Application and Effect of Micropeptide miPEP164c on Flavonoid Pathways and Phenolic Profiles in Grapevine “Vinhão” Cultivar

Abstract

Climate change increasingly challenges viticulture, demanding innovative and sustainable strategies to preserve grapevine productivity and grape quality. MicroRNA-encoded peptides (miPEPs) have emerged as natural regulators of gene expression, providing a novel mechanism for fine-tuning plant metabolism. Here, we evaluated whether exogenous application of miPEP164c, previously shown to repress VviMYBPA1 in vitro, can modulate flavonoid pathways in field-grown grapevines (Vitis vinifera L. cv. Vinhão). Grape clusters were sprayed with 1 µM miPEP164c before and during véraison, and molecular, biochemical, and metabolomic analyses were performed at harvest. miPEP164c treatment significantly upregulated pre-miR164c transcripts, leading to post-transcriptional silencing of VviMYBPA1 and strong downregulation of the proanthocyanidin-related genes VviLAR1, VviLAR2, and VviANR. Correspondingly, LAR and ANR activities were reduced by up to 75%, and total proanthocyanidin content decreased by nearly 30%. Metabolomic profiling showed reduced flavan-3-ols and moderate shifts in phenolic acids and stilbenoids, while anthocyanins increased slightly. Overall, miPEP164c reprogrammed flavonoid metabolism under vineyard conditions, selectively lowering tannin biosynthesis without affecting other key phenolics. These findings establish miPEPs as promising biostimulants for precise modulation of grape berry composition, offering new tools for urgently needed sustainable and precision viticulture and improved wine quality under climate change and the increasing environmental challenges it poses.

1. Introduction

The quality and yield of grape berries are strongly influenced by grapevine adaptability to changing environmental conditions [1,2,3]. A crucial aspect in berry development and stress resilience is the specialized metabolism, which plays a pivotal role in determining the quality of the berries through the production of a diverse array of phenolic compounds, the most abundant class of specialized metabolites in grapevines [4,5,6]. The phenolic compounds of the grape are divided into two different classes: nonflavonoid (hydroxybenzoic acids, hydroxycinnamic acids, volatile phenols, and stilbenes) and flavonoid compounds (flavones, flavonols, flavanones, flavan-3-ols, and anthocyanins) [3]. Besides their contribution to wine color, aroma, texture, and stability, phenolic compounds play key protective roles against biotic and abiotic stresses in plants [7].

Transcription factors (TFs) serve as key regulators that integrate external environmental signals to coordinate the complex network of biochemical pathways governing specialized metabolism across diverse plant species. Several TF families are involved in plant development and physiological processes, such as defense, metabolism, and stress response. Among these, the MYB TF family has been described across various plant species as a major regulator of phenolic and flavonoid synthesis [8,9]. In grapevines, various TFs have been identified as either positive regulators (activators) or negative regulators (repressors) of specific branches of the flavonoid pathway. For instance, the anthocyanin-related TFs MYBA1/A2 and the proanthocyanidin-related TFs MYBPA1/A2 function as activators of anthocyanin and proanthocyanidin synthesis, respectively [10,11]. Conversely, MYBC2-L1 plays a negative regulatory role in both anthocyanin and proanthocyanidin biosynthesis by competing with these transcription activators [12]. Therefore, the coordinated interplay between activators and repressors exerts significant control over the biosynthesis of specialized metabolites, both in response to environmental stimuli and to fulfill the plant’s intrinsic metabolic needs during the different developmental stages [13]. Understanding the intricate mechanisms by which transcription factors modulate specialized metabolism in grapevines is essential for improving grape quality, nutritional value, and stress resilience.

Specialized metabolic pathways are also regulated post-transcriptionally by microRNAs and their encoded peptides (miPEPs). These miPEPs offer a promising strategy to modulate plant secondary metabolism and enhance resilience against environmental stresses intensified by climate change. These small peptides, translated from non-mature microRNA precursors, enhance the transcription and accumulation of their corresponding primary miRNA, leading to an increased downregulation of the miRNA-targeted genes [14]. Therefore, they play crucial regulatory roles in plant development, stress responses, and specialized metabolite biosynthesis. For instance, in Medicago truncatula, overexpression of miPEP171b induced miR171b accumulation, associated with lateral root formation, resulting in significant changes in root development [14]. The exogenous application of miPEP172c in Glycine max promoted nodule formation, boosting nitrogen fixation and therefore reducing the need for nitrogen fertilizers [15]. In Arabidopsis thaliana, miPEP858 modulated the expression of genes involved in plant growth and development [16]. Similarly, miPEP171d1 identified in V. vinifera promoted adventitious root formation, offering of promising approach to improve clonal propagation in grapevines [17]. More recently, treatments with miPEP172b and miPEP3635b increased cold tolerance in grape plantlets, enhancing cold stress resistance [18].

