1. Introduction
Zinc is the second-most-common transition metal in living organisms [
1], being essential for the metabolism of humans and crops. Zinc has several physiological functions, namely in enzyme kinetics, cell membrane integrity, control of oxy radicals, and synthesis of sugars and chlorophylls [
2,
3]. Regarding enzymes, Zn is a cofactor of carbonic anhydrase, carboxypeptidase, RNA polymerase, and Zn-superoxide dismutase [
2,
4]; these enzymes are involved in the proteosynthesis and metabolism of carbohydrates, lipids, and nucleic acids [
4]. Zinc ions are also involved in the transcription factor family—called zinc fingers, controlling the proliferation and differentiation of cells [
4].
Optimal growth of most crops requires Zn concentrations ranging between 15 and 20 mg kg
−1 DW [
1,
2]. Nevertheless, when the Zn threshold is surpassed, symptoms of toxicity can occur, such as decrease in roots and shoots growth, metabolism deviation, and oxidative damage [
5,
6]. Chlorosis develops in the younger leaves extending to the other leaves if the toxicity persists, and an inhibition of photosystems I and II can occur reversibly if not subjected to a constant stress [
6]. Interference with the ionic balance can also occur [
7]. Among different plant species, an approximate value for the threshold of Zn toxicity in leaves is about 300 μg/g
DW [
1].
Regarding human health, Zn is found in several tissues, such as the muscles, bones, liver, and brain (i.e., synaptic vesicles) [
8]. Moreover, Zn deficiency has a foreshadowed effect in the epidermal, nervous, gastrointestinal, skeletal, immune, and reproductive systems. Likewise, its deficiency triggers many human health problems, for instance an inhibition in growth, a weakened immunity system, and an increase in the risk of infections, disorders of the gastrointestinal or urinary tract, and cancer [
8,
9,
10].
According to estimates, 17.3% of the world’s population is at risk of inadequate Zn intake. Country-specific estimated prevalence of inadequate Zn intake was correlated negatively with the total energy, the Zn contents of the national food supply, and the percentual value of Zn derived from foods of animal origin and was correlated positively with the phytate:zinc molar ratio of the food supply [
11]. This inadequacy may be fairly common, particularly in Sub-Saharan Africa and South Asia, allowing inter-country comparisons and existing primarily where Zn deficiency is more acute.
In this context, biofortification programs of different crops mostly used in human consumption have been implemented worldwide. Regarding rice, four different Zn forms were applied as a foliar treatment on three cultivars under a field trial, and Zn bioavailability was assessed by in vitro digestion/Caco-2 cell model [
12]. It was observed that foliar Zn fertilization promote grain Zn concentration and Zn bioavailability among the rice cultivars, especially in the case of Zn-amino acid and ZnSO
4. On average, Zn-amino acid and ZnSO
4 increased Zn concentration in polished rice up to 24.04% and 22.47%, respectively. Furthermore, foliar Zn application could maintain grain yield and the protein and minerals (Fe and Ca) quality of the polished rice. [
12]. Moreover, through the construction of plant transformation vectors and transgenic indica rice plants, it was possible to reach approximately 30% of the estimated average requirement (EAR) of Fe and Zn in the human diet, without a yield reduction, when it is common to detect concentrations of approximately 2 μg g
−1 Fe and 16 μg g
−1 Zn in polished grains of different rice varieties. The HarvestPlus breeding programs for biofortified rice target 13 μg g
−1 Fe and 28 μg g
−1 Zn levels, c.a. 30% of the EAR [
13].
The biofortification of
Pisum sativum through Zn application in soils at concentrations of 4 and 8 mg ZnSO
4·7H
2O kg
−1 and foliar Zn application of two sprays of 0.25% or 0.5% (
w/v) ZnSO
4·7H
2O before flowering and at early grain-filling stage was studied. Foliar application prompted increases above 60 mg Zn kg
−1 in grain, while during the cooking process a decrease of
c.a. 30% in grain Zn concentration was noted [
14]. The
Triticum aestivum L. biofortification program at the International Maize and Wheat Improvement Center (CIMMYT) led to a partnership to breed competitive wheat varieties with 40% higher Zn concentration (>12 mg/kg) over the commercial varieties in the target regions of South Asia [
15,
16]. Biofortification of high-yielding maize varieties in Zn is of great importance to the health of those whose diets are mostly based on this staple crop, with the USA and China being the world’s biggest maize consumers. Clinical studies have so far indicated that genetically biofortified maize increased Zn absorption in human bodies [
17], which is quite relevant regarding the adequate daily Zn intake.
