Implications of exogenous allantoin in enhancing growth and development of Vitis vinifera L. cultivars under semi-arid condition

Abstract

Elucidating the physiological enhancement mechanisms of grapevines through biostimulant applications is essential for developing optimized viticulture practices in modern agriculture. However, comprehensive knowledge about how foliar allantoin supplementation modulates growth parameters, fruit quality, and secondary metabolite profiles across different cultivars remains limited. We investigated reproductive growth, photosynthetic capacity, and biochemical dynamics in Vitis vinifera L. cultivars Sultana and Merlot subjected to graduated allantoin concentrations (0, 10, 20, and 30 mM), followed by detailed morphological and metabolomic profiling. Data were analyzed using two-way ANOVA followed by LSD and Duncan’s tests (p < 0.01 and p < 0.05). Cultivar and allantoin application significantly influenced all measured parameters (p < 0.01), with notable interaction effects observed for panicle weight, berry development, and chlorophyll content among morphological traits, and phenolic compounds, anthocyanins, and carbohydrates among biochemical parameters. Allantoin-supplemented plants consistently demonstrated superior performance metrics, maintaining higher reproductive success (panicle weight increases up to 151% in Sultana), enhanced berry quality parameters (TSS improvement reaching 23.9 °Brix), and more robust photosynthetic capacity under optimal treatment compared to controls. Enhancement of secondary metabolite biosynthesis was also more pronounced in treated vines, with better accumulation of phenolic compounds, anthocyanins, and improved antioxidant status reflected by reduced MDA levels. Metabolic profiling revealed distinct cultivar-specific response patterns, with Sultana demonstrating superior responsiveness in reproductive and vegetative parameters, while Merlot showed enhanced secondary metabolite accumulation and stress tolerance indicators. Correlation analysis explained strong relationships between morphological traits (berry length-berry weight r = 0.94), demonstrating coordinated developmental responses to allantoin treatment. We conclude that allantoin foliar application significantly enhances grapevine productivity and berry quality through comprehensive improvement of growth, physiological, and biochemical pathways. These findings have direct applications for viticulture management, particularly in semi-arid regions where optimizing yield and fruit quality through bio stimulant application can enhance vineyard productivity without extensive modifications to existing practices. The differential responses between cultivars provide insights for variety-specific bio stimulant strategies, while the quantitative enhancement profiles identified offer valuable parameters for optimizing vineyard management practices under diverse environmental conditions.

Introduction

Among the diverse array of grape cultivars, Sultana stands out as particularly noteworthy for raisin and table grape production. Indeed, this seedless white variety occupies the largest cultivation area globally, with extensive production in the United States, Australia, Turkey, Greece, and notably Iran¹. It is well established that over half of the world’s raisin production originates from this single cultivar, which demonstrates optimal performance in warm, semi-arid climates². Morphologically, Sultana produces large panicles with medium-sized, oval berries displaying white to light golden coloration and thin skin structure³. Furthermore, this mid-season cultivar typically reaches harvest maturity in September and finds application in raisin processing, syrup production, and fresh consumption². On the other hand, the Merlot cultivar traces its origins to the prestigious Bordeaux regions of France and has achieved widespread global cultivation due to its exceptional versatility for wine production⁴. This prominent red grape variety is particularly valued for high-quality wine production and demonstrates notable adaptability to diverse growing environments. The berries are characterized by their dark red to black coloration, thin skin structure, and distinctive sweet, fruity flavor profile⁵. Notably, the elevated concentrations of anthocyanins and polyphenolic compounds in Merlot grapes serve as critical determinants of wine color development and aromatic complexity, contributing to the wine’s characteristic deep hue and sophisticated sensory attributes⁶.