The discovery of new bio-sustainable, safe, and cost-effective molecules is increasingly important in the current context of climate change, with the consequent urgent need to develop and implement adaptive strategies to mitigate adverse effects on vineyard productivity and grape quality. Such biostimulants can be applied exogenously to modulate grapevine metabolism and improve productivity and/or berry cluster quality [19]. By targeting key components of specialized metabolic pathways, miPEPs can fine-tune the biosynthesis of bioactive compounds such as phenolics and flavonoids, which are essential for plant adaptation to changing environmental conditions. Moreover, they can modulate plant defense responses by enhancing the production of metabolites involved in biotic stress resistance, such as stilbenes, glucosinolates, and alkaloids, which possess insecticidal or antimicrobial properties [20,21].

Therefore, by modulating metabolic pathways, miPEPs offer a versatile mechanism for optimizing plant performance under stress conditions, enhancing crop yield, quality, and resilience. They also offer a sustainable alternative to chemical pesticides by promoting natural pest resistance, particularly in vineyards. Ongoing research into miPEP-mediated regulation of specialized metabolism holds great promise for the development of innovative biotechnological strategies to improve crop adaptability and sustainability in a changing climate scenario.

In a previous study, we identified miPEP164c and demonstrated that, in suspension-cultured grape berry cells (Gamay Freaux cv.), miPEP164c treatment significantly downregulated the proanthocyanidin biosynthetic pathway by repressing transcription activator VviMYBPA1 through miRNA-mediated post-transcriptional silencing [6]. This resulted in a downregulation of key genes and enzymes of the proanthocyanidin biosynthetic pathway and a significant decrease in proanthocyanidin content. Simultaneously, miPEP164c indirectly increased anthocyanin biosynthesis, reflecting the competitive allocation of carbon between the anthocyanin and proanthocyanidin branches. The most significant changes induced by miPEP164c in grape berry cells were achieved at a concentration of 1 µM.

Thus, based on our previous findings, this study aimed to evaluate whether the exogenous application of 1 µM miPEP164c directly to grape berries in vineyards, before véraison (thus, before anthocyanin synthesis and accumulation), could replicate the effects observed in vitro, thereby evaluating its potential as a tool to enhance agronomic traits and grape resilience.

2. Materials and Methods

2.1. Vineyard Conditions, Treatment, and Sampling

Grape berry samples were harvested from a vineyard of “Vinhão”cv. located in the Portuguese DOC region of “Vinhos Verdes” with coordinates: 41°28′28″ N latitude, 8°34′59″ W longitude, 165 m altitude. The plants were planted from southwest to northeast, with a spacing of 2.2 m between rows and 1.0 m within the row. They were trained on a vertical shoot position trellis system and uniformly pruned using a unilateral Royat Cordon method.

Grapevine berry clusters of 21 plants distributed over three side-by-side vineyard rows (to ensure similar edaphoclimatic conditions) from each of three 0.25 ha plots randomly located throughout the vineyard were sprayed with an aqueous solution of 1 µM miPEP164c (with 0.05% Tween-20 as a surfactant for adhesion). In each of the three other plots of 0.25 ha, the clusters of 21 plants from three vineyard rows were sprayed with 0.05% Tween-20 as the control condition. Both control and miPEP164c-treated grapevines were healthy, cultivated under the same microclimate, and grown using standard cultural decisions as applied by commercial farmers. Precautions were adopted to prevent the applied solutions from reaching the soil. The first application was performed in the late green phase before the beginning of véraison (EL-32), and the second application was conducted during the véraison (EL-35) stage. At harvest time, three independent sets of berries (70–100 berries per set) were collected from each experimental condition (miPEP164c-treated and control), with the precaution of representing sun-exposed and non-sun-exposed parts of the clusters. Each set was obtained from each of the three experimental plots per condition, and each set (from each plot) represented a biological replicate. From each set, approximately half of the berries were ground into a fine powder under liquid nitrogen and stored at −80 °C. The remaining half of each set of berries was used for exocarp isolation, by gently separating the berry skin from the mesocarp, immediately grinding it to a fine powder, and then storing it at −80 °C. Both samples were then freeze-dried.

