, the accumulation of chlorophyll in grapevines fluctuates according to their phenological stage and generally experiences a rapid increase during leaf expansion until flowering, then stabilizes and may gradually decline as ripening approaches [5]. Moreover, in the assessment of two potted grapevines “Touriga Nacional” (TN) and “Trincadeira” (TR) to different stress treatments (heat, drought, and light), each genotype exhibits a specific adaptive response to such environmental conditions. In TN, photosynthesis remains unaltered under light stress, and this cultivar (cv.) maintains the stomata open. This feature is advantageous not only for photosynthesis but also for heat dissipation through evaporative cooling (indicated by high stomatal conductance), demonstrating TN’s adaptation to warmer conditions [6]. Earlier research indicates an optimum temperature range for photosynthesis in potted “Thompson seedless” grapevine plants under a glasshouse between 25 °C and 30 °C [7]. The physiological activities of grapevines differ among cultivars. For example, “Sugraone” exhibits 12% more chlorophyll and a 30% higher photosynthetic rate compared to “Kings Ruby”. Likewise, higher photosynthetic and transpiration rates have been determined at the berry set stage, while water-use efficiency (WUE) peaks around blooming [1].

Regarding the patterns of change in plant hormones, it is well known that ABA plays a crucial role in regulating diverse plant development and physiological processes, including vegetative growth, leaf senescence and abscission, stomatal opening, photosynthetic activity, and as a signaling mediator for plants’ adaptive responses to environmental stress [8,9,10]. In different grapevine genotypes, throughout the progressive drought conditions, negative correlations between stomatal conductance and foliar ABA concentrations have been reported [11,12,13]. In addition, the role of ABA is emphasized as a connection between the berry ripening process and the grapevine’s response to stress. Since the stress response is predominantly mediated by ABA, applying exogenous ABA to grapevine organs is reviewed as a strategy to enhance grape quality and manage abiotic stress [14].

Salicylic acid (SA) is a fundamental endogenous hormone and signaling molecule identified in most plant species. It influences various physiological processes, including seed germination, photosynthesis, ion uptake, and transport, thereby regulating plant growth and development [15,16]. SA can promote the accumulation of phenylalanine (PAL) mRNA, the synthesis of new PAL proteins, and enhance PAL activity under high-temperature stress. Additionally, SA treatment increases the levels of phenolic compounds in grape berries [17].

Jasmonic acid (JA) plays a central role in regulating various plant defense mechanisms, including the detoxification of reactive oxygen species and the rise of osmoprotectants against abiotic stress. Recent evidence suggests that plant tolerance is linked to the modulation of JA responses to stress. This modulation promptly influences JA metabolism and responses by upregulating the expression of genes involved in JA biosynthesis and signaling [18,19,20,21]. The interaction between JA and ABA to modulate the response to abiotic stress has been suggested [22,23]. Specifically, the concentrations and gene expression of ABA increase in response to JA, and in contrast, ABA induces the expression of JA biosynthetic genes and contents [24,25].

Regarding the involvement of auxin in grapevine growth and physiological processes, it has been shown that exogenous naphthalene acetic acid (NAA) increases growth, proline production, total soluble sugars contents, maintains photosynthetic activity, and enhances water-use efficiency, as well as IAA and ABA levels. In addition, its application induces a high antioxidant enzyme activity, and it decreases hydrogen peroxide and malondialdehyde content [10]. Exogenous application of IAA at preharvest induces changes in sugar content, acidity, and color in grape berries [26]. Other treatments, such as ethephon, promote IAA biosynthesis [27], and kaolin increases IAA and ABA concentrations in grapevines [28,29,30].

On the other hand, the uptake of mineral nutrients can differ between the genotypes of Vitis vinifera. Mainly, the accumulation of N, P, and Zn²⁺ displays only slight variations. Nevertheless, the absorption of K⁺, Ca²⁺, Mg²⁺, and Cl⁻ shows major changes. Particularly, by using several cvs. (“Airén”, “Cencibel”, “Garnacha”, “Cabernet Sauvignon”, and “Chardonnay”), the results indicate that the uptake of Ca²⁺ is high in “Cencibel”, P is low in “Airén”, and K⁺ significantly differs in almost all of the cvs. [31]. Under a hydroponic culture experiment, it has been indicated that the uptake of K⁺ by “Dattier de Beiruth” grafted on SO4 rootstock is high, and the accumulation of Mg²⁺ is inhibited by the rise of the K⁺:Mg²⁺ ratio due to the antagonism between both nutrients [32]). Potassium within the plant is mostly found in a free form that can quickly be redistributed to growing organs like berries, or it can be stored in branches and roots [33]. Potassium is crucial for plants, activating enzymes in photosynthesis and respiration and maintaining cellular osmotic potential [34]. The variations in temperature and evaporation affect soil water availability for grapevines and impact internal water movement, influencing nutrient absorption and mobilization [35].

