4. Discussion
The effects of Ca on plant nutrition and yield and firmness of many fruits have been widely studied [
21,
22]. However, the effects of Ca as a biostimulant have received little attention. Our results demonstrated that the foliar application of CaCO
3-rich industrial residues, as a Ca source, on ‘Shiraz’ vines affected the performance of plants and the quality of grapes and their wine. This treatment increased the fruit yield, by increasing the weights of clusters, berries, and fruit skin and pulp. This effect has also been reported in several cultivars of table grapes (‘Crimson Seedless’, ‘Perlette’, and ‘Kings Ruby’) treated in preharvest with CaCl
2, causing increases in weight of bunches and individual berries [
20,
21,
22,
23]. CaCl
2 also increased the yield in pomegranates [
22]. Overall, the increases in the weights of clusters, berries, and fruit skin and pulp are mainly attributed to Ca due to the high content of this mineral element in the test solution. Similar effects have been reported for Ca in other grape varieties and plant foods (e.g., sugar beet, tomatoes, blueberries, etc.) presumably due to their actions on the promotion of the uptake and mobility of minerals in plants, the inhibition of cell-wall hydrolytic enzymes caused by the down-regulation of
PG1 and
PG2 gene expression, the promotion of sugar accumulation in fruits, and the Ca-mediated signaling that allows the expression of
VIT_02s0012g02190,
VIT_16s0039g02020, and
VIT_18s0072g00370 genes, which cause increases of cellulose content in fruit [
8,
13,
15]. Additionally, Ca could promote the expression of genes involved in fruit development and ripening, including the expansin genes
VIT_03s0038g03430 and
VIT_08s0007g00440, responsible for cell expansion and fruit growth, or the
FaCDPKs, gen of CDPKs involved in fruit development, as reported in grapes and other berries [
11,
13]. Ca could also either induce the biosynthesis of ABA, which is a phytohormone accelerating grape development or act as a stressing factor, such as ABA, inducing responses influencing fruit growth, as reported previously for ‘Cabernet Sauvignon’ grapes [
11,
24,
25,
26].
The treatment of vines with CaCO
3 did not affect the ripening rate of grapes, according to their values of TSS, pH, and TA. Similarly, the foliar application of CaCl
2 and ABA on pomegranate plants and vines, respectively, affected some quality attributes of fruit but SST, pH, and TA remained unaltered in fruit [
8,
22,
27]. However, the pH and TA of wines seemed to be slightly affected by the treatment of vines with CaCO
3. This effect might be related to other compositional changes in fruit induced by the treatment with CaCO
3, with these changes altering the performance of fermentative microorganisms and enzymes during fermentation and, consequently, retarding the stabilization of the content of organic acids (e.g., malic acid) in the must, as observed in ’Muscadine’ wines [
28].
The treatment with CaCO
3 also increased the firmness of grapes, as reported for other grape varieties (e.g., ‘El-Bayadi’ and ‘Vinhão’) cultivated in other regions and treated in preharvest with CaCl
2 [
10,
21]. This increase in fruit firmness might be attributed to the higher intracellular availability of Ca and its subsequent association with pectin chains, favoring cellular cohesion and fruit firmness [
29]. Although the increase in firmness has not been considered an important factor in wine grapes, Ca-mediated firmness could favor the prevention of diseases by decreasing the expression of cell wall hydrolytic enzymes and improving the resistance of the middle lamella to the action of these enzymes, as demonstrated in ‘Vinhão’ table grapes treated with CaCl
2 [
15]. This increase of firmness in CaCO
3-treated grapes could also influence the release of grape juice and phenolic compounds during the wine making process, causing higher content of phenolic compounds in wine. Similarly, the treatment of pears with CaCl
2 caused increases in fruit firmness and release of juice [
30].
Grapes from CaCO
3-treated vines showed a darker color, characterized by lower
L* and
b* values than those of fruit from untreated vines. Similarly, others have observed that the preharvest treatment of vines with ABA favored color development in ‘Crimson Seedless’ table grapes and fruit from some wine grape varieties (‘Yan’, ‘Cabernet Sauvignon’, ‘Malbec’, and ‘Merlot’) [
7,
8,
31,
32]. Thus, the effect of Ca on grape color could be a consequence of the Ca-mediated increase of anthocyanin biosynthesis [
13]. As expected, wines made with Ca-treated grapes showed higher color intensity and tonality, as compared to control wine. This effect of Ca resembles that of methyl jasmonate (MeJA) in ‘Tempranillo’ fruit and wine, which showed higher color values than those from untreated vines [
27]. MeJA acts as a stress factor in grapes, activating the phenylpropanoid pathway as a defense response and promoting the biosynthesis of anthocyanins and stilbenes [
33,
34]. Thus, the effect of CaCO
3 on the biosynthesis of phenolic compounds might be related to its effect as a stress factor and direct interaction with enzymes involved in the phenylpropanoid pathway, improving the color of grapes and wine [
13].