2.2. Solubilization of miPEP164c

After the in silico identification of miPEP164c [6], the micropeptide was obtained as 1 mg aliquots from Smart Bioscience. The solubilization process of this micropeptide followed the recommended guidelines provided by Smart Bioscience Peptide Solubility Guidelines. To solubilize the miPEP, each aliquot was dissolved in 1 mL of pure water, resulting in a final concentration of 1 mg mL⁻¹. Subsequently, the solubilized miPEP was sterilized by filtration to ensure its purity before further use. The specific sequence of miPEP164c was as follows: miPEP164c—MEKQGTCITSSCTTNQ.

2.3. Anthocyanin Quantification

The quantification of anthocyanins was carried out according to the method established by Conde et al. [19]. Anthocyanins were extracted from 100 mg of samples from each experimental condition with 1 mL of 90% methanol. After 30 min of vigorous shaking, the suspensions were centrifuged at 18,000× g for 20 min. One hundred µL of each supernatant was mixed with 900 µL of 25 mM KCl at pH 1.0. Absorbance readings were taken at 520 nm and 700 nm using a Shimadzu UV-160A spectrophotometer (Kyoto, Japan). Total anthocyanin quantification was expressed in terms of cyanidin-3-glucoside equivalents using the formula:

$$[\mathrm{Total} \mathrm{anthocyanins}] \left(\mathrm{mg}/\mathrm{g} \mathrm{DW}\right) = {\displaystyle \frac{({\mathrm{A}}_{520} - {\mathrm{A}}_{700}) \times \mathrm{MW} \times \mathrm{DF} \times 1000}{\epsilon \times 1}}$$

where MW represents the molecular weight of cyanidin-3-glucoside (449.2 g mol⁻¹), DF signifies the dilution factor, and ε denotes the molar extinction coefficient of cyanidin-3-glucoside (26,900 M⁻¹ cm⁻¹). The resulting concentration was subsequently expressed per milligram of dry weight (DW) of the cellular material used.

2.4. Proanthocyanidin Quantification

Proanthocyanidins were measured using the DMACA assay, based on the protocol optimized by Wallace and Giusti [22]. For proanthocyanidin extraction, 100 mg of samples from each experimental condition was treated with 1 mL of 90% methanol, followed by 30 min of vigorous shaking and centrifugation at 18,000× g for 15 min. The DMAC reagent (4-dimethylaminocinnamaldehyde) was freshly prepared by dissolving 2% (w/v) DMAC in a 1:1 mixture of methanol and 6N H2SO4 (v/v), and the solution was kept protected from light due to DMAC’s sensitivity. The assay mixture included 1.175 mL of methanol and 50 µL of the 2% DMAC reagent, with 10 µL of each sample added to initiate the reaction. After incubation for 15 min at room temperature under low-light conditions, the absorbance was measured at 640 nm using a Shimadzu UV-160A spectrophotometer. A fresh standard curve was generated for each assay using (+)-catechin and DMAC, with concentrations ranging from 50 to 500 µg mL⁻¹. The proanthocyanidin content was expressed as catechin equivalents per gram of DW.

2.5. Total Phenolic Quantification

The concentration of total phenolics was quantified by the Folin-Ciocalteu colorimetric method as described in Conde et al. [19], with slight modifications. Total phenolics were extracted from 20 mg of samples from each experimental condition in 750 μL of 90% (v/v) methanol. The homogenates were vigorously shaken for 30 min and subsequently centrifuged at 18,000× g for 20 min. Twenty μL of each supernatant was added to 1.58 mL of deionized water and 100 μL of Folin reagent, shaken, and then incubated for 5 min in the dark before adding 300 μL of 2 M sodium carbonate. After 2 h of incubation in the dark, the absorbances were measured at 765 nm (Shimadzu UV-160A spectrophotometer, Kyoto, Japan). Total phenolic concentrations were determined using a gallic acid calibration curve and represented as gallic acid equivalent (GAE).

