Zeolite in Vineyard: Innovative Agriculture Management Against Drought Stress

Abstract

Discovering, analyzing, and finding a key to understanding the physiological and biochemical responses that Vitis vinifera L. undertakes against drought stress is of fundamental importance for this profitable crop. Today’s considerable climatic fluctuations force researchers and farmers to focus on this issue with solutions inclined to respect the ecosystem. In this academic work, we focused on describing the drought stress consequences on several parameters of secondary metabolites on Vitis vinifera leaves (quercetins, kaempferol, resveratrol, proline, and xanthophylls) and on some ecophysiological characteristics (e.g., water potential, stomatal conductance, and leaf temperature) to compare the answers that diverse agronomic management techniques (i.e., irrigation with and without zeolite, pure zeolite and no application) could instaurate in the metabolic pathway of this important crop with the aim to find convincing and thought-provoking responses to use this captivating and versatile mineral, the zeolite known as the “magic rock”. Stressed grapevines reached 56.80 mmol/m²s gs at veraison and a more negative stem Ψ (+10.63%) compared to plants with zeolite. Resveratrol, in the hottest season, fluctuated from 0.18–0.19 mg/g in zeolite treatments to 0.37 mg/g in stressed vines. Quercetins were inclined to accumulate in response to drought stress too. In fact, we recorded a peak of quercetin (3-O-glucoside + 3-O-glucuronide) of 11.20 mg/g at veraison in stressed plants. It is interesting to note how the pool of metabolites was often unchanged for plants treated with zeolite and for plants treated with water only, thus elevating this mineral to a “stress reliever”.

1. Introduction

The introductory statement of the International Organisation of Vine and Wine’s Annual Report delivers a clear message: global wine production in 2024 is projected to decrease even further compared to the already low levels recorded in the previous year. Adverse climatic conditions affecting vineyards in both hemispheres continue to be the primary cause of this ongoing reduction in worldwide yields. Vitis vinifera L. holds a central role in global viticulture and enology, with an estimated 7.2 million hectares under cultivation in 2023 and an annual wine production volume of approximately 237 million hectoliters [1]. From research on the Scopus website, it is highlighted how in recent years the publications concerning grapevine and climate change have exponentially increased their trend. Problems such as increases in the incidence of pathogens [2] and changes in the composition of phenolic compounds in berries [3] are addressed with care and detail.

Climate change has affected water security through increased temperatures, altered precipitation patterns, the reduction and loss of cryospheric components, and the rising frequency and intensity of extreme weather events [4], thereby hindering endeavors to fulfill the homeostatic balance of the plant. In the 21st century, as compared to the age from 1850 to 1900, around 1.60 °C enhancement in land mass temperature and around 0.90 °C enhancement in ocean surface temperature have been observed [5]. In general, crops have shown several adaptive replies to counterbalance stress effects; for instance, during drought periods they seal stomata and gather compatible solutes to preserve a low water potential and escape dehydration [6]. Stomatal conductance [7], vessel anatomy [8], and channel proteins (aquaporins) [9] regulate water flow in grapevines to the leaves and from the leaves to the atmosphere.

In particular, regarding drought stress rejoinder, grapevine varieties were categorized as near-isohydric (e.g., Montepulciano) or near-anisohydric (e.g., Sangiovese) [10]. This classification is thought to be owing to their divergence in the perception of an important chemical signal arising from the roots: the abscisic acid [11]. Isohydric cv can keep an invariable midday leaf water potential (Ψ leaf) and above critical thresholds (i.e., cavitation = −1.5 MPa) careless of ground H₂O availability or atmospheric H₂O demand by means of stomatal conductance (gs) depletion, while anisohydric ones retain their stomata open also under a lowing Ψ leaf [12]. Nevertheless, these two behaviors are not always recognizable, and plants that normally show off an anisohydric attitude could manifest constrained gs under determined conditions [13].

Water stress signaling is mediated through both abscisic acid (ABA)-dependent and ABA-independent pathways, which collectively modulate the transcriptional activation of gene networks involved in drought tolerance and adaptive responses. The resulting gene products are implicated in the biosynthesis and accumulation of osmoprotectants, activation of cellular detoxification mechanisms, repair or replacement of stress-damaged proteins, and amplification of stress-related signaling cascades [14,15]. Proline [16], glycine betaine (e.g., N,N,N-trimethyl glycine) [17], and mannitol [18] (three important osmoprotectants) have been found to be crucial for stress furtherance tolerance directly or indirectly via ROS scavenging system. In fact, the over-production of non-toxic soluble substances, in general, characterized by low molecular weight and important solubility, represents habitual stress strategies in plant [19]. These include C₁₂H₂₂O₁₁ and (CHOH)_nH₂, respectively, sucrose and sugar alcohols [20]. Quercitol (C₆H₁₂O₅), pinitol (C₇H₁₄O₆), and quebrachitol (C₇H₁₄O₆) are three cyclitols identified as “compatible solutes”, lightly different respect primary metabolites, discovered in drought-tolerant woody plants [21,22] (i.e., leaf and root of Eucalyptus species [23]). Moreover, it was indicated that cyclitols such as sugar alcohols and inositols can imitate water structure and preserve a hydration sphere on every side of macromolecules [24]. Ca²⁺, released from some inositol byproducts, can squarely promote the expression of stress-response genes, following in improved stress tolerance [25,26].

Water stress combined with high temperatures forces the plant to protect itself from photoinhibition that occurs when the rate of excitation energy shift from the antennae to the photochemical reaction center surpasses the electron transport one [27]. Reductions in photosynthetic capacity, damage to thylakoids, trimming in ribulose 1, 5-bisphosphate carboxylase/oxygenase, and elevation of reactive oxygen species (i.e., peroxol, dioxidan-2-idylide, and hydridooxygen) are consequences of high irradiance stress [28,29,30]. In fact, when leaves are subjected to light going too far in the utilization capacity by net photosynthesis (deriving from a high photon flux density or deriving from a lack of net carbon dioxide assimilation by water stress), a diminishment in chlorophyll fluorescence yield at maximum fluorescence degree happens, named non-photochemical quenching [31]. Plants have developed strategies to thwart reactive oxygen species that are of enzymic and non-enzymic manner [32]. Carotenoids preserve photosynthetic apparatus in two keyways: (a) β-Caroten quenches chlorophyll (Chl) triplet and singlet oxygen; (b) chlorophyll singlet is quenched from xanthophyll cycle in chloroplasts (i.e., reversible changeover from violaxanthin to zeaxanthin via the intermediary antheraxanthin) [27]. Emerging evidence indicates that zeaxanthin contributes to the dynamic turnover of the D1 protein in photosystem II (PSII), enhancing the proteolytic degradation of damaged D1 proteins by FtsH proteases [33,34]. The importance of acidic lumen pH in non-photochemical quenching of Chl a fluorescence was noted in Arabidopsis [35] (low lumen pH starts up the violaxanthin de-epoxidase, originating the antheraxanthin and zeaxanthin line-up that it is considered to be implicated in the fluorescence quenching activity [31]). The acidification of the thylakoid lumen and the activation of the xanthophyll cycle represent key biochemical mechanisms underlying non-photochemical quenching, thereby contributing critically to photoprotective responses in plants [35]. Moreover, leaf values less than 0.83 Fv/Fm (i.e., photosystem II photochemical capacity) have been discovered in plants subjected to stress levels [36].