This research aimed to investigate the physiological responses of three grapevine genotypes, focusing on changes in phytohormones, mineral nutrients, growth, leaf temperature, and stomatal conductance. By revealing these traits under natural conditions, the study may identify the common and specific attributes of these genotypes, as well as their potential for cultivation in determined crop regions.

2. Materials and Methods

2.1. Plant Material and Experimental Conditions

V. vinifera “Castellana Negra” (“Cas”), “Negramoll” (“Neg”), and “Tintilla” (“Tin”) are traditionally cultivated in several viticulture areas, including subtropical Canary Island conditions with great agronomical potential and productivity. The plants were propagated from cuttings taken from shoots pruned in field-grown vineyards. Each cutting, with four buds, was placed in 1 L plastic bags filled with substrate, leaving two buds exposed above the surface, while the other two were buried to promote rooting. After 6 months of growth, the plants were transferred to 5 L plastic pots. Once the plants reached 1 year of age, they were pruned, maintaining two-to-three buds on each of the two shoots per plant. Pruning was performed on February 15, and at this stage, the length of the stems containing the buds was around 15 cm. Plants were grown in 5 L plastic pots filled with a peat substrate (Leader potting soil, Düren, Germany) under a greenhouse. Such substrate contained the following amounts of N (200 mg L⁻¹), P₂O₅ (200 mg L⁻¹), and K₂O (300 mg L⁻¹), and 2 weeks before pruning was added to each pot 60 g of granular fertilizer (NPK fertilizer containing Mg with trace elements: 18-9-10 + 2MgO + Te (Osmocote Pro 5-6M, ICL Specialty Fertilizers, Murcia, Spain)). The experimental system was conducted for 102 days, and the climatic variables recorded, such as temperature that ranged an average value between 16 and 33 °C, relative humidity varied in an interval of 50–90%, and maximum photosynthetically active radiation (PAR) registered around 1200 µmol m⁻² s⁻¹.

2.2. Plant Growth and Sampling

Plant growth expressed as functional leaf number per plant was determined along three dates (22, 76, and 102 days) after the beginning of bud sprouting. At each date, the total of functional leaves per plant and three plants per treatment were harvested, frozen in liquid nitrogen, freeze-dried, and used for the analyses of mineral nutrients and phytohormones.

2.3. Stomatal Conductance

Stomatal conductance was determined by means of a steady-state porometer (SC-1 Leaf Porometer, Meter Group Inc., Pullman, WA, USA) on fully expanded leaves, typically between the third and fifth leaves from the plant apex. Measurements were usually taken between 08:30 and 11:00 a.m., with leaf chamber temperatures averaging around 25.7 ± 0.8 °C and humidity at approximately 59.4 ± 4.0%.

2.4. Analyses of Phytohormones

The levels of ABA, JA, SA, and IAA were analyzed in leaf tissues following the procedure described previously [36]. Thus, 50 mg of dry and powdered leaves were homogenized in 2 mL of ultrapure water with a mill ball equipment (MillMix 20, Domel Železniki, Slovenija) together with the addition as internal standards of 25 ng of [²H₆]-ABA, [¹³C₆]-SA, dehydrojasmonic acid (DHJA), and 2.5 ng of [²H₅]-IAA. After centrifuging at 4000× g and 4 °C for 10 min, the supernatants were collected, and the pH was adjusted to 2.8–3.2 using 80% acetic acid. The extract was partitioned twice against 2 mL of diethyl ether (Fisher Scientific, Hampton, NH, USA), and the organic layer was recovered and dried under vacuum using a centrifuge concentrator (Speed Vac, Jouan, Saint Herblain Cedex, France). When dried, the pellet was resuspended in 0.5 mL of a 10:90 methanol:H₂O solution by sonication. The mixture was filtered through 0.22 µm of polytetrafluoroethylene membrane syringe filters (Albert S.A., Barcelona, Spain) and injected into an ultraperformance LC system (Acquity SDS; Waters Corp., Milford, MA, USA). Chromatographic separations were conducted on a reversed-phase C18 column (Gravity, 50 × 2.1 mm, 1.8 µm particle size; Macherey–Nagel GmbH, Düren, Germany) by means of methanol:H₂O gradient (both supplemented with 0.1% acetic acid) at a flow rate of 300 µL min−1. The studied phytohormones were quantified with a TQS triple quadrupole mass spectrometer (Xevo TQ-S, Waters Corp., Milford, MA, USA) through an orthogonal Z-spray electrospray ion source. Standard curves were performed by injecting commercial standards into the UPLC–MS system and used to quantify hormone concentrations in the samples. Results were processed with MassLynx v4.1 software. Data represent the average of three biological replicates for each experimental group.