2.6. Protein Extraction

Protein extraction was performed following the method described by Badim et al. [23]. Grape berry cells were mixed with an extraction buffer at a 1:2 (v/v) ratio of cellular powder to buffer. The buffer consisted of 50 mM Tris-HCl (pH 8.9), 5 mM MgCl2, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM dithiothreitol (DTT), 0.1% (v/v) Triton X-100, and 1% (w/v) polyvinylpolypyrrolidone (PVPP). The mixtures were vortexed vigorously for 20 min and then centrifuged at 18,000× g for 20 min at 4 °C. The supernatants were kept on ice and used for subsequent enzymatic assays. Total protein concentrations were determined, with bovine serum albumin serving as the standard.

2.7. Enzymatic Activity Assays

The activity of UDP-Glucose-3-O Glucosyltransferase (UFGT) was assessed using the method of Lister et al. [24], with modifications by Conde et al. [19]. The reaction mixture consisted of 300 mM Tris-HCl buffer (pH 8), 200 µL of enzyme extract, 1 mM UDP-glucose, and 1 mM quercetin as the substrate (saturating concentration), in a final volume of 500 µL. Each mixture was incubated in the dark with gentle agitation for 30 min. Absorbance at 350 nm was measured before (t = 0) and after (t = 30) the incubation by diluting 100 µL of the reaction mixture with 900 µL of Tris-HCl buffer, allowing for monitoring of quercetin 3-glucoside production (ε = 21,877 M⁻¹ cm⁻¹).

Leucoanthocyanidin reductase (LAR) activity was determined by spectrophotometrically tracking the conversion of dihydroquercetin to (+)-catechin, based on the method of Gagné et al. [25] with some modifications. The reaction mixture contained 1.7 mL of 0.1 M Tris-HCl buffer (pH 7.5), 300 µL of protein extract, 2 µL of 100 mM NADPH, and 1 µL of dihydroquercetin (10 mg/mL in DMSO), which initiated the reaction. (+)-Catechin production (ε = 10,233 M⁻¹ cm⁻¹) was monitored at 280 nm for 30 min.

The activity of anthocyanidin reductase (ANR) was measured following the procedure of Zhang et al. [26]. The assay mixture included 1.5 mL of 0.1 M PBS buffer (pH 6.5), 100 µL of enzyme extract, 1 mM ascorbic acid, 0.07 mM cyanidin chloride, and 1 mM NADPH to initiate the reaction. The reaction was tracked by measuring the oxidation rate of NADPH (ε = 6.22 mM⁻¹ cm⁻¹) at 340 nm over 20 min at 45 °C. A control experiment, without cyanidin chloride, was conducted under the same conditions. All enzyme activities were measured using a Shimadzu UV-160A spectrophotometer (Kyoto, Japan).

2.8. Metabolomics Analysis of Specialized Metabolome by Ultra-Performance Liquid Chromatography Coupled to Mass Spectrometry (UPLC-MS)

The extraction of metabolites from freeze-dried complete grape berry and exocarp samples was performed, as in [19], using 1.5 mL of 80% (v/v) methanol per 25 mg of dry weight, with three replicates for each condition. Following maceration and 30 min of sonication, samples were incubated overnight at 4 °C in the dark. The samples were then centrifuged at 18,000× g for 10 min to collect the supernatants. Ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS) was conducted using an ACQUITY UPLC system integrated with a photodiode array detector and a Xevo TQD mass spectrometer (Waters, Milford, MA, USA), equipped with an electrospray ionization source controlled by Masslynx 4.2 software. Analyte separation was achieved using a Waters Acquity HSS T3 C18 column (150 × 2.1 mm, 1.8 μm) at a flow rate of 0.4 mL/min and a temperature of 55 °C. The injection volume was 5 μL. The mobile phase consisted of solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid in acetonitrile). Chromatographic separation was performed, as in [19], over an 18-min linear gradient from 5% to 50% (v/v) solvent B. MS detection was conducted in both positive and negative modes, with a capillary voltage of 3000 V and sample cone voltages of 30 and 50 V. Relative quantification was determined for the compounds as described in Supplementary Table S1.