Nowadays, strategies to protect the vine from these concrete risks, ensuring the well-being of the plant, a translocation of solutes, and a correct source/sink balance are increasingly sought after by winemakers in the areas at risk. In general, emergency irrigation is one of the most widespread practices to mitigate water shortages and radiation increases. However, the use of this means of relief is not always possible or easy to implement [37]. Even legislators are aware of the need to adopt different strategies for sustainable management of climate change problems, funding research aimed in this direction [38].

Zeolites, initially characterized by Cronstedt in the 18th century, are classified within the tectosilicate framework and comprise a group of over 50 mineral species. These minerals are structurally defined as hydrated aluminosilicates incorporating alkali and alkaline earth cations [39]. Thanks to their unique properties (i.e., reversible dehydration, strong and selective CEC or molecular absorption, and so on), they manifest themself in a multitude of usages in agriculture [40]. Zeolite’s uses as a foliar treatment, as an implement against pests (i.e., Tribolium confusum and Callosobruchus maculatus [41]) or pathogens [42], and as a leaf temperature reducer [43] are widespread. On Vitis vinifera, the effects of zeolites have so far been recorded, for example, on the activity of all antioxidant enzymes and leaf chlorophyll amount [44], against berry sunburn damage [45], as a controller of grapevine downy mildew [46], and on flavonoid gene expression and interactions with epiphytic microorganisms [47].

In organic grape production, where the use of synthetic fertilizers is restricted, zeolite can function as a buffering agent, improving soil water-holding capacity, nutrient availability, and overall rhizosphere stability. Its slow-release behavior aligns well with the principles of organic farming, promoting gradual nutrient supply without harmful runoff. Moreover, zeolite is a permitted input in organic farming under EU regulations (EC 889/2008), provided it is naturally mined and not chemically modified. Its inert and non-toxic nature ensures environmental safety and compatibility with ecological viticultural practices [48,49,50].

Identifying new agronomic approaches capable of improving the management of water resources within vineyards, or even replacing it completely, is an urgent need considering climate change. Undoubtedly, the choice to resort to irrigation is widely adopted in the viticultural context and its beneficial effects have been widely studied in viticulture. However, very little is known about how zeolite applied to the soil interacts with the ecophysiological and biochemical aspects of the grapevine. Can it be a substitute for open-field irrigation to overcome the problems related to climate change?

In fact, as soil conditioners, their impact on the biochemistry and ecophysiology of leaves should be assessed as a basis for analyzing the positive or negative repercussions they may have on production. The intimate and intricate relationship between the canopy and the ripening of the berry has led this research to focus on the repercussions that water stress and high temperatures create on leaf biochemistry. This research aimed to focus attention on the root of the problem, that is, on the delicate biochemical mechanisms that can be established in the plants without any adjuvant, and therefore more subject to water stress (abiotic stress), in the ones marked by zeolite treatment as a soil amendment, in others with a standard tool against climate change (i.e., irrigation), and finally in the ones with both soil management (i.e., zeolite and irrigation).

2. Materials and Methods

2.1. Vineyard, Experimental Design, and Soil Treatments

The trial took place in a nine-year-old vineyard of Sangiovese, located in Italy (San Casciano V. di Pesa, 43°67′ N, 11°17′ E) characterized by the following parameters: 775P rootstock, counter-espalier training, Guyot pruning, and 2.5 m × 0.8 m spacing. The vineyard was made up of clay soil (i.e., 37%) with medium-high content of organic matter (i.e., 1.8%) and high total limestone (i.e., 21%). The vineyard was managed under standard organic farming practices, including mechanical weed control, organic-certified fertilization, and the exclusion of synthetic pesticides and herbicides, following EU Regulation 2018/848.

The observations were conducted over 2 years (2022–2023) on 40 test grapevines (three blocks per treatment where N = 10). The two master factors were soil management (zeolite treatment) and irrigation. Processings were (a) irrigated and treated plants (WWZeo), (b) irrigated plants (WW), (c) treated plants (Zeo), and plants naturally exposed to abiotic factors without treatments (WS). Italian clinoptilolite-rich zeolite (BIG-Zeo, Pisa, Italy) was buried at a dose of 1 kg per vine and covered with ground mixed with sphagnum peat moss (all chemical–physical characteristics are visible at the following link, https://www.agricolainternazionale.it/prodotti/big-zeo-granulare/ (accessed on 15 June 2025)). BIG-Zeo was distributed in February, 39 DOY-2022 and 40 DOY-2023, during the dormancy stage, at approximately 20 cm depth around the planting perimeter of each vine.

The water supply to rows, through a precision drip irrigation system, was calibrated using water potential thresholds [51] until veraison and until ripening, −1.4 and −1.6 MPa, respectively. WWZeo and WW were intentionally given the same amount of water of 10 L per vine, from flowering (i.e., eighteen irrigations during 2022 and thirteen during 2023).

Meteorological data on air temperature and precipitation were collected from a Davis Vantage Vue weather station (USA and Canada) installed in proximity to the vineyard, covering the period from 1 April to 31 September.

2.2. Evaluation of Chlorophyll Fluorescence, Water Potential, Stomatal Conductance, and Temperature of the Leaf

As temperatures continue to rise, from June, midday water potential (Ψ stem) with a Scholander-type pressure chamber was evaluated on ten mature leaves for each soil management × irrigation treatment arrangement. These measurements were accomplished on seven separate days (i.e., 20 June or 171 DOY, 3 July or 184 DOY, 15 July or 196 DOY, 20 July or 201 DOY, 28 July or 209 DOY, 16 August or 228 DOY, 24 August or 236 DOY 2022 and 15 June or 166 DOY, 3 July or 184 DOY, 17 July or 198 DOY, 1 August or 213 DOY, 8 August or 220 DOY, 16 August or 228 DOY, 1 September or 244 DOY 2023) after wrapping the leaf in a plasticized aluminum folio for one hour in advance of the measurements.

At the same time, from June, leaf temperature, with the help of the Ciras3 PP System was evaluated on ten mature leaves for each soil management × irrigation treatment arrangement. These measurements were accomplished on seven separate days (i.e., 20 June, 3 July, 15 July, 20 July, 28 July, 16 August, 24 August 2022 and 15 June, 3 July, 17 July, 1 August, 8 August, 16 August, 1 September 2023).

The evaluation of chlorophyll fluorescence was made by Hansatech Fluorometer during pre-bunch closure (184 DOY 2022 and 184 DOY 2023), veraison (209 DOY 2022 and 213 DOY 2023), and maturation (228 DOY 2022 and 228 DOY 2023) [52]. The clips were placed between 1:00 PM to 2:00 PM on ten leaves per tagged grapevine, collocated on the set-out southeast side of the canopy to the sunlight.

The gauge of stomatal conductance (N = 10) was made by Ciras3 PP system during the same phenological periods of chlorophyll fluorescence detection.

2.3. Evaluation of Phenolic Compounds in Leaves

The assessment of phenolic compounds in leaves was made during pre-bunch closure (184 DOY 2022 and 184 DOY 2023), veraison (209 DOY 2022 and 213 DOY 2023), and maturation (228 DOY 2022 and 228 DOY 2023) [52] with an HPLC system (Agilent, Sanra Clara, CA, USA) (N = 6). Briefly, to determine phenolic compounds, 2 g of lyophilized sample were pulled out twice with 20 mL of 3% formic acid in methanol under agitation for 45 min. After centrifugation, the supernatants were unified and brought to a final volume of 50 mL. The extract was analyzed by HPLC-DAD following the technique cataloged by Gómez-Alonso et al. [53].