At the physiological level, it is well established that plants respond to environmental stress through the activation of key enzymatic systems, which accelerate the accumulation of specific metabolites, thereby enhancing their tolerance to adverse conditions^7,15. Although numerous stress-responsive molecules have been identified and characterized, the specific role of allantoin in mediating plant stress responses has only recently begun to be understood^8,9,12. Allantoin, a nitrogen-rich compound derived from purine catabolism, plays a fundamental role in nitrogen transport and recycling mechanisms within plant systems⁹. In plant tissues, allantoin and its hydrolysis product allantoate, collectively known as ureides, function as vital nitrogen carriers characterized by their optimal 1:1 carbon-to-nitrogen ratio and efficient long-distance transport capabilities from source to sink tissues¹⁰. Notably, ureide metabolic pathways demonstrate superior efficiency for nitrogen mobilization compared to amide transfer mechanisms, as allantoin catabolism yields four ammonium ions per molecule¹¹. This metabolic efficiency enables plants to utilize allantoin as a primary nitrogen source during periods of temporary soil nitrogen deficiency, while the potential for foliar application has been increasingly highlighted as a strategy to enhance crop yield and quality in grapevine cultivation¹².

In viticultural applications, exogenous allantoin supplementation has been demonstrated to improve critical yield parameters including panicle size, berry number, and individual fruit weight¹³. Moreover, allantoin promotes anthocyanin accumulation in cellular tissues and grape skins through the stimulation of ABA signaling pathways and enhancement of anthocyanin biosynthetic routes. While certain field studies report minimal direct impacts on panicle weight or berry size, allantoin consistently accelerates skin pigmentation processes and elevates anthocyanin concentrations. Beyond grape cultivation, allantoin facilitates recovery following nitrogen deficiency and is associated with the activation of jasmonic acid signaling pathways, which contributes to improved disease resistance and stress tolerance¹⁴. Additionally, allantoin supports comprehensive plant antioxidant defense systems, encompassing both enzymatic and non-enzymatic components that collectively reduce oxidative stress damage under drought or salinity conditions^12,15. Despite extensive research demonstrating that allantoin enhances crop yield, fruit quality, and stress resilience, there still remains a significant gap in our current understanding of the underlying biochemical and molecular mechanisms involved in these processes. Although allantoin’s beneficial effects are widely recognized in various crop systems, its practical application as a yield-enhancing agent has been insufficiently explored in grapevine cultivation, particularly under semi-arid conditions. Moreover, few studies have comprehensively analyzed the differential responses of key cultivars such as Sultana and Merlot to exogenous allantoin applications using integrated physiological and biochemical approaches. While previous studies demonstrated allantoin’s effects on anthocyanin accumulation during grape berry ripening, no previous study has systematically evaluated dose-dependent responses across multiple morphological, physiological, and biochemical parameters in contrasting cultivars under field conditions representative of semi-arid viticulture^9,15. The semi-arid conditions prevalent in many viticultural regions present unique challenges characterized by limited water availability, high evapotranspiration rates, and temperature fluctuations that significantly impact vine physiology and fruit development. Under these environmental constraints, grapevines experience increased oxidative stress and altered nitrogen metabolism, which directly affect yield components and berry quality parameters^12,15. While previous studies have examined allantoin’s effects under controlled conditions or in other crop systems, the specific physiological and biochemical responses of commercially important cultivars such as Sultana and Merlot to graduated allantoin concentrations under semi-arid viticulture remain poorly characterized. This knowledge gap is particularly critical given the increasing adoption of biostimulant-based approaches in sustainable viticulture and the need for cultivar-specific management strategies that can optimize both productivity and fruit quality under water-limited conditions.

Therefore, this study aims to: (i) evaluate the individual and interactive effects of graduated allantoin concentrations on comprehensive yield and quality parameters in Sultana and Merlot grape cultivars grown under semi-arid field conditions, (ii) investigate the modulatory role of allantoin in enhancing grapevine physiological performance, photosynthetic capacity, and stress tolerance indicators under the chronic water limitation characteristic of semi-arid viticulture, and (iii) characterize the specific biochemical and metabolic changes in grape berries and plant tissues in response to different allantoin treatment regimes, with particular emphasis on antioxidant systems and secondary metabolite accumulation.