2.9. RNA Extraction and cDNA Synthesis

Total RNA was extracted following the method outlined by Reid et al. [27], with additional purification steps using the GRS Total Plant RNA extraction kit. Approximately 200 mg of each sample was used for RNA extraction. The extraction buffer was customized and contained 2% (w/v) cetyltrimethylammonium bromide (CTAB), 2% (w/v) polyvinylpyrrolidone (PVP K-30), 300 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, and 40 mM dithiothreitol (DTT). After RNA extraction, DNase I treatment was performed directly on the column using Thermo Scientific™ DNase I. cDNA (Waltham, MA, USA) synthesis was then carried out using 1 µg of total RNA and the Xpert cDNA Synthesis Mastermix Kit (Grisp, Porto, Portugal), following the manufacturer’s protocol. The RNA concentration and purity were determined using a Nanodrop spectrophotometer, and RNA integrity was verified via electrophoresis on a 1% agarose gel stained with SYBR Safe (Invitrogen™, Life Technologies, Carlsbad, CA, USA).

2.10. Transcriptional Analyses by Real-Time qPCR

Quantitative real-time PCR (qPCR) was conducted using Xpert Fast SYBR Blue (Grisp, Porto, Portugal) on a CFX96 Real-Time Detection System (Bio-Rad, Hercules, CA, USA). Each reaction contained 1 µL of cDNA in a final volume of 10 µL. Primer pairs specific to each target gene are listed in Supplementary Table S2. A melting curve analysis was performed to ensure the specificity of the amplification. The reference genes VvACT1 (actin) and VvGAPDH (glyceraldehyde-3-phosphate dehydrogenase) were selected based on their proven stability and suitability for normalization in grapevine qPCR assays [27]. Three independent runs were conducted for each experimental condition, with triplicate reactions per run. Gene expression was normalized against the average expression of the reference genes using the Pfaffl method [28], and data were analyzed with Bio-Rad CFX Manager software, version 3.1.

2.11. Statistical Analyses and Data Presentation

Statistical analysis was performed using Student’s t-test in Prism version 9 (GraphPad Software, Inc., San Diego, CA, USA). Data are presented as mean ± standard deviation (SD). Statistical significance between conditions was indicated by asterisks: * for p < 0.05, ** for p < 0.01, *** for p < 0.001, and **** for p < 0.0001. At least three biological replicates were included for each experimental condition. Principal component analysis (PCA) and heatmap of metabolomic data were performed in R software version 4.1.0 using the built-in functions, and its output visualization was achieved with Factoextra version 1.0.5. The heatmap was constructed using heatmaply (v1.3.0) using log2 fold change (miPEP164c/control).

3. Results

Following the application of a 1-µM miPEP164c spraying solution on field-grown grape berries at the late-green phase (EL-32) and at the beginning of véraison (EL-35), a significant upregulation was observed in the transcript levels of pre-miR164c, the precursor form of miR164c, increasing by 68% in whole berries and 88% in the exocarp extracted from treated grape berries, as depicted in Figure 1A. The expression of VviMYBPA1, the in silico predicted target of miPEP164c, was indeed downregulated both in whole berries and in the exocarp of grape berries subjected to miPEP164c treatment, with a significant decrease of 48% and 30%, respectively, compared to non-treated samples, as demonstrated in Figure 1B.

The expression levels of both VviLAR1 and VviLAR2 were downregulated in grape berries and on grape exocarps following miPEP164c treatment. VvLAR1 transcript abundance significantly decreased by 58% in grape berries and 25% in grape exocarps, while VviLAR2 was downregulated by 42% in grape exocarps, with no significant changes in grape berries detected (Figure 2A,B). Concordantly, the overall LAR enzyme activity (V_max) was strongly downregulated with a decrease of 54% in grape berries and 75% in treated grape berry exocarps, as demonstrated in Figure 2C.

The transcript abundance of VviANR (a well-described target of transcription activator VviMYBPA1, together with VviLAR1/2) was also negatively affected, decreasing by 45% in grape berries and 43% in grape exocarps (Figure 3A). Similarly, the enzyme activity of ANR was strongly downregulated by 69% and 84% in grape berries and grape exocarps, respectively, as demonstrated in Figure 3B.

In accordance with the transcriptional and biochemical analysis, the total content of proanthocyanidins exhibited a significant reduction of 22% and 28% in whole berries and in the exocarp, respectively, compared to the non-treated samples (Figure 4).