2.4. Evaluation of Proline, Zeaxanthin, Lutein, βcarotene, Chlorophyll a, and Chlorophyll b

Proline content (N = 10) was determined in the lyophilized sample using the ninhydrin reaction method as described by Carillo and Gibon [54] during the following days 184 DOY 2022 and 184 DOY 2023, 209 DOY 2022 and 213 DOY 2023, as well as 228 DOY 2022 and 228 DOY 2023.

For the determination of photosynthetically active compounds, 250 mg of powder were extracted in 2.5 mL of acetone for 5 min in an ultrasonic bath. The sample was centrifuged, and the supernatant was transferred to a 5 mL volumetric flask. The pellets were re-extracted, and the combined supernatants were brought to a final volume of 5 mL.

The extract was analyzed by HPLC-DAD for carotenoid determination (N = 6) as described by Beckett et al. [55]. Chlorophyll content (N = 6) was determined spectrophotometrically on the same acetone extract, following the method described by Lichtenthaler [56]. Carotenoid (zeaxanthin, lutein, and βcarotene) and chlorophyll content (a and b types) were sampled during the following days 184 DOY 2022 and 184 DOY 2023, 209 DOY 2022 and 213 DOY 2023, as well as 228 DOY 2022 and 228 DOY 2023.

2.5. Statistical Analysis

Two-way ANOVA, worked out by RStudio software (2021.09.1+372), was employed for all ecophysiological and biochemical measures in V. vinifera L. processed with or without clinoptilolite (T/T), under two irrigation (IR) regimens (WW, irrigation; WS, no irrigation). Values are the data mean (Tukey’s HSD) [57] of each leaf parameter, considering clinoptilolite (T and T) and IR as factors (p ≤ 0.05). Data normality was assessed using the Shapiro–Wilk test, and homogeneity of variances was confirmed with Levene’s test prior to ANOVA. A Principal Component Analysis (PCA), obtained with the Ggplot2 package for R (RStudio software), was performed to identify the underlying relationships among some quantitative traits assessed in this work (i.e., proline, lutein, zeaxanthin, βcarotene, and a-b chlorophylls). To statistically validate the separation observed in the PCA plot, a PERMANOVA (Permutational Multivariate Analysis of Variance) was performed using the adonis2() function from the vegan R package (R version 4.1.2), based on a Euclidean distance matrix and 999 permutations. The model tested the effect of the treatment on the multivariate dataset, and the proportion of variance explained was reported as R². In addition, the homogeneity of multivariate dispersions (a key assumption of PERMANOVA) was assessed using the betadisper() function in the same package. The non-significant result (p > 0.05) confirmed that the observed differences between groups were not due to unequal within-group variances.

3. Results

3.1. Weather Station Data

The climate trends from April to September are shown in Figure 1.

Rains (April–September) were higher in 2022 (556 mm) as opposed to 2023 (440 mm), primarily owing to the heavy downfalls (36.9 mm fell during 217 DOY and 43.1 mm fell during 224 DOY) that occurred in August 2022. However, as can be seen from the graph, the distribution of the entire rainfall sum is significantly different in the two years. In the 2022 test year, rainfall was scarce in days from pre-bunch closure to veraison and the only significant occurrence (7 mm) was certified on DOY 188. While in the 2023 test year, more intensive rainfall occurred throughout the period from pre-bunch closure to veraison, and the most notable occurrences (14.4, 5.8, 17, and 5.3 mm) were observed on DOY 195, 196, 206, and 207. The 2022 summertime was distinguished by the happening of vehement and prolonged heatwaves that in the interval from fruit set to maturation resulted in max air temperatures to thresholds higher than 35.0 °C (i.e., 147, 165–169, 177–178, 183–188, and the longest and most important from 194 to 219 DOY). On the contrary, in 2023, the prohibitive utmost temperatures were reached toward the end of summer with a short period of recovery given by the cooling of the temperature (28.4 °C on 207 DOY) due to a rain event at the end of July.

3.2. Chlorophyll Fluorescence and Stomatal Conductance Results

Chlorophyll fluorescence (Fv/Fm) and stomatal conductance changed both in 2022 between different management treatments, whereas the Fv/Fm parameter did not differ between the zeolite and/or irrigation according to the data displayed in Figure 2.

Specifically, focusing on stomatal conductance, the following reductions were found for WS treatment compared to WWZeo, Zeo, and WW at veraison in 2022, −57.86%, −36.11%, and −57.89%, respectively. The following abatements were represented in the figure for WS treatment compared to WWZeo, Zeo, and WW at maturation in 2023, −58.93%, −56.00%, and −58.87%, respectively.

Changes in chlorophyll fluorescence were found only at veraison and ripening in 2022, where treatments with zeolite and/or water recorded significantly higher values than WS.

3.3. Stem Water Potential and Leaf Temperature Results

The assessment of water potential, conducted from June to the final part of August 2022 (171–236 DOY) and from June to early September 2023 (166–244 DOY) by including the main phenological periods of the vine (pre-cluster closure, veraison, ripening, and harvest), made known that, as expected, automatic irrigation and clinoptilolite or their merged application triggered positive answers in tagged-vines (Figure 3 and Figure 4).

The trends, as clearly visible in the figures, followed the climatic trend (temperatures and precipitations). Often, there are no significant differentiations between WW and Zeo. Only in the moments of greater stress (209 DOY/veraison 2022 and 228 DOY/maturation 2023), the four soil managements strengthened the divergences among them. Specifically, on the DOYs indicated, the following positive increases in potential were found compared to the drought-affected plants: WWZeo + 46.15%, Zeo + 18.75%, WW + 32.27%, and WWZeo + 18.63%, Zeo + 11.69%, WW + 7.91%, respectively.

Leaf temperature trends closely mirror maximum air temperatures and are strongly influenced by them (Figure 5 and Figure 6).

Overall, in 2022, leaf temperatures fluctuated from a rock-bottom of 29.11 °C to a maximum of 40.72 °C (recorded respectively by WWZeo treatment on 24 August and by the WS treatment on 3 July). Instead, in 2023, leaf temperatures fluctuated from a rock-bottom of 26.13 °C to a maximum of 37.14 °C (recorded respectively by Zeo treatment on 15 June and by the WS treatment on 1 August). In 2022, two main peaks in the trend are noticeable, DOYs 184 and 201. During both of these hot days, the leaf temperature of WS plants was more elevated than all other treatments. In particular, the following increases were found: +7.29% compared to WWZeo, +6.79% compared to Zeo, +8.81% compared to WW during DOY 184 and +5.65% compared to WWZeo, +5.65% compared to Zeo, and +2.99% compared to WW during DOY 201. In 2023, two main peaks in the trend are noticeable, DOYs 198 and 228. During both of these hot days, the leaf temperature of WS plants was higher than all the others. In particular, the following increases were found: +5.78% compared to WWZeo, +5.27% compared to Zeo, +2.42% compared to WW during DOY 184 and +5.40% compared to WWZeo, +7.95% compared to Zeo, and +2.91% compared to WW during DOY 201.