Materials and methods

Experimental design and plant material

The experiment was conducted during the 2023 growing season at the research vineyard of the University of Maragheh, Maragheh, Iran (37°23′N, 46°16′E, elevation 1,485 m). The experimental site experiences mean annual precipitation of 320 mm, predominantly occurring during winter and spring months, with mean annual temperature of 12.5 °C, summer maximum temperatures reaching 35–38 °C, and relative humidity ranging from 35 to 45% during the growing season. These semi-arid conditions impose significant physiological constraints on grapevine cultivation, characterized by limited water availability during critical growth periods, high vapor pressure deficit (typically 2.5-4.0 kPa during midday in summer), and elevated evapotranspiration rates exceeding 6 mm day⁻¹ during summer months. During the experimental period (April-September 2023), total precipitation was 78 mm, concentrated in April and May, while reference evapotranspiration (ET₀) totaled approximately 850 mm. Soil moisture content in the root zone (0–60 cm depth) was monitored bi-weekly using a theta probe soil moisture sensor, with values ranging from 12 to 18% volumetric water content, which corresponds to moderate water deficit conditions for grapevines. Under such conditions, vines experience periodic water stress that can limit vegetative growth, reduce berry size, and alter secondary metabolite profiles. Irrigation was applied according to standard regional practice (deficit irrigation strategy providing approximately 50% of crop evapotranspiration requirements), delivered through a drip irrigation system with emitters spaced 0.75 m apart along the vine row. The combination of high solar radiation (average 2,800 h annually, with daily photosynthetically active radiation reaching 1,800-2,200 µmol m⁻² s⁻¹ during summer) and moderate water deficit typically enhances anthocyanin and phenolic accumulation but may compromise yield without appropriate management interventions. The study was designed as a 2 × 4 factorial experiments based on a randomized complete block design with three replications. The two experimental factors were: (1) Cultivar (two levels: Sultana and Merlot), and (2) Allantoin concentration (four levels: 0, 10, 20, and 30 mM). This yielded eight treatment combinations (2 cultivars × 4 concentrations), with each combination replicated three times, resulting in 24 experimental units (vines) in total. Twenty-four uniform, healthy grapevines (Vitis vinifera L.) were selected as experimental material: 12 vines of cv. Sultana (white, seedless, table grape) and 12 vines of cv. Merlot (red, wine grape). All vines were 8 years old, grafted onto 41B rootstock, trained on a vertical trellis system with bilateral cordon, and spaced 2.5 m × 3.0 m. Each treatment combination (cultivar × allantoin concentration) was applied to three biological replicates, with each replicate consisting of one vine. Allantoin (≥ 98% purity, Sigma-Aldrich, CAS Number: 97-59-6) was applied as a foliar spray at four concentrations: 0 mM (control, distilled water only), 10 mM, 20 mM, and 30 mM. Foliar applications were performed at the fruit set stage (BBCH 71, when approximately 50% of flowers had set into small berries) and repeated 15 days later at the berry development stage (BBCH 75). At this phenological stage, berries are undergoing rapid cell division and are highly responsive to nitrogen supplementation, which can significantly influence final fruit size and quality parameters. The second application timing (BBCH 75) corresponds to the phase of intensive cell expansion and early sugar accumulation, where metabolic activity is maximal and nutrient demand is elevated. Applications were made in the early morning (06:00–08:00 h) using a hand-held pressure sprayer to ensure complete coverage of leaf surfaces until runoff, with approximately 500 mL of solution applied per vine. Throughout the experimental period, all treated vines were monitored weekly for potential phytotoxicity symptoms including leaf necrosis, chlorosis, burn symptoms, or premature leaf abscission. No visible phytotoxic effects were observed at any allantoin concentration (10, 20, or 30 mM) during the entire growing season, indicating good tolerance of both cultivars to the applied doses under the semi-arid field conditions of this study. A non-ionic surfactant (Tween-20, 0.05% v/v) was added to all solutions including controls to ensure uniform coverage. A non-ionic surfactant (Tween-20, 0.05% v/v) was added to all solutions including the 0 mM control to ensure uniform coverage and droplet adhesion. Since all treatments (including the control) received identical surfactant concentrations, any observed effects can be attributed specifically to allantoin rather than surfactant influence. This approach is consistent with standard practice in foliar biostimulant research where surfactants are maintained constant across all treatments to isolate the active compound’s effects. However, we acknowledge that the absence of a positive control (e.g., conventional urea application or commercial biostimulant) limits direct benchmarking of allantoin efficacy relative to established nitrogen supplementation practices. Future comparative studies incorporating standard nitrogen sources (urea, ammonium nitrate) and commercial biostimulant formulations would provide valuable context for evaluating the relative cost-effectiveness and performance advantages of allantoin in viticulture applications. Fully expanded, healthy leaves from the middle portion of shoots and mature berries at commercial harvest maturity (BBCH 89, approximately 120 days after full bloom for Sultana and 135 days for Merlot) were collected from each experimental vine. Samples were immediately placed on ice, rapidly transported to the laboratory within 30 min, wrapped in aluminum foil to prevent light exposure, frozen in liquid nitrogen, and stored at -80 °C until biochemical analysis.