2.5. Determination of Foliar Mineral Elements

Ground leaf tissues (0.2 g) collected at d 22, 76, and 102 were used to determine the ion contents. Thus, the powder was cremated in a muffle oven at 450 °C and the ashes were dissolved in an aqueous HCl solution for analyzing the mineral elements. Nitrogen was determined using Kjeldahl digestion in sulphuric medium with copper and selenium as catalysts, gathering in boric acid and titration with a 0.05 N aqueous sulphuric acid solution (Sigma-Aldrich, Madrid, Spain) [37,38]. Phosphorus was analyzed by using the vanadium phosphomolybdate method [39]. The cations, such as K⁺, Ca²⁺, Mg²⁺, Na⁺, Fe²⁺, Mn²⁺, Cu²⁺, and Zn²⁺) by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Lastly, boron was measured via the azomethine H colorimetric [40].

2.6. Statistical Analyses and Experimental Design

A total of 81 plants (27 per cultivar) were distributed in three blocks with 9 plants per cultivar in each one. Three plants per cultivar and block were used for measuring leaf number and stomatal conductance, as well as three plants per treatment and block were sampled to analyze their concentrations in minerals and phytohormones in leaf tissues at each stage of growth. Data were statistically analyzed by using the software IBM^® SSPSS^® Statistics version 25 for Windows (IMB Corporation, Armonk, NY, USA). All the parameters were examined through one-way ANOVA, and post hoc Duncan test was used for mean separation. The significance level was indicated at p < 0.05.

3. Results and Discussion

3.1. Plant Growth

To assess the growth of Vitis vinifera plants, the number of leaves was counted at three stages following initial bud sprouting: on days 22, 76, and 102 (Figure 1). As expected, the number of functional leaves increased progressively in proportion to plant age, except for “Cas”, which maintained the same number of leaves on both d 76 and d 102. Therefore, “Cas” showed an average of 9 leaves at the beginning of the experiment and 22 by the end, while “Neg” and “Tin” exhibited similar leaf numbers at each stage, ranging from 10–12 leaves on d 22 to 35–32 leaves on d 102. Compared to “Cas”, both “Neg” and “Tin” had a higher number of leaves per plant (25% and 20% more on d 76, and 38% and 33% more on d 102), respectively. These cultivars are widely cultivated in wide regions of vineyards and are particularly well-suited to subtropical conditions in many regions of the Canary Islands; however, they differ in their growth and development characteristics [41]. It is also important to note that, under our experimental conditions, “Cas” appears to exhibit slower growth in terms of leaf emergence and shoot development compared to “Tin” and “Neg”, at least during the early stages. However, Rodríguez-Torres [41] suggests that adult “Cas” plants in the field may be significantly more vigorous than the other genotypes mentioned. Therefore, since plants grew in non-stressful conditions the differences in leaf number observed between these cultivars, specifically the lower number in “Cas” and the higher in “Neg” and “Tin” may be related to the regional microclimate and the specific behavioral traits of the genotypes. In previous research, it has been reported that the temperature variations affect shoot growth in “Semillon”, leading to the largest canopies at 35 °C and the smallest in those at 40 °C, showing a two-fold dissimilarity [42]. In general, global warming can negatively affect grapevine development through the disruption of phenology, growth, yield, and physiological processes [1,2]. In several viticulture locations worldwide midday air temperatures often exceed 40 °C in summer, impacting grapevines along berry ripening, and leading to yield decrease and consequently vineyards productivity [3,4].