3.4. Leaves Phenolic Compounds Results

The phenolic compounds of the leaves are reported in

Table 1. Two-way ANOVA on 3 July 2022 for leaf biochemical compounds in V. vinifera L. processed with or without clinoptilolite (T/T), under irrigation (IR) regimens (WW, irrigation; WS, no irrigation). Reported value is data mean of each leaf biochemical parameter, assuming clinoptilolite (T/T) and IR as factors. Significance difference is reported as an alphabet letter (mean ± SE, n = 5); in the last three rows, significance Pr(>F) is noted (Significance codes: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). Abbreviations: procyanidin B1 (PB1), epigallocatechin (Epigc), caftaric acid (Caft), c-coutaric acid (cCout), t-coutaric acid (tCout), fertaric acid (Fert), resveratrol 3-O-glucoside (Resv), myricetin (Myr), kaempferol (Kaem), isorhamnetin (Isorh), quercetin-3-O-galactoside+quercetin-3-O-rutinoside (Qga+r), quercetin-3-O-glucoside+quercetin-3-O-glucoronide (Qgl+glc).

	3 July 2022
	PB1	Epigc	Caft	cCout	tCout	Fert	Resv	Myr	Kaem	Isorh	Qga+r	Qgl+glc
	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
Zeolite Treatment (T)
WWZeo	1.22 ± 0.23 a	0.65 ± 0.10 ab	1.13 ± 0.17 ab	0.39 ± 0.05 a	0.25 ± 0.06 a	0.87 ± 0.20 ab	0.23 ± 0.05 a	1.83 ± 0.27 a	2.24 ± 0.24 b	0.41 ± 0.21 b	0.43 ± 0.03 b	6.43 ± 0.32 b
Zeo	0.81 ± 0.08 b	0.46 ± 0.08 c	1.04 ± 0.04 ab	0.22 ± 0.02 c	0.21 ± 0.01 a	0.68 ± 0.03 b	0.21 ± 0.01 ab	1.43 ± 0.05 b	1.74 ± 0.45 b	0.27 ± 0.02 b	0.38 ± 0.03 b	5.92 ± 0.30 b
Irrigation Regime (IR)
WW	0.86 ± 0.03 b	0.53 ± 0.05 bc	0.95 ± 0.06 b	0.35 ± 0.07 a	0.23 ± 0.03 a	0.74 ± 0.07 ab	0.14 ± 0.01 b	1.32 ± 0.14 b	1.62 ± 0.22 b	0.26 ± 0.03 b	0.37 ± 0.03 b	5.77 ± 0.42 b
WS	1.13 ± 0.12 a	0.79 ± 0.11 a	1.26 ± 0.12 a	0.28 ± 0.03 bc	0.26 ± 0.02 a	0.91 ± 0.05 a	0.21 ± 0.01 b	1.95 ± 0.05 a	2.86 ± 0.35 a	0.60 ± 0.07 a	0.57 ± 0.03 a	7.21 ± 0.44 a
Significance Pr(>F)
T	0.843	0.032 *	0.722	0.743	0.608	0.334	0.011 *	0.971	0.152	0.028 *	0.002 **	0.127
IR	0.343	0.448	0.075	0.000 ***	0.613	0.857	0.179	0.182	0.041 *	0.016 *	0.000 ***	0.025 *
T × IR	0.000 ***	0.000 ***	0.004 **	0.062	0.092	0.006 **	0.016 *	0.000 ***	0.000 ***	0.000 ***	0.000 ***	0.000 ***

(pre-cluster closure 2022),

Table 2. Two-way ANOVA on 28 July 2022 for leaf biochemical compounds in V. vinifera L. processed with or without clinoptilolite (T/T), under irrigation (IR) regimens (WW, irrigation; WS, no irrigation). Reported value is data mean of each leaf biochemical parameter, assuming clinoptilolite (T/T) and IR as factors. Significance difference is reported as an alphabet letter (mean ± SE, n = 5); in the last three rows, significance Pr(>F) is noted (Significance codes: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). Abbreviations: procyanidin B1 (PB1), epigallocatechin (Epigc), caftaric acid (Caft), c-coutaric acid (cCout), t-coutaric acid (tCout), fertaric acid (Fert), resveratrol 3-O-glucoside (Resv), myricetin (Myr), kaempferol (Kaem), isorhamnetin (Isorh), quercetin-3-O-galactoside+quercetin-3-O-rutinoside (Qga+r), quercetin-3-O-glucoside+quercetin-3-O-glucoronide (Qgl+glc).

	28 July 2022
	PB1	Epigc	Caft	cCout	tCout	Fert	Resv	Myr	Kaem	Isorh	Qga+r	Qgl+glc
	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
Zeolite Treatment (T)
WWZeo	0.63 ± 0.07 b	0.65 ± 0.04 b	1.81 ± 0.12 b	0.11 ± 0.03 a	0.32 ± 0.02 ab	0.93 ± 0.07 b	0.16 ± 0.03 b	1.49 ± 0.25 b	1.43 ± 0.44 b	0.80 ± 0.03 b	0.39 ± 0.06 b	7.10 ± 0.58 b
Zeo	1.62 ± 0.86 a	0.79 ± 0.10 b	1.83 ± 0.07 b	0.14 ± 0.2 a	0.30 ± 0.01 b	0.89 ± 0.02 b	0.17 ± 0.01 b	1.60 ± 0.13 b	1.35 ± 0.27 b	0.70 ± 0.05 b	0.46 ± 0.04 b	7.81 ± 0.10 b
Irrigation Regime (IR)
WW	0.71 ± 0.07 b	0.61 ± 0.19 b	1.84 ± 0.10 b	0.14 ± 0.01 a	0.29 ± 0.03 b	0.83 ± 0.07 b	0.11 ± 0.03 c	1.34 ± 0.19 b	1.02 ± 0.24 b	0.80 ± 0.05 b	0.43 ± 0.05 b	7.35 ± 0.35 b
WS	1.49 ± 0.08 a	1.25 ± 0.01 a	2.07 ± 0.12 a	0.11 ± 0.01 a	0.37 ± 0.04 a	1.26 ± 0.18 a	0.23 ± 0.01 a	2.14 ± 0.10 a	2.82 ± 0.10 a	1.13 ± 0.13 a	0.67 ± 0.04 a	10.06 ± 0.55 a
Significance Pr(>F)
T	0.933	0.002 **	0.029 *	0.759	0.249	0.022 *	0.824	0.049 *	0.002 **	0.000 ***	0.000 ***	0.000 ***
IR	0.000 ***	0.000 ***	0.032 *	0.537	0.038 *	0.001 **	0.000 ***	0.000 ***	0.000 ***	0.009 **	0.000 ***	0.000 ***
T × IR	0.654	0.000 ***	0.074	0.049 *	0.001 **	0.000 ***	0.000 ***	0.005 **	0.000 ***	0.000 ***	0.002 **	0.000 ***

(veraison 2022),

Table 3. Two-way ANOVA on 16 August 2022 for leaf biochemical compounds in V. vinifera L. processed with or without clinoptilolite (T/T), under irrigation (IR) regimens (WW, irrigation; WS, no irrigation). Reported value is data mean of each leaf biochemical parameter, assuming clinoptilolite (T/T) and IR as factors. Significance difference is reported as an alphabet letter (mean ± SE, n = 5); in the last three rows, significance Pr(>F) is noted (Significance codes: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). Abbreviations: procyanidin B1 (PB1), epigallocatechin (Epigc), caftaric acid (Caft), c-coutaric acid (cCout), t-coutaric acid (tCout), fertaric acid (Fert), resveratrol 3-O-glucoside (Resv), myricetin (Myr), kaempferol (Kaem), isorhamnetin (Isorh), quercetin-3-O-galactoside+quercetin-3-O-rutinoside (Qga+r), quercetin-3-O-glucoside+quercetin-3-O-glucoronide (Qgl+glc).