Measurement of panicle and berry weight

To measure the weight of grape panicles and individual berries, mature fruits at the final stage of development were detached from the shoots. Their weights were determined using a precision scale with an accuracy of 0.01 g.

Measurement of fruit firmness

The firmness of the grape berries was measured using a digital penetrometer (FR-5120).

Measurement of total soluble solids (TSS)

The total soluble solids of grape berries were measured using a Brix refractometer (Erma, Tokyo, Japan).

Measurement of panicle and berry morphological characteristics

To measure the length of panicles and berries, as well as the length and width of leaves, three samples were randomly selected from each plant. Measurements were taken using a ruler and caliper, and the average values were recorded in cm. Berry diameter was measured using a caliper, and the diameters were recorded in mm. To assess leaf greenness in a non-destructive manner, a SPAD device can be employed. For this purpose, before sampling and harvesting, the leaf greenness was measured using a SPAD chlorophyll meter (Model SPAD-502 Plus, Japan). Specifically, three leaves were randomly selected from each plant, and their chlorophyll content was measured using the device. The mean value of these samples was then recorded.

Measurement of photosynthetic pigments

For the measurement of photosynthetic pigments, 0.2 g of the grapevine plant sample was powdered in a porcelain mortar using liquid nitrogen. Then, 20 mL of 80% acetone was added to digest the samples. The mixture was subsequently centrifuged at 6000 rpm for 10 min. The absorbance of the samples was read using a spectrophotometer at wavelengths of 663 nm for chlorophyll a, 645 nm for chlorophyll b, and 470 nm for carotenoids. Finally, the concentrations of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll were calculated using the following formulas and expressed as mg g^-1 FW of the sample¹⁶.

V = volume of the filtered solution (the supernatant obtained from centrifugation), A = absorbance at wavelengths of 663, 645, and 470 nm, W = fresh weight of the sample (in g).

Measurement of total soluble carbohydrates

In this method, 0.2 g of fresh leaf tissue were powdered using liquid nitrogen and thoroughly homogenized with 10 mL of 96% ethanol. The mixture was then incubated in a water bath at 80 °C for one hour. Subsequently, 1 mL of the obtained extract was mixed with 1 mL of 0.5% phenol and 5 mL of 98% sulfuric acid. The absorbance was measured using a spectrophotometer (Shimadzu, model UV 1800, Kyoto, Japan) at a wavelength of 483 nm. The total soluble carbohydrate concentration was determined based on a standard curve and expressed as mg g^-1 FW¹⁷.

Quantification and calibration procedures

For all spectrophotometric measurements, standard calibration curves were prepared using appropriate reference compounds. Gallic acid (Sigma-Aldrich, ≥ 98% purity) was used as the standard for total phenolic content determination, with concentrations ranging from 0 to 500 µg mL⁻¹ (r = 0.998). Anthocyanin content was expressed as cyanidin-3-glucoside equivalents using authentic cyanidin-3-glucoside standard (Sigma-Aldrich, ≥ 96% purity) with concentrations from 0 to 200 µg mL⁻¹ (r = 0.997). Total soluble carbohydrates were quantified using glucose as the standard (0–1000 µg mL⁻¹, r = 0.999). Bovine serum albumin (BSA, Sigma-Aldrich, ≥ 98% purity) was used for protein quantification (0–1000 µg mL⁻¹, r = 0.996), and proline content was determined using L-proline standard (0–100 µg mL⁻¹, r = 0.998). All calibration curves exhibited correlation coefficients (r) greater than 0.995, ensuring reliable quantification of the measured compounds.