Anthocyanin-related transcription activators MYBA1 and MYBA2 were also studied. As depicted in Figure 5, after miPEP164c treatment, VviMYBA1 transcript levels were only affected in the grape exocarp, with a reduction of 39%, while VviMYBA2 was not significantly affected in both samples tested.

Similarly, VviUFGT1, the gene encoding the key and final enzyme in anthocyanin synthesis, was also downregulated only in the exocarps of treated grape berries, displaying a decrease of 21% when compared to non-treated exocarps (Figure 6A). A tendency was also observed in the enzyme activity of UFGT, which was slightly downregulated in the exocarps of treated berries, decreasing by 19% when compared to non-treated berry exocarps. A slight increase in UFGT activity was observed in whole berries, although with no statistical significance (Figure 6B). However, the concentration of anthocyanins in whole berries increased by 30%, while in the grape exocarps they were reduced by 7%, in comparison to non-treated berries and exocarps (Figure 6C).

Flavonoid structural genes VviDFR and VviLDOX, leading to both anthocyanin and proanthocyanidin synthesis, were not significantly affected by miPEP164c treatment (Figure 7). VviMATE1, responsible for the transport of proanthocyanidins and anthocyanin-acylglucosides, was slightly downregulated in whole berries, decreasing by 39% when compared to non-treated grape berries (Figure 7A). VviABCC1 belongs to the ATP-binding cassette (ABC) family and transports glycosylated anthocyanins into the vacuole in a glutathione-dependent manner. This transporter was significantly upregulated in both grape berries and grape exocarps, with an increase of two-fold and 41% in transcript abundance, respectively (Figure 7B). However, VviGST4 encoding for a protein that promotes the stabilization of anthocyanins during transport to the vacuole by promoting the conjugation of anthocyanins and glutathione, protecting them from oxidation, was not affected by miPEP164c treatment (Figure 7C).

Modulation of Specialized Metabolites Induced by miPEP164c Exogenous Application in Grape Berry Clusters—An UPLC-MS-Based Metabolomics Analysis

Through a comprehensive UPLC-MS-based metabolomics investigation of miPEP164c-treated whole berries and exocarps, we successfully identified a diverse array of specialized metabolites. Specifically, our analysis revealed the presence of five phenolic acids, six stilbenoids, 10 flavonols, 13 flavan-3-ols, and 15 anthocyanins. A Principal Component Analysis (PCA), where a clear separation of the samples is observed, is represented in the Supplementary Material (Figure S1). The concentrations of the total phenolics are depicted in Figure S2. The alterations in the relative abundances of the identified metabolites are visually represented in the heatmap presented in Figure 8. In whole grape berries, there was an overall strong downregulation in flavan-3-ols quantities, with a significant decrease in all flavan-3-ols except epigallocatechin, whose content was not altered by miPEP164c treatment. Although there was a slight decrease observed in the quantities of some anthocyanins in whole berries, none were statistically significant. Regarding other compounds, in the stilbenoid group, only E-ε-viniferin was downregulated, and in the flavonol group, out of 10 flavonols, only myricetin-glucoside was upregulated. In the exocarps of treated grape berries, levels of phenolic acids, particularly gallic, coutaric, and fertaric acid, were increased. Regarding the content of flavan-3-ols, only eight out of 13 were detected, and only procyanidin B1 and epicatechin were significantly downregulated. Epigallocatechin was upregulated, and the other five were not influenced by miPEP164c treatment in the exocarps. Regarding anthocyanin content, there was an overall significant downregulation of these compounds in the exocarps treated, except for peonidin, which was upregulated. Flavonols were also significantly downregulated in treated exocarps, except for monogalloyl glucose laricitrin, which was upregulated. Finally, stilbenoids piceatannol and t-resveratrol were also significantly downregulated in the exocarps. Overall, in a way that is important to the overall quality of grape wine, as further discussed in the Discussion section, miPEP164c treatment modulated grape berry secondary metabolism, downregulating most flavan-3-ols and specific stilbenoids, and preserving anthocyanin composition, while increasing phenolic acids and selectively upregulating compounds such as peonidin and epigallocatechin.

4. Discussion

We previously stated that miRNA-encoded peptides (miPEPs) are a class of endogenous regulatory molecules with the potential to fine-tune gene expression through amplification of their corresponding miRNA pathways, thereby offering a conceptually “precision” alternative to conventional biostimulants.