	16 August 2022
	PB1	Epigc	Caft	cCout	tCout	Fert	Resv	Myr	Kaem	Isorh	Qga+r	Qgl+glc
	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
Zeolite Treatment (T)
WWZeo	0.88 ± 0.24 b	0.62 ± 0.20 ab	1.17 ± 0.04 b	0.28 ± 0.06 ab	0.30 ± 0.03 a	0.95 ± 0.06 a	0.19 ± 0.03 b	1.94 ± 0.28 ab	1.86 ± 0.27 b	0.37 ± 0.03 a	0.39 ± 0.03 b	6.99 ± 0.22 b
Zeo	0.63 ± 0.03 b	0.42 ± 0.09 b	1.34 ± 0.09 a	0.31 ± 0.04 a	0.28 ± 0.03 a	0.81 ± 0.05 b	0.18 ± 0.03 b	1.64 ± 0.21 b	1.67 ± 0.29 b	0.34 ± 0.02 a	0.38 ± 0.03 b	6.47 ± 0.17 c
Irrigation Regime (IR)
WW	0.81 ± 0.05 b	0.65 ± 0.23 ab	1.27 ± 0.12 ab	0.32 ± 0.01 a	0.29 ± 0.02 a	0.89 ± 0.04 ab	0.26 ± 0.03 b	1.77 ± 0.22 ab	1.79 ± 0.23 b	0.33 ± 0.03 a	0.42 ± 0.05 b	6.61 ± 0.32 bc
WS	1.42 ± 0.09 a	0.95 ± 0.25 a	1.40 ± 0.03 a	0.21 ± 0.03 b	0.31 ± 0.02 a	1.00 ± 0.08 a	0.37 ± 0.09 a	2.22 ± 0.28 a	3.28 ± 0.25 a	0.36 ± 0.06 a	0.59 ± 0.05 a	8.50 ± 0.24 a
Significance Pr(>F)
T	0.000 ***	0.014 *	0.065	0.199	0.673	0.043 *	0.000 ***	0.125	0.000 ***	0.681	0.000 ***	0.000 ***
IR	0.013 *	0.612	0.002 **	0.093	0.832	0.490	0.089	0.589	0.000 ***	0.863	0.004 **	0.000 ***
T × IR	0.000 ***	0.028 *	0.606	0.003 **	0.221	0.000 ***	0.050	0.009 **	0.000 ***	0.223	0.001 **	0.000 ***

(maturation 2022),

Table 4. Two-way ANOVA on 3 July 2023 for leaf biochemical compounds in V. vinifera L. processed with or without clinoptilolite (T/T), under irrigation (IR) regimens (WW, irrigation; WS, no irrigation). Reported value is data mean of each leaf biochemical parameter, assuming clinoptilolite (T/T) and IR as factors. Significance difference is reported as an alphabet letter (mean ± SE, n = 5); in the last three rows, significance Pr(>F) is noted (Significance codes: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). Abbreviations: procyanidin B1 (PB1), epigallocatechin (Epigc), caftaric acid (Caft), c-coutaric acid (cCout), t-coutaric acid (tCout), fertaricacid (Fert), resveratrol 3-O-glucoside (Resv), myricetin (Myr), kaempferol (Kaem), isorhamnetin (Isorh), quercetin-3-O-galactoside+quercetin-3-O-rutinoside (Qga+r), quercetin-3-O-glucoside+quercetin-3-O-glucoronide (Qgl+glc).

	3 July 2023
	PB1	Epigc	Caft	cCout	tCout	Fert	Resv	Myr	Kaem	Isorh	Qga+r	Qgl+glc
	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
Zeolite Treatment (T)
WWZeo	0.06 ± 0.02 a	0.13 ± 0.02 a	1.71 ± 0.37 a	0.02 ± 0.00 a	0.04 ± 0.01 a	0.13 ± 0.03 a	0.01 ± 0.00 a	0.22 ± 0.01 ab	0.17 ± 0.04 ab	0.02 ± 0.01 a	0.43 ± 0.04 b	6.75 ± 0.35 a
Zeo	0.06 ± 0.01 a	0.11 ± 0.03 a	1.89 ± 0.46 a	0.02 ± 0.00 a	0.04 ± 0.01 a	0.13 ± 0.02 a	0.01 ± 0.00 a	0.20 ± 0.03 b	0.25 ± 0.06 b	0.02 ± 0.00 a	0.42 ± 0.15 b	7.14 ± 0.39 a
Irrigation Regime (IR)
WW	0.04 ± 0.00 a	0.11 ± 0.02 a	1.51 ± 0.28 a	0.02 ± 0.00 a	0.04 ± 0.01 a	0.12 ± 0.02 a	0.01 ± 0.00 a	0.16 ± 0.02 b	0.21 ± 0.02 b	0.02 ± 0.00 a	0.31 ± 0.01 b	6.98 ± 1.39 a
WS	0.08 ± 0.04 a	0.11 ± 0.03 a	2.27 ± 0.54 a	0.03 ± 0.01 a	0.05 ± 0.02 a	0.17 ± 0.05 a	0.02 ± 0.01 a	0.29 ± 0.05 a	0.44 ± 0.20 a	0.04 ± 0.01 a	0.62 ± 0.06 a	10.06 ± 2.91 a
Significance Pr(>F)
T	0.957	0.496	0.668	0.985	0.451	0.466	0.194	0.449	0.055	0.199	0.391	0.072
IR	0.122	0.580	0.045 *	0.044 *	0.495	0.192	0.983	0.010 *	0.012 *	0.324	0.003 **	0.050
T × IR	0.110	0.581	0.192	0.299	0.243	0.241	0.305	0.000 ***	0.186	0.038 *	0.002 **	0.119

(pre-cluster closure 2023),

Table 5. Two-way ANOVA on 1 August 2023 for leaf biochemical compounds in V. vinifera L. processed with or without clinoptilolite (T/T), under irrigation (IR) regimens (WW, irrigation; WS, no irrigation). Reported value is data mean of each leaf biochemical parameter, assuming clinoptilolite (T/T) and IR as factors. Significance difference is reported as an alphabet letter (mean ± SE, n = 5); in the last three rows, significance Pr(>F) is noted (Significance codes: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). Abbreviations: procyanidin B1 (PB1), epigallocatechin (Epigc), caftaric acid (Caft), c-coutaric acid (cCout), t-coutaric acid (tCout), fertaric acid (Fert), resveratrol 3-O-glucoside (Resv), myricetin (Myr), kaempferol (Kaem), isorhamnetin (Isorh), quercetin-3-O-galactoside+quercetin-3-O-rutinoside (Qga+r), quercetin-3-O-glucoside+quercetin-3-O-glucoronide (Qgl+glc).