Measurement of leaf and berry phenolic compounds

Initially, 0.2 g of plant sample were homogenized with 2 mL of 0.01% acidic methanol. The samples were then centrifuged at 12,000 rpm for 10 min at 4 °C. The reaction mixture was prepared by combining 20 µL of the extract, 1,590 µL of distilled water, 100 µL of Folin-Ciocalteu reagent, and 300 µL of 7.5% sodium carbonate. After incubating the reaction mixture for 2 h in the dark, the phenol content was measured using a spectrophotometer at 765 nm. The phenolic content was calculated based on a gallic acid standard curve¹⁸.

Measurement of leaf and fruit anthocyanins

To determine anthocyanin content, one gram of fresh plant tissue was taken and thoroughly ground in 10 mL of acidic methanol (methanol: hydrochloric acid, 99:1) using a mortar and pestle to obtain the extract. The resulting extract was homogenized and then centrifuged at room temperature at 6,000 rpm for 10 min. The absorbance of the supernatant was measured at 530 nm by a spectrophotometer, using acidic methanol as the blank¹⁹.

Measurement of total soluble protein

First, 2 mg of BSA (bovine serum albumin) was dissolved in 1 mL of distilled water. Protein standards with concentrations of 0.2, 0.4, 0.6, 0.8, and 1 mg/mL were then prepared, to each of which 100 µL of Bradford reagent was added. For example, to prepare the 0.2 mg/mL standard, 100 µL of BSA, 100 µL of Bradford reagent, and 800 µL of distilled water or potassium phosphate buffer were combined in a microtube. Next, the 1× Bradford reagent was prepared by dissolving 100 mL of 95% ethanol, 200 mL of phosphoric acid, and 350 mg of Coomassie Brilliant Blue dye completely, filtering it through filter paper, and storing it in a dark glass container at room temperature. Subsequently, 20 mL of the stock solution was diluted with distilled water to a final volume of 100 mL. Finally, 1,000 µL of the 1× Bradford reagent was added to 50 µL of the extracted sample, and, together with the standards, the absorbance was measured at 595 nm using a spectrophotometer²⁰.

Determination of proline content in leaf samples

Proline content was measured based on the reaction of amino acids with ninhydrin, in which proline forms a red formazan complex that dissolves in toluene and can be quantified spectrophotometrically. Briefly, 0.5 g of plant tissue was powdered in liquid nitrogen, mixed with 10 mL of 3% sulfosalicylic acid, and centrifuged at 10,000 rpm for 20 min at 4 °C. Two milliliters of the supernatant were combined with 2 mL of ninhydrin reagent and 2 mL of glacial acetic acid, then incubated in a hot water bath for 1 h and subsequently cooled on ice. Four milliliters of toluene were added, and the mixture was vortexed. The absorbance was measured at 520 nm using a spectrophotometer (UV-1800 Shimadzu, Japan), and proline concentration was calculated using a standard curve²¹.

Measurement of malondialdehyde (MDA) concentration

MDA, as a product of lipid peroxidation, was quantified according to the method of Heath, and Packer²². Briefly, 0.5 g of leaf tissue was homogenized in 1.5 mL of 0.1% TCA. The homogenate was centrifuged at 1,000 rpm for 10 min at 4 °C. Then, 0.5 mL of the supernatant was mixed with 1 mL of 0.1% (w/v) thiobarbituric acid (TBA) in 20% (w/v) trichloroacetic acid (TCA). The mixture was heated at 95 °C for 30 min in a water bath and then quickly cooled in ice for 30 min. Absorbance was measured at 532 nm and 600 nm using a spectrophotometer, and the MDA content was calculated (nmol/g fresh weight) using the standard formula.