3.2. Stomatal Conductance and Leaf Temperature

Stomatal conductance (gs) remained consistent throughout the different growth stages, ranging from 220 to 314 mmol m⁻² s⁻¹ in the leaves of all genotypes; however, the only exception was on d 76, when “Neg” showed the lowest value (162 mmol m⁻² s⁻¹) (Figure 2A). At this date, compared to “Neg” this variable was 35% and 26% higher in “Cas” and “Tin”, respectively, although, by d 76, leaf temperature was similar in the different cvs. as well as throughout the experimental period (Figure 2B). The decrease in gs at d 76 coincided with the highest leaf temperature (around 30 °C), suggesting that ‘Ne’ may be slightly sensitive to temperature equal or higher to 30 °C. Nevertheless, the determined gs value in “Neg” seems adequate for supporting the photosynthesis activity. Overall, the data suggest that a leaf temperature range (20–30 °C) is suitable for maintaining stomatal function in these grapevine genotypes. Similar findings show that the optimal temperature for photosynthesis in potted ”Thompson” grapevine plants under glasshouse generally ranged between 25 °C and 30 °C [7]. On the other hand, it has been reported in adult plants that the increasing temperatures lead to a decrease in photosynthesis, with an optimal rate at 30 °C under saturated light conditions. Stomatal closure contributed to just 15–30% of this decline [43,44]. Particularly, in “Semillon” leaves, the decline in photosynthesis caused by high temperatures is primarily non-stomatal, contrasting with the response perceived during short periods of high-temperature exposure [45]. In photosynthesis in “Chardonnay” leaves after fruit removal, optimal photosynthesis occurs at 30 °C, with increased rates at higher temperatures, aligning with current climate conditions. Additionally, stomatal limitation of photosynthesis varies from 20% at low to 45% at high temperatures [46]. For “Semillon”, photosynthesis and carbon allocation exhibit a high temperature threshold at 33 °C, with extended exposure to higher temperatures proving detrimental to overall performance [46]. Under our experimental conditions, temperature did not seem to significantly impact stomatal conductance, leaf number, or plant growth of the different genotypes.

3.3. Plant Hormones Changes

The foliar ABA concentrations showed a similar pattern of change in the studied cultivars throughout the experimental period (Figure 3A). A slight increase in ABA content was observed from the initial to the final sampling dates This increase was particularly noticeable between d 22 (93 and 68 ng.ng⁻¹ DW) and 102 (137 and 115 ng.g⁻¹ DW) in “Cas” and “Tin”, respectively. At each stages of growth, “Cas” and “Neg” displayed similar levels of ABA; however, “Tin” accumulated reduced contents of this phytohormone than “Cas” at d 22 and d 102. These small differences between genotypes seem to occur temporary, but generally under non-stressful environments similar amounts of ABA would be accumulated in these cvs. The current scientific literature extensively highlights the role of ABA in regulating several physiological processes, including plant growth, leaf senescence, abscission, stomatal closure, photosynthetic activity, among others. ABA also plays a crucial role as a signaling mediator in plant responses to abiotic stress [8,9,10]. In grapevines, stress-induced ABA movement within the leaf mediates the stomatal response, regulating both transpiration and CO₂ assimilation [47].

Nevertheless, JA concentration peaked during the early growth stage (d 22), with the highest levels observed in “Neg” followed by “Tin” and then “Cas” (Figure 3B). Afterwards, JA levels in “Neg” progressively decreased by around 76% and 92% at d 76 and d 102, respectively compared to d 22. “Tin” leaves also accumulated their maximum concentrations at the first stage of growth and displayed lower levels later. In “Cas” leaves, JA exhibited a slightly different pattern compared to the other genotypes, keeping contents steady at 22 as 102 days of growth, with a small decrease observed at d 76. By comparing d 22 and d 102, the levels of JA and ABA in all the genotypes showed an antagonistic change probably linked to plant age. While JA was higher and lower at d 22 and d 102, respectively, ABA displayed the opposite trend. These results are compatible with previous scenarios where the interaction between JA and ABA modulates the plant responses to environmental stress in various experimental systems [22,23]. JA also regulates plant tolerance mechanisms through the detoxification of reactive oxygen species and synthesis of osmoprotectants under abiotic stress conditions. In addition, this modulation rapidly affects JA responses by upregulating the expression of genes involved in JA biosynthesis and signaling [18,19,20,21].