	1 August 2023
	PB1	Epigc	Caft	cCout	tCout	Fert	Resv	Myr	Kaem	Isorh	Qga+r	Qgl+glc
	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
Zeolite Treatment (T)
WWZeo	1.07 ± 0.11 b	0.99 ± 0.13 a	1.81 ± 0.05 b	0.13 ± 0.02 a	0.31 ± 0.03 c	1.03 ± 0.09 c	0.07 ± 0.01 c	1.81 ± 0.11 a	1.72 ± 0.43 b	0.58 ± 0.08 a	0.28 ± 0.02 b	9.58 ± 0.78 b
Zeo	1.37 ± 0.06 a	0.64 ± 0.15 b	2.05 ± 0.16 ab	0.14 ± 0.02 a	0.39 ± 0.01 b	1.36 ± 0.03 b	0.10 ± 0.01 b	1.65 ± 0.13 a	2.36 ± 0. 25 ab	0.48 ± 0.01 a	0.35 ± 0.03 ab	9.70 ± 0.21 b
Irrigation Regime (IR)
WW	1.35 ± 0.11 a	0.71 ± 0.26 ab	2.25 ± 0.22 a	0.12 ± 0.01 ab	0.46 ± 0.01 a	1.53 ± 0.07 a	0.08 ± 0.01 bc	1.96 ± 0.27 a	1.53 ± 0.16 b	0.57 ± 0.05 a	0.30 ± 0.02 b	9.58 ± 0.78 b
WS	1.42 ± 0.12 a	0.51 ± 0.05 b	2.11 ± 0.09 a	0.08 ± 0.01 b	0.40 ± 0.02 b	1.43 ± 0.06 ab	0.12 ± 0.01 a	1.65 ± 0.04 a	3.11 ± 1.32 a	0.55 ± 0.04 a	0.42 ± 0.08 a	11.20 ± 0.19 a
Significance Pr(>F)
T	0.006 **	0.027 *	0.003 **	0.006 **	0.000 ***	0.000 ***	0.026 *	0.401	0.462	0.244	0.171	0.021 *
IR	0.003 **	0.005 **	0.458	0.252	0.963	0.003 **	0.000 ***	0.011 *	0.008 **	0.036	0.001 *	0.006 **
T × IR	0.044 *	0.381	0.020 *	0.012 *	0.000 ***	0.000 ***	0.562	0.344	0.224	0.153	0.392	0.021 *

(veraison 2023), and

Table 6. Two-way ANOVA on 16 August 2023 for leaf biochemical compounds in V. vinifera L. processed with or without clinoptilolite (T/T), under irrigation (IR) regimens (WW, irrigation; WS, no irrigation). Reported value is data mean of each leaf biochemical parameter, assuming clinoptilolite (T/T) and IR as factors. Significance difference is reported as an alphabet letter (mean ± SE, n = 5); in the last three rows, significance Pr(>F) is noted (Significance codes: *** p ≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). Abbreviations: procyanidin B1 (PB1), epigallocatechin (Epigc), caftaric acid (Caft), c-coutaric acid (cCout), t-coutaric acid (tCout), fertaric acid (Fert), resveratrol 3-O-glucoside (Resv), myricetin (Myr), kaempferol (Kaem), isorhamnetin (Isorh), quercetin-3-O-galactoside+quercetin-3-O-rutinoside (Qga+r), quercetin-3-O-glucoside+quercetin-3-O-glucoronide (Qgl+glc).

	16 August 2023
	PB1	Epigc	Caft	cCout	tCout	Fert	Resv	Myr	Kaem	Isorh	Qga+r	Qgl+glc
	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g	mg/g
Zeolite Treatment (T)
WWZeo	1.51 ± 0.25 a	0.41 ± 0.08 a	1.23 ± 0.17 b	0.11 ± 0.01 b	0.26 ± 0.02 b	0.68 ± 0.09 c	0.06 ± 0.01 a	0.96 ± 0.14 c	1.27 ± 0.33 b	0.36 ± 0.02 b	0.24 ± 0.05 b	6.88 ± 1.19 a
Zeo	1.70 ± 0.71 a	0.53 ± 0.15 a	1.29 ± 0.06 b	0.17 ± 0.06 ab	0.27 ± 0.02 b	0.93 ± 0.10 bc	0.09 ± 0.04 a	1.28 ± 0.11 b	1.84 ± 0.09 a	0.69 ± 0.22 ab	0.28 ± 0.03 ab	7.43 ± 0.16 ab
Irrigation Regime (IR)
WW	1.48 ± 0.16 a	0.46 ± 0.09 a	1.34 ± 0.09 ab	0.12 ± 0.02 b	0.40 ± 0.06 a	1.25 ± 0.12 a	0.06 ± 0.02 a	1.44 ± 0.17 ab	1.66 ± 0.18 ab	0.94 ± 0.16 a	0.27 ± 0.04 ab	7.89 ± 0.13 ab
WS	1.86 ± 0.12 a	0.52 ± 0.07 a	1.58 ± 0.13 a	0.20 ± 0.01 a	0.34 ± 0.07 ab	1.07 ± 0.24 ab	0.10 ± 0.03 a	1.68 ± 0.15 a	2.01 ± 0.29 a	0.95 ± 0.26 a	0.34 ± 0.01 a	8.60 ± 0.41 a
Significance Pr(>F)
T	0.750	0.781	0.007 **	0.348	0.000 ***	0.000 ***	0.764	0.000 ***	0.039 *	0.000 ***	0.038 *	0.003 **
IR	0.162	0.118	0.030 *	0.000 ***	0.408	0.631	0.043 *	0.001 **	0.001 **	0.098	0.027 *	0.065
T × IR	0.641	0.574	0.199	0.585	0.194	0.012 *	0.769	0.576	0.377	0.111	0.543	0.804

(maturation 2023).

Overall, no significant differences of note were found for procyanidin B1, and epigallocatechin. In contrast, quercetin and kaempferol showed clear trends as water stress progressed in both seasons. At veraison 2022 (DOY 209), the following increases in quercetin-3-O-galactoside+quercetin-3-O-rutinoside were found +71.79%, +45.65%, and +55.81% in stressed plants compared to WWZeo, Zeo, and WW, respectively. Furthermore, at the same stage, the following increases in quercetin-3-O-glucoside+quercetin-3-O-glucoronide were seen +41.69%, +28.81%, and +36.87% in stressed plants compared to WWZeo, Zeo, and WW, respectively. As regards the veraison of 2023, the increases recorded for Qga+r and Qgl+glc were +50.00%, +20.00%, +40.00%, and +16.91%, +15.46%, +16.91%. In the pre-cluster sampling conducted in 2023, the majority of the evaluated parameters did not exhibit statistically significant variations, with the exception of myricetin, kaempferol, and Qga+r, which showed lower values in plants’ leaves subjected to a water regime and/or soil applications of clinoptilolite. During 2022 vintage, t-coutaric acid at DOY 184, c-coutaric acid at DOY 209, t-coutaric acid, and isorhamnetin at DOY 228 were unaffected by any of the factors underrating. In addition to what has already been reported for 2023, isorhamnetin was not exerted influence by treatments even at the DOY 213 sampling. Interestingly, the treatments led to changes in the resveratrol content in WWZeo and Zeo leaves likened to the stressed ones. Focusing on veraison time (DOY 209 2022 and DOY 213 2023), WS plants showed the following percentage bumps up +43.75%, +35.29%, +109.00%, compared to WWZeo, Zeo, and WW in 2022 and the following +71.42%, +20.00%, +50.00% in 2023.

3.5. Principal Component Analysis

To give an overview of the trial, Figure 7 presents the analysis of the main components with the following parameters: proline, lutein, zeaxanthin, βcarotene, and the two chlorophylls.