Statistical data analysis

This study was conducted as a factorial experiment in a completely randomized design. The experimental data were analyzed using SAS software (version 9) and the means were compared using the LSD test and Duncan’s multiple range test at the 1% and 5% probability levels. Graphs were created using Microsoft Office 2016 software.

Results

Variance analysis of treatment effects

Our findings revealed that replication had no significant effect on any of the evaluated traits (Table 1). Variety had significant effects on panicle weight, leaf length, chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, total soluble carbohydrates, leaf phenolic compounds, fruit phenolic compounds, leaf anthocyanin, fruit anthocyanin, total soluble protein, proline, and MDA (p ≤ 0.01), while its effect on total soluble solids was significant at the 5% probability level (p ≤ 0.05). Allantoin application significantly influenced panicle weight, berry weight, total soluble solids, panicle length, berry length, leaf length, leaf width, chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, total soluble carbohydrates, leaf phenolic compounds, fruit phenolic compounds, leaf anthocyanin, fruit anthocyanin, total soluble protein, proline, and MDA (p ≤ 0.01). No significant effect was observed on berry diameter, fruit firmness, or SPAD index. The interaction between variety and allantoin was statistically significant for panicle weight, berry weight, panicle length, leaf length, chlorophyll a, chlorophyll b, total chlorophyll, carotenoids, total soluble carbohydrates, leaf phenolic compounds, fruit phenolic compounds, leaf anthocyanin, fruit anthocyanin, total soluble protein, proline, and MDA (p ≤ 0.01). The experimental design utilized 12 vines per cultivar (n = 12 total per cultivar), with three biological replicates per treatment combination (cultivar × allantoin concentration), where each replicate represented measurements from one individual vine. Although both Sultana and Merlot encompass varietal diversity, the vines used in this study were clonally propagated and maintained under identical management practices for 8 years, ensuring genetic uniformity within each cultivar group. Preliminary analysis of variance revealed no significant differences among individual vines within cultivar-treatment combinations for any measured parameter, validating the pooling approach. The pooled data analysis was performed by averaging measurements from three biological replicates (individual vines) for each treatment combination, which is a standard approach in viticulture research and provides sufficient statistical power to detect treatment effects while accounting for biological variation. This pooling strategy was statistically justified as the within-cultivar variation was substantially lower than the between-treatment variation for all parameters examined.

Table 1.

Analysis of variance (ANOVA) of morphological, physiological, and biochemical traits of Merlot and Sultana grape cultivars treated with allantoin.

Source of variation	df	Panicle weight	Berry weight	TSS	Panicle length	Berry length	Berry diameter	Leaf length	Leaf width	Chl a	Chl b
Replication (R)	2	27.486ns	3.790ns	0.951ns	9.698ns	0.002ns	0.339ns	0.138ns	3.558ns	0.604ns	12.874ns
Variety	1	1770.231**	16.452ns	3.270*	1.265**	0.004ns	0.401ns	4.270**	4.731ns	36.442**	74.239**
Allantoin	3	12512.210**	5332.294**	95.281**	8.001**	6.985**	2.843ns	64.419**	30.743**	4.181**	5.851**
Variety × Allantoin	3	1413.618**	21.963**	1.311ns	12.358**	0.008ns	0.570ns	15.250**	1.237ns	1.968**	0.225**
Error	14	79.241	22.587	1.025	0.321	0.004	0.486	0.347	3.571	0.068	0.078

Source of variation	df	Total Chl	Carotenoids	Total soluble carbohydrate	Leaf phenolics	Berry phenolics	Leaf anthocyanin	Berry anthocyanin	Total soluble protein	Proline	MDA
Replication (R)	2	11.915ns	4.114ns	0.002ns	0.000ns	0.000ns	0.000ns	0.000ns	0.000ns	0.000ns	0.026ns
Variety	1	52.445**	0.236**	22.83**	87.925**	85.040**	42.863**	19.808**	11.68**	532.16**	6.31**
Allantoin	3	8.637**	0.019**	12.56**	15.057**	95.735**	23.540**	31.835**	1.70**	42.19**	0.43**
Variety × Allantoin	3	2.105**	0.004**	2.28**	3.014**	62.527**	18.357**	13.569**	0.45**	46.20**	0.78**
Error	14	0.097	0.08	0.01	0.741	1.954	1.157	0.691	0.05	0.46	0.02

df = degree of freedom; R = replication; ** = significant at the 1% probability level; * = significant at the 5% probability level; ns = non-significant.