On the other hand, SA was the plant hormone that exhibited higher leaf content with respect to the other analyzed hormones (Figure 3C). At d 22, “Cas” leaf tissues accumulated around 1280 ng.g⁻¹ DW and subsequently its concentration increased over 44% and 38% at d 76 and d 102, respectively. In this genotype, the accumulation of SA seems to increase with plant age, in contrast to JA. In addition, the concentrations in “Cas” leaf tissues were greater at d 76 (2.8–2.2-fold) and d 102 (2.3–2.2-fold) compared to those in “Neg” and “Tin”, respectively. Finally, the average of SA content in “Neg” and “Tin” leaves were similar throughout the experimental period. It is now well established that SA regulates plant growth and physiological processes, through enhancing photosynthesis, ion uptake and transport, proline and soluble sugar levels, the antioxidant system, and reducing oxidative stress [15,16]. In addition, SA can enhance phenylalanine activity and promote the accumulation of phenolics substances in grape berries under high-temperature field conditions [17]. Leaf IAA content showed a similar trend in all genotypes during the three growth stages, with a slight accumulation on d 22, followed by a moderate increase thereafter (Figure 3D). The IAA levels were consistently low during the growth period, ranging from 1.5 to 4.2 ng.g⁻¹ DW. These foliar levels seem to be suitable for plant growth and physiology in these genotypes under natural conditions, as no stress was applied in this experimental system. The involvement of IAA in grape quality was reported by Deytieux-Belleau et al. [26], showing that the supply of IAA at preharvest induces changes in sugar content, acidity and color in grape berries. Other experiments indicate that kaolin increases IAA and ABA contents in grapevines [28,29,30]. In addition, another auxin (NAA) increases IAA and ABA concentrations, water use efficiency, proline, total soluble sugars, plant growth, and maintains photosynthetic activity [10].

3.4. Leaf Mineral Nutrients Changes

Foliar N levels remained similar in all cvs. at each date, ranging from 3–3.2% on d 22 to 2.4–2.5% of DW on d 102 (

Table 1. Contents of foliar macronutrients in grapevines cultivars: Castellana, Negramoll and Tintilla at three dates during a period of 102 days after the onset of sprouting. Data are means ± standard errors obtained from the total of leaves per plants (average of 9 plants per genotype). For each date, different letters designate significant differences at p < 0.05, analyzed by ANOVA and Duncan test.

Days After Initial Bud Sprouting
Macronutrients	22	76	102
“Cas” N (%DW)	3.02 ± 0.01 a	2.66 ± 0.14 a	2.53 ± 0.17 a
“Neg” N (%DW)	3.17 ± 0.05 a	2.80 ± 0.15 a	2.51 ± 0.07 a
“Tin” N (%DW)	3.11 ± 0.19 a	2.66 ± 0.08 a	2.36 ± 0.08 a
“Cas” P (%DW)	0.20 ± 0.01 a	0.38 ± 0.09 a	0.51 ± 0.07 a
“Neg” P (%DW)	0.24 ± 0.02 ab	0.27 ± 0.04 a	0.38 ± 0.04 a
“Tin” P (%DW)	0.29 ± 0.02 b	0.31 ± 0.06 a	0.43 ± 0.04 a
“Cas” K+ (%DW)	0.85 ± 0.02 a	0.87 ± 0.06 a	0.75 ± 0.05 a
“Neg” K+ (%DW)	0.84 ± 0.03 a	0.91 ± 0.05 a	0.94 ± 0.05 a
“Tin” K+ (%DW)	0.78 ± 0.05 a	0.72 ± 0.06 a	0.83 ± 0.07 a
“Cas” Ca2+ (%DW)	0.90 ± 0.08 a	1.57 ± 0.13 a	1.84 ± 0.09 b
“Neg” Ca2+ (%DW)	0.96 ± 0.07 a	1.19 ± 0.14 a	1.37 ± 0.02 a
“Tin” Ca2+ (%DW)	1.20 ± 0.04 b	1.51 ± 0.18 a	1.81 ± 0.11 b
“Cas” Mg2+ (%DW)	0.12 ± 0.01 a	0.15 ± 0.01 a	0.16 ± 0.02 a
“Neg” Mg2+ (%DW)	0.14 ± 0.01 a	0.15 ± 0.01 a	0.15 ± 0.01 a
“Tin” Mg2+ (%DW)	0.17 ± 0.01 b	0.18 ± 0.01 a	0.20 ± 0.00 b