It is well-established that foliar spraying is the best approach to increase the level of Zn, as well as the levels of other nutrients, in edible plant tissues, when compared with soil fertilization [
12,
14,
18]. Plus, foliar spraying does not depend upon root-to-shoot translocation [
19,
20]. Nevertheless, some authors use a double fertilization through the foliar application of organic fertilizer, without significantly affecting the vine vigor and fruit quality of grapes, while impacting the ionome and phenolic compounds [
21]. Accordingly, Zn biofortification of grapes of
Vitis vinifera L. variety Fernão Pires through foliar spraying with ZnSO
4 and ZnO prompt this study, further motivation being to assess the physicochemical attributes of the fruits and winemaking.
4. Discussion
The effects of climatic conditions and soil type on grape ripening and wine quality in two Cabernet Sauvignon vineyards under the same climate, but on distinct soils, revealed that soil type is determinant in wine phenolic composition and tasting characteristics [
42]. In fact, the soil influences vine development and grape ripening through soil temperature, water supply, and mineral supply. Soil temperature has a significant effect on vine phenology, while a shortage in the water supply restricts shoot and berry growth, which is critical for reaching a suitable grape composition to produce high-quality red wines [
43]. In our case, the vineyard with variety Fernão Pires expands through a slight slope with low or moderate surface drainage (i.e., classes 1 and 2), with a homogeneous water distribution, but with a higher propensity for accumulation and/or infiltration of surface water in the SW–NE direction. Unfortunately, the current severe drought in mainland Portugal might well affect grape sugar, as noted by Van Leeuwen et al. [
44], although phenolic compounds and particularly anthocyanins could be increased in grape skins [
45].
Soil fertility and soil physical and chemical characteristics are of great importance regarding to grape quality. For example, the presence of soil carbonates in European vineyards probably leads to deficiencies in some nutrients such as Mn, reducing the availability to vines, and affecting the color in red grapes [
46]. In the same context, Bramley et al. [
47] claimed that the availability of Fe and Mn in adequate quantities is very important for producing high-quality wines, although emphasizing the importance of N fertilization, which is responsible for both a vine´s vigor and yield, when applied at moderate levels, since the excess of N slows down the process of maturation, producing juices with fewer sugars and phenolic compounds.
Our current levels of Mn in soil vineyards (299 mg kg
−1) are compared with other data from the Iberian Peninsula, in which higher levels were observed in the Castilla-La Mancha vineyards, despite the huge variation found in the soils, i.e., 380 mg kg
−1 ± 740 [
48]. The mean concentration in vineyards soils from northwest Romania was closer to ours i.e., 250 mg kg
−1 [
48]. Regarding Fe, the concentrations of both origins were almost identical—22 g kg
−1 and 21 g kg
−1 from Spain and Romania, respectively [
48,
49], while from the present study only 3.5 g kg
−1 was detected. Similar concentrations were also found between Ca levels in Romanian soils [
49] and the average value observed by us, i.e., 5.3 g kg
−1 and 5.4 g kg
−1, respectively.
Evaluation of total macro- and micronutrients in soils is not a good predictor of the concentration in the plants [
50], since the plant-available fraction is a function of soil characteristics, mainly the pH and organic matter. In this context, several authors use the available levels of N, P, and K, mainly, among other elements, for establishing relationships with the elemental composition of the grapes and wines [
51,
52]. For example, Mackenzie and Christy [
53], when studying the elemental composition of soil vineyards, observed that grape juice properties such as Baumé/Brix and titratable acidity are clearly correlated with several plant-available trace elements in the soil, mainly Ca, Sr, Ba, Pb, and Si. Nevertheless, some criticism exists about the multiple extraction-procedure methods to determine the available fraction [
54]. Despite this, different authors consider that the mineral composition pattern is transferred through the soil–wine system, and differences observed for soils are reflected in grape musts and wines, though not for all elements [
48,
49,
55]. In this framework, it must be emphasized that evaluation of soil macro- or micronutrients as soil quality indicators is not recommended in Australia, because current industry practice utilizes petiole analysis for macro- and micronutrient testing rather than soil tests, and negligible soil data are available [
56]. It is well-known that petiole sampling is useful for the posterior analysis and diagnosis of the nutritional state of vineyards at field level [
46].