Effects of allantoin on reproductive growth parameters

The results indicated that foliar application of allantoin significantly affected panicle weight, berry weight, panicle length, and berry length in both Sultana and Merlot cultivars (Fig. 1). For panicle weight, the control treatments yielded 49.2 ± 3.8 g in Sultana and 58.8 ± 4.2 g in Merlot. Application of 10 mM allantoin increased panicle weight to 68.5 ± 5.1 g in Sultana and 78.3 ± 6.2 g in Merlot, representing increases of 39.2% and 33.2%, respectively. The 20 mM treatment further elevated panicle weight to 98.7 ± 7.3 g in Sultana and 105.4 ± 8.1 g in Merlot (increases of 100.6% and 79.3%, respectively). The highest panicle weight was observed with 30 mM allantoin, reaching 123.6 ± 9.2 g in Merlot and 120.8 ± 8.9 g in Sultana, corresponding to increases of 151.3% in Sultana and 110.3% in Merlot compared to their respective controls. Statistical analysis revealed significant main effects of both cultivar (p < 0.01) and allantoin concentration (p < 0.01), as well as a significant cultivar × allantoin interaction (p < 0.01). Berry weight exhibited a dose-dependent response to allantoin application. Control vines produced berries weighing 1.13 ± 0.09 g in Sultana and 1.41 ± 0.11 g in Merlot. The 10 mM concentration increased berry weight to 1.40 ± 0.12 g in Sultana and 1.69 ± 0.15 g in Merlot. At 20 mM, berry weight reached 2.15 ± 0.17 g in Sultana (90.3% increase) and 2.45 ± 0.19 g in Merlot (73.8% increase). The maximum berry weight was achieved at 30 mM allantoin: 2.92 ± 0.23 g in Sultana (158.3% increase) and 2.93 ± 0.22 g in Merlot (107.8% increase). The cultivar × allantoin interaction was highly significant (p < 0.01). Panicle length measurements showed similar enhancement patterns. Control panicles measured 10.2 ± 0.8 cm in Sultana and 8.5 ± 0.6 cm in Merlot. Progressive increases were observed with 10 mM (12.9 ± 0.9 cm Sultana, 10.1 ± 0.7 cm Merlot), 20 mM (14.1 ± 1.1 cm Sultana, 12.8 ± 0.8 cm Merlot), and 30 mM (15.3 ± 1.2 cm Sultana, 12.6 ± 0.9 cm Merlot. Berry length in Sultana demonstrated particularly strong responsiveness, increasing from 0.58 ± 0.04 cm in controls to 0.73 ± 0.06 cm (10 mM), 0.92 ± 0.07 cm (20 mM), and 1.06 ± 0.08 cm (30 mM), corresponding to a maximum increase of 82.8%. Merlot berries showed more modest but still significant increases from 0.71 ± 0.05 cm (control) to 0.98 ± 0.07 cm (30 mM), representing a 38.0% enhancement.

Fig. 1.

Effects of allantoin treatments on panicle weight, berry weight, panicle length, and berry length of Merlot and Sultana grape cultivars. Data are presented as mean ± standard error. Different letters (a, b, c, d, e) above bars indicate statistically significant differences according to Duncan’s multiple range test (p < 0.05, n = 3).