). This indicates a slightly higher levels at the beginning of the trial compared to the end. In contrast, leaf P content followed an opposite trend, although in this nutrient the levels were low, initiating at 0.2–0.3% on d 22 and increasing to 0.4–0.5% of DW by d 102. Leaf K⁺ concentrations were initially consistent around 0.8% of DW on d 22 (Table 1), with a non-significant decrease in “Tin” compared to “Cas” and “Neg” by d 76. Later, on d 102, “Cas” leaves had slightly lower K⁺ levels compared to “Neg”. Throughout the entire growth period, K⁺ concentrations in all genotypes ranged from 0.7% to 0.9% of DW (Table 1). Overall, the concentrations of the measured macronutrients (N, P and K⁺) appeared optimal for all genotypes during these growth stages, as no deficiency symptoms were observed under the experimental conditions. Tagliavini and Scandellari [33] indicate that accurately determining K⁺ contents in leaves is often challenging, as most K⁺ exists in a free form that can quickly be redistributed to growing organs, like berries or be stored in reserve organs, including branches and roots. It has also been reported that the fluctuation of temperature and evaporation can influence the internal water movement processes within the grapevine, impacting nutrient uptake and mobilization [35]. As well, potassium can activate enzymes involved in photosynthesis and respiration, and maintaining cellular osmotic potential [34].

Foliar Ca²⁺ levels progressively increased during the experimental period in all cvs., ranging from 0.9 to 1.8% of DW, regardless of the date (Table 1). The data pointed out that Ca²⁺ content in “Tin” was approximately 25% and 21% higher at d 22 compared to its concentration in “Cas” and “Neg” leaves, respectively. In addition, the diagnosed levels of Ca²⁺ at d 102 were about 25% lower in “Neg” in comparison with “Cas” and “Tin”. Notably, Ca²⁺ exhibited the second-highest major higher level in leaf tissues in all cvs., following N, while K⁺ ranked third. The average of these concentrations was relatively higher compared to those reported by Amiri et al. [48], which were around 0.88% for “Qarah Shani” and 0.86% for “Thompson Seedless”.

The foliar levels of Mg²⁺ were statistically similar in “Cas” and “Neg” at each date throughout the experimental period (Table 1). However, “Tin” accumulated higher concentrations of this element compared to the other genotypes at d 22 (29% and 18%) and d 102 (25%), respectively. The foliar concentrations of this nutrient were markedly lower than those determined in “Qarah Shani” and “Thompson Seedless”, which were about 1.95% and 2.01%, respectively [48]. In an experimental system using Vitis vinifera “Dattier de Beiruth” grafted on SO4 rootstock under a hydroponic culture, the results show that the uptake of K⁺ is high, and the buildup of Mg²⁺ is inhibited by the increase of K⁺:Mg²⁺ ratio indicating an antagonism between both nutrients [32].

The foliar concentrations of Na⁺ in the three cvs. varied between 0.02% and 0.06% DW throughout the different stages of growth, and such levels appear to be appropriate for these genotypes since no ion toxic symptoms were detected in the leaves (

Table 2. Contents of foliar micronutrients in grapevines cultivars: Castellana, Negramoll and Tintilla at three dates during a period of 102 days after the onset of sprouting. Data are means ± standard errors obtained from the total of leaves per plants (average of 9 plants per genotype). For each date, different letters designate significant differences at p < 0.05, analyzed by ANOVA and Duncan test.