On the other hand, the treatment with CaCO
3 caused increases in the contents of total and individual phenolic compounds in grapes. The effect of Ca on these response variables has been previously reported for some fruits, including ‘Sweetheart’ and ‘Lapins’ cherries and strawberries treated in preharvest with CaCl
2 [
34,
35]. ‘Malbec’ grapes from ABA-treated vines also showed higher TPC than control fruit [
7]. Currently, there is no information about the effect of the preharvest treatment of vines with CaCO
3 on the content of total and individual phenolic compounds in aged wine. Our study demonstrated that the treatment of vines with CaCO
3 improved the content of these compounds in wine. This finding is supported by studies with ‘Yan73′, ‘Merlot’, and ‘Cabernet Sauvignon’ wines made with fruit from vines treated with ABA in preharvest, which showed a higher TPC than control wines [
8,
32].
In our study, grapes from CaCO
3-treated vines and their wine showed higher contents of malvidin-3-
O-glucoside and pelargonidin-3-
O-glucoside. Similar results have been observed in table grapes and strawberries from plants treated with CaCl
2 in preharvest and cherries treated with CaCl
2 in postharvest [
13,
34,
35]. ABA also produced similar effects in ‘Isabel’, ‘Manicure Finger’, and ‘Beihong’ grapes [
9,
35]. Interestingly, the CaCO
3-treated grapes contained pelargonidin 3-
O-glucoside, an anthocyanin that has been reported in ‘Cabernet Sauvignon’ and ‘Pinot Noir’ grapes from vineyards established at an altitude above 2000 m.a.s.l. [
36]. The positive effect of CaCO
3 on malvidin-3-
O-glucoside was lower in wine than in grapes, probably due to CaCO
3 inducing the synthesis of other polyphenols not identified that copigmented with this anthocyanin, decreasing the concentration of its free form. As stated above, there is no information about the effect of the preharvest application of Ca on the composition of phenolic compounds in aged wine. In our study, the treatment of grapes with CaCO
3 caused increases in the anthocyanin content in wine. This Ca-mediated effect was similar to that reported for ‘Yan73’, ‘Cabernet Sauvignon’, ‘Malbec’, ‘Tempranillo’, and ‘Graciano’ wines made with grapes from vines treated with ABA or MeJA in preharvest [
6,
32,
33]. Several mechanisms might be involved in the positive effect of CaCO
3 on the anthocyanin content in grapes and, consequently, in wines. Ca activates directly the biosynthesis of ethylene, ABA, and other phytohormones, which influence the biosynthesis of anthocyanins [
24]. However, it also could upregulate the expression of Ca sensors for CAMs (
VIT_08s0040g00470 and
VIT_14s0006g01400) and CDPKs (
VIT_04s0023g03420), causing increases in the expression of some genes involved in the synthesis of
phenylalanine ammonia-lyase (
PAL, 13 genes),
4-coumarate CoA ligase (
4CL, 1 gene),
UDP-glucose: flavonoid 3-O-glucosyltransferase (
UFGT, 4 genes), which play an important role on anthocyanin biosynthesis in grape skin [
13]. Ca, through CaMs, could also favor the transcription and expression of genes encoding for other enzymes involved on anthocyanin biosynthesis, including chalcone synthase, chalcone isomerase, stilbene synthase, flavanone 3-hydroxylase, flavonoid 3′-hydroxylase, flavonoid 3-
O-glucosyltransferase, dihydroflavonol 4 reductase and anthocyanin synthase, as reported in some fruits [
10,
13,
34,
37,
38]. Finally, Ca, through CaMs, could also regulate the expression of some families of transcription factors (R2R3 MYB TFs, bHLH TFs, etc.) for genes (
MdMYB308L, MdbHLH33, MdDFR, etc.) involved in anthocyanin biosynthesis, as reported in apples [
37,
39,
40,
41]. CaCO
3 also favored the biosynthesis of
trans-cinnamic acid in grapes, which is a precursor of anthocyanins [
1]. The increases in phenolic compounds in grapes and wines might also be a consequence of the CaCO
3-mediated increase of skin mass in grapes, with skin being the major accumulation site for anthocyanins [
15].
The treatment of vines with CaCO
3 favored the accumulation of some flavonols in wine, but they were not observed in grapes. Gil-Muñoz et al. [
42] did not observe increases of flavonols in grapes (‘Syrah’ and ‘Monastrell’) treated in preharvest with MeJA, however, the wines made with these grapes had higher contents of flavonols as compared to control wines. These results suggest that the alcohol in wine is needed to the release of flavonols from grape tissues during the wine making process [
43]. Quercetin was abundant in wine from CaCO
3-treated grapes. Others have also observed that the treatment of vines (‘Graciano’, ‘Yan 73’, and ‘Cabernet Sauvignon’) with MeJA or ABA caused increases of quercetin in wines [
32,
33]. This flavonol is highly valorized in wines because it associates with anthocyanins, favoring especially the copigmentation with malvidin and the stabilization of wine color [
44]. However, further research is needed to understand how CaCO
3 favored the accumulation of quercetin in the tested samples since the accumulation of this flavonol is mainly influenced by exposure of fruit to light, especially to UV-B light [
6,
43].