Figure 7 provides a synthesized overview of leaf biochemical parameters associated with water stress, highlighting the predominant interrelationships among these markers. The PCA relationship proclaimed an explicit separation between WS treatment and WWZeo/Zeo/WW. All the components involved played a significant role in the subdivision into ellipses (i.e., correlations between variables and factors on F1 and F2).

The PCA performed on the leaf biochemical traits reveals clear patterns of separation among the treatments based on multivariate variation. The first principal component (Dim1) explains 57.9% of the total variance, while the second component (Dim2) accounts for 16.4%, for a cumulative variance of 74.3%.

The score plot shows distinct clustering of the WS (Water Stress) samples (orange circles) on the right side of the plot, clearly separated along Dim1 from the other treatments (WW, WWZeo, Zeo), which are more overlapped in the central-left region of the plot. This indicates that Dim1 is the main driver of the treatment differentiation, especially distinguishing the water-stressed plants without zeolite (WS) from the other conditions.

According to the loading vectors and correlation coefficients (table inset), the variables most strongly associated with Dim1 are: β-carotene (r = −0.788), lutein (r = −0.747), chlorophyll a (r = 0.847), chlorophyll b (r = 0.809). These compounds drive the major variance and thus the separation of WS samples, which show higher accumulation of proline, zeaxanthin, and carotenoids, and lower levels of chlorophylls, compared to well-watered or zeolite-treated plants.

Dim2, which explains a smaller portion of variance (16.4%), appears to contribute less to treatment separation but still helps distinguish subtle differences within the well-watered and zeolite-treated groups. Among the variables, lutein (r = 0.582) and β-carotene (r = 0.503) also contribute moderately to this axis.

The multivariate effect of treatment was statistically validated via PERMANOVA (adonis2, Euclidean method, 999 permutations), which revealed a significant group effect (F = 6.75, R² = 0.126, p = 0.001). Importantly, a test of multivariate dispersion (betadisper) confirmed no significant differences in group dispersion (p = 0.941), supporting the conclusion that the observed separation is due to treatment effects rather than variance heterogeneity.

Overall, the PCA highlights how water stress, especially without mitigation (WS), induces a pronounced shift in leaf biochemical composition, while zeolite application appears to buffer these changes, maintaining a biochemical profile more similar to well-watered plants.

In addition, at veraison, the averages analyzed were 264.36 ± 41.63 WWZeo, 334.93 ± 5.45 WW, 371.30 ± 10.35 Zeo, 478.15 ± 53.77 WS (lutein 2022); 230.01 ± 56.55 WWZeo, 234.15 ± 90.31 WW, 257.17 ± 44.93 Zeo, 286.06 ± 67.48 WS (lutein 2023); 22.37 ± 8.14 WWZeo, 25.00 ± 10.17 WW, 41.35 ± 5.21 Zeo, 64.08 ± 11.91 WS (zeaxanthin 2022); 13.01 ± 3.65 WWZeo, 18.10 ± 0.62 WW, 18.13 ± 1.29 Zeo, 24.95 ± 3.47 WS (zeaxanthin 2023).

4. Discussion

This study investigated the impact of drought stress on secondary metabolite profiles and selected ecophysiological traits in Vitis vinifera L. leaves, intending to compare the plant responses induced by four distinct agronomic management strategies. Particular attention was given to elucidating the potential modulatory effects of zeolite—often referred to as the “magic rock”—on metabolic pathways, aiming to provide scientifically grounded and innovative insights into its application in viticulture.

In our research, in WS grapevines, nearly severe drought stress was reached during 2022 veraison (gs = 56.80 mmol/m²s) and during 2023 ripening (gs = 52.20 mmol/m²s). The answer of a plant to a water stress can be subdivided into three steps: absence of stress (stomatal conductance greater than 150 mmol/m²s), modest (stomatal conductance between 50 and 150 mmol/m²s), and acute drought stress (stomatal conductance lower than 50 mmol/m²s) [58]. However, we hypothesize a disablement of ATP synthesis and a slowdown in ribulose 1,5-bisphosphate activity in WS plants already in pre-cluster closure in 2022 (at gs = 111.4 mmol/m²s), as demonstrated by Flexas and Medrano [59]. During the 2022 summer, the event of intense and prolonged heat waves from fruit set to maturation joined with water stress (max air temperatures to thresholds higher than 35.0 °C) certainly had an impact on the fluorescence of chlorophyll by affecting the optimal range temperature of a C3 plant. Thermal stress is presumed to disrupt the functional integrity of photosystem II by impairing both the plastoquinone electron acceptor complex and the oxygen-evolving complex on the donor side. This disruption likely leads to the inactivation of key stromal enzymes involved in the Calvin–Benson cycle, ultimately resulting in an overaccumulation of reactive oxygen species and enhanced oxidative damage in WS plants [60]. Under this stress association (39.4 °C max air temperature and −2.09 MPa stem water potential), the steady-state level of the D1 protein (D1p, thought of as the extremely sensitive protein to photosystem II stress), as demonstrated by Balfagón et al. [61], was probably decreasing, suggesting that photodamage to photosystem II could surpass biosynthesis and fixing of D1p. Moreover, in the aforementioned year, the confirmed photodamage was accompanied by a drop in photosystem II activity (Fv/Fm) and the incapability of WS grapevines to regain this task following recovery from unfavorable circumstances (i.e., WS Fv/Fm at veraison = 0.68 and WS Fv/Fm at maturation = 0.66). This is also confirmed by Zhou et al. [62], who found degeneration on the chloroplast ultrastructure of Solanum lycopersicum pots after drought + heat combination for four/six days (lower quantum yield of PSII). On the contrary, the addition of clinoptilolite improved the ability of the soil to remain hydrated for long periods and, in general, made progress in the nutrient status of clay-based root zones, especially the selective storage of NH⁺⁴ and K⁺ cations [63]. In fact, during the pre-cluster closure and ripening periods, both the stomatal conductance and the Fv/Fm ratio of plants treated with zeolite alone were largely comparable to those of irrigated plants and plants irrigated with zeolite, confirming the ability of this mineral (applied in purity) to dehydrate and hydrate following rainfall events reversibly [64]. This property certainly had a positive influence on soil-plant water relations [65] by triggering biochemical reactions in plants, that will be discussed below. A positive effect of zeolite (10 t/ha) on the physiologic characteristics of Brassica napus was also found [66].

Maintaining a leaf temperature in treated plants within the risk thresholds (below 40 °C) may have certainly influenced sugar metabolism avoiding reductions in leaf starch content and not unbalancing the sucrose–starch balance. In fact, it was found that shrinkage in triose phosphate utilization ability via sucrose synthesis cut down the total carbohydrate levels in leaves with temperatures above 40 °C of Moros alba (i.e., uridine diphosphate sucrose synthase alteration) [67]. With water reservoir problems and leaves temperature at 40 °C in Cineraria maritima L., Capsicum annuum L., and Catharanthus roseus L., a depletion of stomatal conductance and net photosynthetic rate occurred [68].