Days After Initial Bud Sprouting
Micronutrients	22	76	102
“Cas” Na+ (%DW)	0.04 ± 0.01 a	0.03 ± 0.00 a	0.03 ± 0.01 a
“Neg” Na+ (%DW)	0.06 ± 0.01 a	0.03 ± 0.00 a	0.04 ± 0.01 a
“Tin” Na+ (%DW)	0.04 ± 0.00 a	0.02 ± 0.00 a	0.04 ± 0.00 a
“Cas” Fe2+ (ppm)	44.47 ± 0.78 a	53.41 ± 2.64 a	0.03 ± 0.01 a
“Neg” Fe2+ (ppm)	47.89 ± 3.13 a	63.43 ± 2.48 bc	0.04 ± 0.01 a
“Tin” Fe2+ (ppm)	48.97 ± 3.37 a	57.31 ± 1.21 ab	0.04 ± 0.00 a
“Cas” Mn2+ (ppm)	36.33 ± 4.63 a	88.70 ± 1.67 b	87.41 ± 10.13 bc
“Neg” Mn2+ (ppm)	40.02 ± 2.66 a	39.85 ± 4.78 a	48.43 ± 11.41 a
“Tin” Mn2+ (ppm)	33.16 ± 1.98 a	39.01 ± 3.00 a	64.41 ± 9.98 ab
“Cas” Cu2+ (ppm)	1.86 ± 0.24 ab	1.14 ± 0.31 a	4.11 ± 1.94 ab
“Neg” Cu2+ (ppm)	2.48 ± 0.19 b	1.25 ± 0.27 a	8.47 ± 1.11 b
“Tin” Cu2+ (ppm)	1.27 ± 0.30 a	1.59 ± 0.34 a	3.53 ± 0.18 a
“Cas” Zn2+ (ppm)	6.08 ± 0.46 b	8.43 ± 1.37 a	7.71 ± 0.82 a
“Neg” Zn2+ (ppm)	7.14 ± 0.40 b	7.38 ± 0.79 a	10.20 ± 1.83 a
“Tin” Zn2+ (ppm)	4.78 ± 0.29 a	8.06 ± 1.69 a	7.25 ± 0.36 a
“Cas” B (ppm)	17.70 ± 1.07 b	26.56 ± 6.34 a	27.40 ± 3.93 a
“Neg” B (ppm)	13.60 ± 0.77 a	18.30 ± 1.19 a	21.99 ± 3.96 a
“Tin” B (ppm)	16.08 ± 1.21 ab	19.58 ± 3.52 a	28.65 ± 8.77 a

). Throughout the trial, no statistical changes in the contents of this micronutrient were observed in any of the genotypes. In this sense, Amiri et al. [48] show similar leaf Na contents in “Qarah Shani” and “Thompson Seedless” under non-stressful conditions.

Regarding Fe²⁺ build up, non-significant variations were observed in the different genotypes during d 22 and d 102 (Table 2). For this micronutrient, only leaves of “Neg” exhibited greater concentration than those of “Cas” at d 76. The contents of this mineral measured in “Qarah Shani” and “Thompson Seedless” were around 308 ppm and 296 ppm, respectively [48]. In productive plants, the determined foliar level of this nutrient range 38–39 ppm in “Flame Seedless” [49], and 171.6 ppm in “Thompson Seedless” grapevines [50]. In previous studies using 12-year-old espalier plants of “Kyoho Early” (V. vinifera × V. labrusca) leaf Fe²⁺ concentrations are around 183.5 ppm [51]. As reported in the distinct cvs., the levels of Fe²⁺ in the leaves seem to vary based on the genotype and growth stage.

Concerning Mn²⁺ variation, the greatest concentrations of this element were measured in “Cas” at d 76 (89 ppm), and then was around 56% higher than those in leaves of “Neg” and “Tin” (Table 2). Afterwards, Mn²⁺ content was kept higher in “Cas” compared to “Neg”; nevertheless, it did not show statistical differences in comparison with “Tin”. It has been reported that the average content of leaf Mn²⁺ in productive plants of “Thompson Seedless” grapevines is around 36.2 ppm [50], and in “Kyoho Early” 33.7 ppm [51]. Mn²⁺ is a crucial micronutrient for plant growth and development, involved in various metabolic functions within different plant cell compartments. It acts as an essential cofactor for the oxygen-evolving complex in photosynthetic machinery [52].

Copper showed the lowest concentrations detected in the foliar analysis of the three genotypes during the trial (Table 2). Minimum levels were measured during the d 22 and d 76 (1.15–2.5 ppm) independently of the cvs. followed by a slight increase reaching about 8.5 ppm in “Neg” at d 102. This genotype accumulated similar levels of Cu²⁺ at each sample date; however, its contents were higher than those of “Tin” by approximately 49% and 58% at d 22 and d 102, respectively. These levels are lower than those determined in adult “Kyoho Early” plants, which display a leaf Cu²⁺ concentration around 15.8 ppm the leaves contain a Cu²⁺ concentration around 15.8 ppm [51].