As previously stated, soil chemical characteristics are of great importance in crop production and, particularly, the effect of soil pH on the availability of nutrients to grapevines. The optimum pH range (measured in water) for nutrient uptake is between 5.5 and 8 [
57], thus, our pH value of 6.85 falls within the ideal range mentioned above. Despite the lack of an adequate threshold of soil organic matter for viticulture, a level of 2% is recommended for Australian soils [
58], although its importance in maintaining soil structure varied vis a vis the type of soil vineyard. Levels of soil organic matter ranging between 1–1.59% are considered as a moderate rate [
58]. The levels observed by us in a previous work [
18] in the same region with two different grape varieties (Moscatel and Castelão) gave us 1.09–1.48%, which is in agreement with the current value observed—1.36%.
The Zn content of grape seeds collected from 50 different locations of Turkey was studied by inductively coupled plasma atomic emission spectrometry. The results show that the average levels varied between a minimum of 6.5 and a maximum of 25.6 mg kg
−1, although the majority of the concentrations range between 10 and 14 mg kg
−1, which encompasses 56% of the samples [
59]. As expected, our biofortified grape seeds exhibit higher Zn values, and our control grape seeds have a level close to the maximum referred above, i.e., 24.6 mg kg
−1.
In the current study, the Zn content of non-biofortified wine (control) is 0.98 mg L
−1, while the maximum average value was 1.56 mg L
−1 observed in the treatment with ZnSO
4, at a foliar spraying rate of 450 g ha
−1. Two different vineyards from the Douro wine district, Portugal, were studied regarding the multielement composition of wines. The first one was from a 10-year-old vineyard (monovarietal grapes) and was used to produce a red table wine, while the second one was from a 60–70-year-old vineyard (polyvarietal) and was used to produce a red fortified wine, similar to the world-famous Port wine [
52]. The authors concluded that the fortified wine has a Zn level of 1.0 mg L
−1, while the red table wine has only 0.43 mg L
−1. In Croatia, the contents of several selected metals in both red and white wine samples (n = 70) were collected from the continental (northeast) and Adriatic areas (near the Adriatic Sea) and were determined by total reflection X-ray analysis fluorescence spectrometry (TXRF). The levels of Zn range between 0.51–1.01 mg L
−1, although it must be stressed that grapes were not submitted to foliar Zn fertilization [
60]. All these values fell in the ranges frequently found in different wines for Zn, i.e., between 0.5 and 3.5 mg L
−1 [
61].
It was reported that Zn is involved in the biosynthesis of chlorophyll, and, under plant stress, the inhibition of the electron transport chain prevails [
10]. In the current study, after three foliar applications, no significant changes on chlorophyll a parameters occurred (Fv/Fm, Y(
NO), Y(
II), Y (
NPQ), qL, qN, and Fv′/Fm′) between treatments, indicating that the threshold of toxicity was not reached, despite our measuring almost 500 mg.kg
−1 in the leaves with the highest concentration of ZnSO
4 (900 g ha
−1), much above the limit of 300 mg.kg
−1, as indicated by [
1], which means that the latter value is an average indication and does not encompass, obviously, the hyperaccumulator Zn plants. However, during fruit development, a higher energy is necessary for biomass production compared to the final stage of the production cycle [
62], as it was observed in general for Y(
II) and qL, when comparing the two monitored dates (
Figure 1). Moreover, no significant variations for qN and Y(
NPQ) were noted, despite that the values tended to increase at the second assessment, which may be related to the production cycle. Indeed, in earlier stages, the photoprotective mechanism of the photosynthetic machinery is more active, as capacity was progressively lost while the berries grow [
61].
Considering the harvest time, vines are more susceptible to additional stresses [
63,
64], namely due to high temperature or drought that can lead to increased synthesis of reactive oxygen species (ROS) and, consequently, to a reduction in photosynthetic CO
2 fixation [
65,
66]. Y(
NPQ) and qN are related to a photoprotective mechanism that prevent photo-oxidative stress in plants, which means that the increase in these values in the second assessment indicates a higher need for energy dissipation, via the xanthophyll cycle, to minimize potential damage to the thylakoid membranes [
67]. After the third application, the content of Zn was higher in treated leaves, as expected, although at harvest part of the Zn load had been mobilized to the grape itself, where seed and skin are included (
Table 2). In fact, Zn is involved in grape development due to the synthesis of growth regulators and chlorophyll, although it is dependent on the ripening stage [
68].