The present work directly tested that conceptual framework in vineyard conditions. Building on our earlier in vitro evidence that miPEP164c increases pre-miR164c accumulation and promotes the post-transcriptional downregulation of the proanthocyanidin (PA)-related transcriptional activator VviMYBPA1, thereby repressing LAR/ANR expression and decreasing PA accumulation, we hypothesized whether the same regulatory process occurred in planta when miPEP164c is applied exogenously to berry clusters. Indeed, the field results support the central mechanistic premise that miPEP164c application could increase pre-miR164c transcript levels in both whole berries and exocarp, simultaneously with decreased VviMYBPA1 expression, reduced expression and activity of key PA enzymes (LAR and ANR), and a consistent reduction in total proanthocyanidins.

4.1. In Planta Application of miPEP164c on Grape Berries Enhances Pre-miR164c Expression, Leading to Reduced Proanthocyanidin Biosynthesis

The exogenous application of a miPEP164c spraying solution to grape berry clusters upregulated the precursor form of miR164c (pre-miR164c) in both whole berries and exocarps, confirming the effectiveness of miPEP164c treatment in promoting the transcription of its corresponding pre-miRNA and ultimately increasing the accumulation of mature miR164c.

As anticipated from the downregulation of VviMYBPA1, miPEP164c treatment led to a significant reduction in proanthocyanidin content in both tissues (Figure 4). This observation is consistent with the downregulation of VviLAR1 and VviANR expression and enzyme activities observed in Figure 2 and Figure 3, which are essential for the biosynthesis of catechins and epicatechins monomers, the key building blocks of proanthocyanidins. Also, VviMATE1, described to have a role in the transport of proanthocyanidins, was slightly downregulated in whole berry samples treated with miPEP164c (Figure 7C). These results confirm the regulatory role of miPEP164c in inducing the miRNA-mediated downregulation of this PA-related transcription activator.

Although epigallocatechin, unlike all other flavan-3-ols, was upregulated in the exocarps of treated berries, this may suggest selective regulation of flavan-3-ol biosynthesis under miPEP164c treatment. However, this observation can also be explained by the distinct hydroxylation pattern on the B-ring that distinguishes epigallocatechin from other flavan-3-ols, a structural feature determined earlier in the flavonoid pathway by the activity of flavonoid 3′,5′-hydroxylase (F3′5′H) (Figure 9).

This difference in structure may result in a preferential metabolic flux, where the downregulated ANR would consider hydroxylated precursors of high priority due to specific enzyme affinities. Also, the decreased activity of the downregulated LAR and ANR may result in higher levels of hydroxylated precursors due to the increase in carbon flow availability to redirect upstream to the F3′5′H pathway. Also of note, unlike most flavan-3-ols, which are more concentrated in the seeds of grape berries, epigallocatechin is found in higher levels in the skins [29]. This is due to the increased expression and activity of F3′5′H in the grape berry skins, which promotes the production of tri-hydroxylated flavan-3-ols like epigallocatechin. The reduction in proanthocyanidin content may have important implications for grape quality, particularly in terms of berry astringency and the mouthfeel of the resulting wine, as proanthocyanidins contribute to these sensory attributes [3]. Higher levels of proanthocyanidin (or tannins) can lead to overly astringent wines and overpower important aromatic components. Lower tannin content may contribute to a smoother and reduced drying sensation on the palate and enable some wines to be appreciated earlier by reducing the need for an extensive aging process to soften the tannins [30,31,32].

4.2. miPEP164c Exogenous Application Modulates Other Flavonoid Compounds in Grape Berries

The impact of miPEP164c on anthocyanin accumulation was less pronounced, with slight increases in whole berries and minor reductions in the exocarps. Accordingly, the transcript levels of VviMYBA1, a gene encoding an anthocyanin-specific transcription activator, were only downregulated in the exocarps, while VviMYBA2 expression remained unchanged in both tissues tested (Figure 5). This suggests that miPEP164c in planta application has a more nuanced effect on anthocyanin biosynthesis than the effect we observed when applied in vitro to grape berry cells, as reported in our previous study with miPEP164c. In a previous study, we observed that by downregulating the proanthocyanidin pathway, miPEP164c in vitro treatment also resulted in an upregulation of anthocyanin synthesis due to the competing nature of these two pathways [6]. This discrepancy observed could be due to several factors. For instance, the complexity of whole plant systems introduces additional layers of regulatory mechanisms that may be overcompensating any indirect effect of miPEP164c on the anthocyanin pathway, which may not be present in isolated grape berry cell cultures. The influence of other metabolites or competing pathways in planta, as well as plant-environment interactions, may induce the plant to counterbalance the miPEP164c effects. Also, the bioavailability and stability of miPEP164c may differ in planta, where it could be degraded or modified more rapidly compared to controlled in vitro conditions.