Zinc displayed the second-lowest foliar contents (4.8–10.2 ppm) among the three genotypes throughout the experimental period, following Cu²⁺ (Table 2). A notable variation in zinc accumulation was observed at d 22, with “Cas” and “Neg” exhibiting concentrations 21% and 33% higher, respectively, than those of “Tin”. However, in other experimental systems involving productive plants, the leaf content of this mineral ranges 11 to 15 ppm in “Flame Seedless” grapevines [49], around 48 ppm and 57 ppm in “Qarah Shani” and “Thompson Seedless”, respectively [48], 35.7 ppm in “Thompson Seedless” [50], and 39.4 ppm in “Kyoho Early” [51]. These reported concentrations are higher than those observed in our study, which may be attributed to several agronomical factors, including soil compositions, fertilizer application rates, genotype traits, and plant age.

Boron contents were similar in the three genotypes mostly over the whole period of study (13.6–28.6 ppm), excepting a higher accumulation of this micronutrient (23%) was noted in the leaves of “Cas” compared to “Neg” (Table 2). In the same range, other experimental systems reported foliar concentrations of this nutrient (21–22 ppm) in adult plants of “Flame Seedless” [49].

Overall, it is worth mentioning that plant nutrient uptake may differ depending on the genotype of Vitis vinifera. For instance, with the use of five cvs. (“Airén”, “Cencibel”, “Garnacha”, “Cabernet Sauvignon”, and “Chardonnay”), it has been found that the buildup of N, P and Zn²⁺ shows only slight variations; however, there are substantial changes in the accumulation of K⁺, Ca²⁺, Mg²⁺, and Cl⁻. In addition, the uptake of the following nutrients can vary according to the cultivar: Ca²⁺ (high in ”Cencibel”, 3.26%), P (low in ”Airén”, 0.09%), and K⁺ significantly differ in almost all the cvs. ranging from 0.47% to 0.8% in ”Cencibel” and “Chardonnay”, respectively [31].

In summary, data presented here showed specific similarities and differences in terms of physiological processes. For instance, the evaluation of plant growth by means of functional leaf number revealed a similar tendency between “Neg” and “Tin” with a progressive increase parallelly to plant age, and although “Cas” exhibited the same growth pattern, its development was slower, and subsequently the emerged leaves were lower than the other genotypes. Regarding stomatal movement, gs measurements were similar between the three genotypes except for “Neg” which demonstrated a lower value at d 76 compared to “Cas” and “Neg”. Such decrease of gs coincides with the highest leaf temperature which might suggest that “Neg” seems to be sensitive to temperatures higher than 30 °C. Concerning the hormonal changes, “Cas” accumulated the highest concentration of SA compared to “Neg” and “Tin”, which both maintained similar contents throughout the experimental period. The data may indicate a possible connection between the elevated SA levels and the fewer leaves in “Cas” plants. To prove this hypothesis, a long-term experiment could be conducted to evaluate the progression of both variables. Relating to ABA, “Cas” and “Neg” accumulated similar levels; however, “Tin” exhibited slightly and temporary reduced concentrations, particularly during the early growth stage (d 22), compared to the other genotypes. JA contents, in turn, fluctuated throughout the stages of growth, displaying a non-uniform pattern of change, and the most apparent observation was likely its high level and the variations among the three genotypes at the initial date. Lastly, IAA contents generally showed similar trends between all the genotypes. In relation to mineral nutrients, similar foliar concentrations of N, P, K⁺ and Na⁺ were accumulated between the distinct genotypes, while the levels of Mg²⁺ were higher in “Tin” than in “Cas” and “Neg” at the beginning and the end of the experiment. Ca²⁺ content was higher in “Tin” compared to “Cas” and ‘Ne’ at d 22, and afterward its content was maintained higher than “Neg” but similar to “Cas” at d 102. The levels of micronutrients fluctuated temporarily between cvs.; however, no constant trend was observed, as the plants did not exhibit any deficiencies in any of the elements.

4. Conclusions

The data presented here illustrates the pattern of change and concentration ranges of phytohormones and mineral nutrients, along with leaf growth and stomatal conductance from the beginning of sprouting to 102 days later in three genotypes of Vitis vinifera. The findings suggest a relationship between high levels of SA and a reduced number of leaves in “Cas” compared to “Neg” and “Tin” throughout the growth stage. Moreover, an antagonistic change was observed in the accumulation of SA and JA, as well as between ABA and JA particularly at the onset and the end of the trial. Consequently, long-term experiments could further explore the involvement of SA and other hormones, and physiological markers in the growth and development of these genotypes under environmental conditions.