The belief in zeolite’s property as a never-ending water reservoir was confirmed by the trend in stem water potentials. This mineral facilitated quick rewetting and lateral water broadcasting around the root zone [69]. In fact, it was seen that soil zeolitic benefit enriched the water disposability of plants by 50.0% [70] and water-holding capacity by 46.0% over the control [71]. This feature clearly helped to avoid photoinhibition damage in the treated plants. Imperishable photoinhibition can occur at RWC between 80% and 50%, even at leaf water potential underneath –1.5 MPa [59]. Stem Ψ, a non-transpiring leaf measurement, represents the most sensitive criterion for grapevine moderate or severe water deficit [72]. The reduction of drought stress generated by these aluminosilicates was described by other authors on several species, for example, Zea mays L. [73], Oryza sativa L. [74], Phaseolus vulgaris L. [75], Phaseolus lanatus L. [76], Satureja hortensis L. [77], and Salvia officinalis L. [78]. Zeolite and irrigation prevented the treated plants from reaching early severe water stress. In fact, on 20 July 2022 (DOY 201) and 8 August 2023 (DOY 220), WS plants recorded stem water potentials of −1.59 MPa and −1.60 MPa, respectively. A severe drought stress on grapevines will negatively affect photosynthetic status, reduce sugar storage and aromatic substances, and, if protracted, will trim impressively the vine lifetime [79]. Even during the first critical moment of the season when the water potential dropped in the WS treatment (3 July, 2022 and 2023), the Zeo treatment performed its mitigating capacity to the best of its ability, reaching the same level as the WWZeo and WW treatments (no significant differences among these three treatments); WS in fact recorded a negative +10.63% stem Ψ and +56.00% stem Ψ compared to Zeo in the two years. The hydrophilicity of the zeolite (12 Å pore diameter) and its release capacity (evaporation of water) after heating explain how the plants could benefit from a greater water-holding capacity [80].

Concerning flavonols, quercetin derivatives exhibited a tendency to accumulate under drought stress conditions. For example, in 2022, quercetin-3-O-galactoside + quercetin-3-O-rutinoside concentration in WS treatment embarked to intensify at the beginning of the stress interval to reach its maximum of 0.67 mg/g at veraison. At the same time, quercetin-3-O-glucoside + quercetin-3-O-glucoronide reached its maximum of 10.06 mg/g at veraison in WS treatment. In the same way, in Chrysanthemum morifolium L. [81], quercetin concentration was found to increase by 1.14 in response to water stress. Modifications in phenolic compound amounts during stress may be correlated to reactive oxygen species production owing to the role of flavonols in plants guardianship against these chemical molecules [82]. This functionality is associated with the specific hydroxylation pattern of flavonoid molecules [83]. Flavonoids featuring an ortho-dihydroxy configuration on the B-ring—such as quercetin—tend to accumulate more extensively compared to those bearing a mono-hydroxyl substitution, and exhibit enhanced superoxide radical scavenging capacity [84]. Studying the chloroplastic antioxidant system showed that quercetin and kaempferol (as treatments against arsenic toxicity on Triticum aestivum L.) made greater the activity of superoxide dismutase, peroxide reductases, and L-ascorbate peroxidase, inducing the ascorbate–glutathione cycle [85]. On potted Calendula officinalis L., quercetin was raised in mild stress condition (59.11 mg/g d.w. at the fruiting phase) [86]. In confirmation of what has been said, well-watered Hypericum brasiliense L. pot grown at 12 °C night/18 °C day temperatures showed the lowest quercetin 3-glucoside and quercetin 3-glucuronide content [87]. Furthermore, in addition to growth in quercetin, increases in kaempferol in Apocynum venetum L. under abiotic stress were found, as in our experiment [88].

Our study could also suggest that high temperatures merged with drought regimes could increase the content of resveratrol in grapevine leaves. In fact, resveratrol fluctuated from 0.18–0.19 mg/g in zeolite treatments (Zeo and WWZeo) to 0.37 mg/g in WS vines at 2022 ripening and from 0.07 mg/g in zeolite treatment (WWZeo) to 0.12 mg/g in WS vines in 2023 veraison. This interesting data is upheld by the work of Chung [89], who detected fluctuations from 2.96 to 38.87 μg/g between unstressed and stressed plants in Rehmannia glutinosa L.

From the further pool of biochemical analyses carried out on the leaves, analyzed by PCA, it is clear that the behavior of plants treated with zeolite alone is halfway between irrigated plants and plants irrigated with zeolite. Zeo showed a significantly different attitude from the WS treatment, highlighting how the magic rock is able to allow plants an excellent equilibrium between plants and the external environment. Applications of zeolite and/or supplemental irrigation were associated with a decline in the accumulation of typical drought-induced stress markers such as proline, zeaxanthin, and lutein, suggesting a mitigation of oxidative and osmotic stress. Concurrently, treatments led to a general increase in chlorophyll a and b concentrations, indicative of improved photosynthetic performance and maintenance of pigment stability under stress-alleviated conditions. These results suggest that zeolite, either alone or in combination with water supply, may contribute to enhancing plant physiological resilience by modulating key metabolic pathways involved in stress adaptation. This is in line with the experiment carried out by Palliotti [90]. They found a reduction in chlorophyl a and b under drought conditions in Sangiovese cv (but non-expressed on a dry weight basis), +22/25% of lutein, and a higher violaxanthin + zeaxanthin + antheraxanthin pool. Proline is synthetized from either glutamate or ornithine pathways, and as was proved, by a predominance in the glutamate pathway in case of osmotic stress situation. In Nicotiana tabacum L. crop, when P5CS enzyme was overexpressed, the plant synthetized around 15 times more proline than the ctrl ones showing tolerance to water shortage [91]. This highlights how, also in our case, this osmolyte was produced to a greater extent in the case of water stress of the plant (WS vines) to achieve osmotic self-protection in the leaf tissues, probably by alleviating cytoplasmic acidosis and maintaining NADP+/NADPH relationship as functional thresholds from a metabolic point of view [92].

5. Conclusions

Discovering, analyzing, and finding a key to understanding the physiological and biochemical responses that Vitis vinifera undertakes against drought stress is of fundamental importance for this profitable crop. Today’s considerable climatic fluctuations force researchers and farmers to focus on this issue with solutions inclined to respect the ecosystem.

In this study, in addition to the clear and evident relationship that exists between water stress and changes in leaf secondary metabolites, the evolution and divergences of these compounds within plants treated with soil applications of zeolites are also shown. Stem water potential, chlorophyll fluorescence, kaempferol, quercetins, proline, and xanthophylls were influenced by zeolite treatments, showing differences when compared to plants kept under abiotic stress without adjuvants (i.e., irrigations). It is interesting to note how the pool of metabolites was often unchanged for plants treated with zeolite and for plants treated with water only, thus elevating this mineral to a “stress reliever”.

6. Future Perspectives

The present findings highlight the promising role of soil-applied zeolite as a sustainable tool to mitigate drought-induced stress in grapevines. Nonetheless, further investigations are required to better understand the long-term implications and potential limitations of its use under diverse pedoclimatic conditions and across different grapevine cultivars. Future research should aim to clarify the molecular signaling pathways modulated by zeolite during water stress, with particular attention to hormone regulation, osmotic adjustment, and antioxidant defense systems. Moreover, the interaction of zeolite with soil microbiota and its potential influence on root exudation and rhizosphere dynamics remain largely unexplored.

From a viticultural management perspective, integrating zeolite application with other sustainable practices—such as deficit irrigation strategies, organic amendments, and canopy microclimate optimization—may enhance resilience to climate change. Multiyear trials and field-scale validations will be essential to confirm the reproducibility of the physiological and biochemical responses observed in this study. In this context, zeolite application could represent a practical and ecological tool to meet the EU’s goals of reducing input dependency and increasing agricultural resilience by 2030.