Additionally, through the UPLC-MS-based metabolomics analysis, we observed changes in other secondary metabolites upstream of the PA and anthocyanin branches, highlighting miPEP164c’s broader regulatory effect on phenolic and flavonoid metabolism. There was an increase in the concentration of some phenolic acids, such as gallic, coutaric, and fertaric acids, in the exocarp, while several flavonoids, such as kaempferol-3-O-glucoside, myrcetin glucoside, and quercetin derivatives (quercetin-3-O-glucuronide, quercetin-3-O-glucoside), were significantly decreased after miPEP164c treatment. The observed increase in phenolic acid content may represent a compensatory response to the reduction in flavonoid compounds, as phenolic acids are important precursors in the biosynthesis of various flavonoid metabolites. Many of these flavonoids play a crucial protective role in plants against abiotic stress due to their strong antioxidant properties and ability to scavenge ROS while also reducing the damage from UV exposure by absorbing UV radiation [33]. This shift in metabolic flow could reflect an adaptive strategy by the plant to maintain defense mechanisms, ensuring plant resilience against environmental stressors. Finally, stilbenoids piceatannol and E-resveratrol were also significantly downregulated in the exocarp, while E-ε-viniferin only decreased in whole berry samples. These changes in phenolic and flavonoid contents could also have important implications for grape quality and the overall sensory attributes of wine, since these compounds contribute to the stability of color, aroma, and astringency while also having key antioxidant properties [30].

Taken together, our results gain more significance when viewed in light of previous studies reporting miPEP-mediated regulation of grape and fruit metabolism in controlled conditions. In addition to our previous findings about the functional role of miPEP164c and miPEP166c, a functional analysis of vvi-miPEP171d1 showed that exogenous application of this miPEP promotes adventitious root formation in grapevine tissue culture by upregulating vvi-miR171d expression, and miPEP3635b increased cold tolerance in grape plantlets, showcasing that miPEPs can also influence developmental and abiotic stress processes [17,18]. Such studies, while conducted under in vitro or controlled conditions, provide important proof-of-concept for the versatility of miPEPs as endogenous regulators. In this context, the present study extends these findings by demonstrating that miPEP164c retains its regulatory specificity under field conditions, validating miPEPs as a feasible and precise class of nature-based molecules capable of modulating grape berry phenolic composition in agronomically realistic settings, as corroborated by Zhang et al. [34] (Figure 10).

5. Conclusions

Overall, miPEP164c application modulates key branches of the flavonoid pathway by selectively downregulating proanthocyanidin synthesis without interfering with other quality-related compounds such as anthocyanins. These findings highlight miPEPs as promising tools for fine-tuning grape berry specialized metabolism, enabling the targeted regulation of stress-related phenolic and flavonoid compounds with key protective roles in plant defense. Such modulation may enhance both grape resilience to environmental changes and the quality attributes of grape berries and resulting wines.

Although the current findings demonstrate the potential of miPEP164c to modulate grapevine’s specialized metabolism, further studies are needed to fully achieve its potential application as a practical biotechnological tool. Optimization of concentrations, timing, and frequency of miPEP applications, as well as exploring other delivery methods of miPEPs in planta, is essential to fully achieve its potential as a regulatory mechanism that can be explored by viticulturists worldwide. Another positive aspect of a miPEP-based approach is its alignment with some of the most innovative environmentally safe and sustainable agricultural practices that are currently being studied, such as, for instance, grass covers in vineyards [35]. As miPEPs are naturally occurring regulatory peptides, their use minimizes the risk of long-term ecological harm. Moreover, their rapid degradation allows for transient, short-term applications that can be employed in response to environmental changes or biotic stresses, thereby increasing plant resilience. Filling these knowledge gaps will enable the transition of miPEPs applications from experimental research to practical biotechnological tools for modern agriculture, particularly in viticulture or other fruit crops, where targeted modulation of specialized metabolism can improve yield and quality.