According to Christensen [
69], Zn is very mobile and its solubility did not influence absorption after foliar spraying in vineyards—low-solubility neutral zinc and ZnO gave responses similar to the other fully soluble compounds, and, at the rates that are normally used, none of the compounds caused visible vine-foliage toxicity. In our case, the highest Zn leaf levels were observed with ZnSO
4, although, when the highest concentration was applied, the concentration in the seed and grape skin decreases. Conversely, the highest level of ZnO did not decrease in both the seeds and skin, despite the accumulation in the leaves being clearly lower. These apparent interesting results can be negatively influenced by the winemaking process (i.e., maceration, extraction, and solubilization during fermentation), where mineral losses occurred, which was confirmed in the present study. Depletion of some elements occurs over time, especially during alcoholic fermentation—for example, the precipitation of K and Ca as tartrate salts begins during alcoholic fermentation and continues during the aging period [
55].
Sucrose, glucose, and fructose are main components in grapes, with an important role in consumers’ acceptability, mostly due to the interaction among the sugar content, synthesis of organic acids and phenolics, sensory properties, alcohol concentration in winemaking, and aroma compounds [
70,
71,
72]. At harvest, the Zn-biofortified grapes of Fernão Pires with ZnSO
4 and ZnO do not present relevant variations of sucrose, glucose, and fructose contents. As sucrose is the final product of photosynthesis, our data further indicate the absence of inhibitory effects in Zn-treated vines. Additionally, the hydrolysis of sucrose, after being produced in leaf photosynthesis and transported through the phloem to the berry, determined the similar levels of the isomers of glucose and fructose. Besides, our data (
Table 3) agree with [
70,
72], where glucose and fructose present higher values compared to sucrose, and the amount of fructose was slightly superior to glucose. In general, grapes treated with ZnSO
4 and ZnO showed a tendency for a higher sugar content (except for ZnO 150 and ZnO 450 g ha
−1 for sucrose and glucose/fructose, respectively), which is an important quality parameter for winemaking, as it affects the fermentation process and alcohol contents [
73], although we must note that a high alcohol content can causes a gustatory disequilibrium affecting wine sensory perceptions, leading to unbalanced wines [
74].
The data from the total fatty acid (TFA’ levels from the current study clearly show that ZnSO
4 has a more pronounced effect on grapes than ZnO, and, in some cases, the levels are even lower than the control grapes (
Table 4), which may be important regarding the profile of the wine. The manipulation of TFA levels is a goal of some researchers through the use of exogenous compounds, such as abscisic acid and methyl jasmonate, in order to promote the enrichment of polyunsaturated fatty acids and/or monounsaturated fatty acids [
75], which was not our goal. Polyunsaturated fatty acids and phenolic compounds are important molecules in grapes, due to their role in the prevention of cardiovascular disease and antioxidant potential, respectively [
76]. Fatty acid composition in grapes of Fernão Pires (
Figure 2;
Table 4) revealed a higher presence of acid linoleic (C18:2), acid palmitic (C16:0), stearic acids (C18:0), and linolenic acid (C18:3), which have a potential influence in the aroma production during winemaking, as observed for the cultivar Cabernet Sauvignon grape [
77].
The colorimetric analyses also did not show visible changes (
Table 5), which indicates that Zn treatments (i.e., 150–900 g ha
−1 of ZnSO
4 and ZnO) did not influence the relative proportions of chlorophylls and carotenoids that determine the final white grape berry color [
78], as well as the color of the wine, which is an important aspect for consumers [
79]. Besides, the physical characteristics of Zn-treated grapes did not reveal an evident negative impact (
Table 6), which is of the utmost importance, since grapes’ size, appearance, texture, and sensory characteristics are important for commercialization [
80]. In fact, sensorially Zn-treated grapes were the most appreciable, pointing to an absence of an undesirable organoleptic response to ZnSO
4 and ZnO (
Figure 3).