The treatment with CaCO
3 caused increases of flavan-3-ols in grapes and wines, including catechin, epicatechin, and the procyanidins B1 and B2. Similar increases of flavan-3-ols have been observed for ‘Yan 73’, ‘Cabernet Sauvignon’, ‘Merlot’, and ‘Monastrell’ grapes and wines from ABA- or MeJA-treated vines [
32,
42]. The flavan-3-ols accumulate in grape seeds [
2], and their release during the wine making process contributes to the organoleptic quality of the final product, especially on wine bitterness (monomeric forms of flavan-3-ols such as catechin and epicatechin) and astringency [
45]. These characteristics are essential to enhance some mouthfeels and the structure of wines [
8].
Grapes and wine from CaCO
3-treated vines showed a higher content of the stilbene resveratrol compared to grapes from untreated vines and their wine. The preharvest treatment of ‘Malbec’ vines with ABA and MeJA also caused increases of stilbene in fruit and wine [
6,
33]. However, MeJA seems to be more effective than ABA to induce the accumulation of stilbenoids in grapes due to it causing a higher activation of plant response to biotic stress [
27,
33]. Interestingly, CaCO
3 and CaCl
2 also increased the stilbenoid content in ‘Shiraz’ and ‘Vinhão’ grapes, allowing the inference that Ca and MeJA act similarly on stilbene biosynthesis [
14,
15]. The induction of the biosynthesis of this stilbene seems to be a response to stress factors and it has been attributed to CaMs, CDPKs, and CLBs proteins that are responsible for mechanisms of adaptability to biotic and abiotic stress in plants with the subsequent synthesis of phytohormones (e.g., ABA or MeJA) involved in this defense mechanism of plants [
11,
13,
24]. Such conditions may activate the expression of the enzyme stilbene synthase, as reported in ‘Beihong’ grapes treated with ABA [
35] and in ‘Vinhão’ grapes treated with CaCl
2 [
10].
The treatment with CaCO
3 favored the biosynthesis of
trans-cinnamic and caftaric acid in grapes. In contrast, Portu et al. [
33] did not find increases of phenolic acids in ‘Tempranillo’ and ‘Graciano’ grapes from vines treated with MeJA. Luan et al. [
32] did not find increases of hydroxycinnamic acids in ‘Yan73′ and ‘Cabernet Sauvignon’ wines made with ABA-treated grapes The high content of
trans-cinnamic acid found in grapes from CaCO
3-treated vines is beneficial from the enological point of view because some studies have demonstrated that this acid is the precursor of other phenolic compounds, including its derivatives [
43]. This increase in the content of phenolic compounds mediated by
trans-cinnamic acid favors the copigmentation of phenolic compounds with flavan-3-ols and the color stabilization of wine during aging [
8]. These beneficial effects might be potentiated by the high content of caffeic acid in wine from CaCO
3-treated vines because it prevents oxidation of wine during aging, favoring its stabilization [
46].
Overall, the increased content of total and individual phenolic compounds in fruit and wine from CaCO
3-treated vines could be attributed to the cascade of Ca-mediated signaling reactions to activate the expression of CaMs, CDPKs, and CLBs proteins, and other mechanisms indicated above. These proteins participate as sensors and transmitters in response to biotic or abiotic stress in plants, as seen in many plants under salinity or drought conditions [
3,
24]. This stress triggers the biosynthesis of ABA, among other phytohormones. ABA activates the expression of PAL and the subsequent synthesis of phenolic compounds [
10,
11]. Thus, CaCO
3 seems to act as a secondary messenger involved in the adaptability responses of vines to biotic and abiotic stress, promoting the synthesis of ABA or JA and, consequently, of phenolic compounds [
4,
13]. CaCO
3 could also induce the synthesis of other enzymes involved in the biosynthetic pathway of phenolic compounds [
13] or their precursors [
1]. The CaCO
3-mediated increase of skin mass in grapes could also be involved in the increases of phenolic compounds [
15]. These increases in the contents of phenolic compounds were probably responsible for the increases in AC in grapes and wine from Ca-treated vines. Similar increases in AC were observed in ‘Perlette’ grapes from CaCl
2-treated vines [
20]. The preharvest treatment of ‘Lapins’ and ‘Sweetheart’ cherries with CaCl
2 and ‘Cabernet Sauvignon’ and ‘Merlot’ grapes with ABA caused increases of AC in fruit and wines, respectively [
8,
12]. Our results demonstrated that CaCO
3, as other elicitors (e.g., ABA), is able to induce the accumulation of phenolic compounds in grapes and wine, causing increases in AC and, probably, in the activity of these foods in the prevention of some chronic degenerative diseases [
3].