Effects of allantoin on berry quality and biochemical composition

Significant differences were observed in total soluble solids (TSS), berry phenolic compounds, total soluble carbohydrates, and total soluble protein following foliar application of allantoin (Fig. 2). For TSS, control vines recorded 22 °Brix in Sultana and 19 °Brix in Merlot. The 10 mM treatment slightly increased TSS to 22.5 °Brix in Sultana and 18 °Brix in Merlot. Maximum TSS values were achieved at 20 mM (23.5 °Brix in Sultana, 21 °Brix in Merlot) and 30 mM (23 °Brix in Sultana, 21 °Brix in Merlot), representing increases of approximately 6.8% in Sultana and 10.5% in Merlot compared to their respective controls. Berry phenolic compounds showed strong dose-dependent increases. Control treatments yielded 175 mg g⁻¹ FW in Sultana and 190 mg g⁻¹ FW in Merlot. Progressive increases were observed with 10 mM (205 mg g⁻¹ FW Sultana, 240 mg g⁻¹ FW Merlot), 20 mM (225 mg g⁻¹ FW Sultana, 285 mg g⁻¹ FW Merlot), and 30 mM (270 mg g⁻¹ FW Sultana, 305 mg g⁻¹ FW Merlot), corresponding to maximum increases of 54.3% in Sultana and 60.5% in Merlot. Total soluble carbohydrates in control vines measured approximately 5.0 mg g⁻¹ FW in Sultana and 4.6 mg g⁻¹ FW in Merlot. Allantoin application progressively increased carbohydrate content, reaching maximum values of 6.1 mg g⁻¹ FW in Sultana (22% increase) and 5.9 mg g⁻¹ FW in Merlot (28% increase) at 30 mM concentration. Total soluble protein exhibited marked enhancement with allantoin treatment. Control vines contained 1.2 mg g⁻¹ FW in Sultana and 1.5 mg g⁻¹ FW in Merlot. Protein content increased progressively with allantoin concentration, reaching 2.2 mg g⁻¹ FW in Sultana (83% increase) and 2.3 mg g⁻¹ FW in Merlot (53% increase) at 30 mM, with Sultana showing greater relative enhancement.

Fig. 2.

Effects of allantoin treatments on total soluble solids, berry phenolic compounds, total soluble carbohydrates, and total soluble protein in Merlot and Sultana grape cultivars. Data are presented as mean ± standard error. Different letters (a, b, c, d, e) above bars indicate statistically significant differences according to Duncan’s multiple range test (p < 0.05, n = 3).

Effects of allantoin on secondary metabolite accumulation

Application of allantoin significantly influenced phenolic and anthocyanin contents in both grape cultivars (Fig. 3). Leaf phenolic compounds in control vines measured 200 mg g⁻¹ FW in Sultana and 230 mg g⁻¹ FW in Merlot. Progressive increases were observed with 10 mM (210 mg g⁻¹ FW Sultana, 235 mg g⁻¹ FW Merlot), 20 mM (220mg g⁻¹ FW Sultana, 245 mg g⁻¹ FW Merlot), and 30 mM (220 mg g⁻¹ FW Sultana, 265 mg g⁻¹ FW Merlot), representing increases of 10% in Sultana and 15% in Merlot. Berry phenolic compounds showed more pronounced enhancement, increasing from control values of 175 mg g⁻¹ FW (Sultana) and 190 mg g⁻¹ FW (Merlot) to maximum levels of 270 mg g⁻¹ FW (Sultana) and 305 mg g⁻¹ FW (Merlot) at 30 mM, corresponding to increases of 54% and 61%, respectively. Leaf anthocyanin content exhibited dose-dependent accumulation, with control values of 320 mg g⁻¹ FW in Sultana and 415 mg g⁻¹ FW in Merlot. Treatment with 10 mM allantoin increased anthocyanins to 330 mg g⁻¹ FW (Sultana) and 440 mg g⁻¹ FW (Merlot), while 20 mM and 30 mM treatments further elevated levels to 350 mg g⁻¹ FW and 350 mg g⁻¹ FW in Sultana, and 445 mg g⁻¹ FW and 505 mg g⁻¹ FW in Merlot, representing maximum increases of 9% in Sultana and 22% in Merlot. Berry anthocyanins displayed the most substantial enhancement, rising from 360 mg g⁻¹ FW (Sultana) and 520 mg g⁻¹ FW (Merlot) in controls to 550 mg g⁻¹ FW (Sultana) and 610 mg g⁻¹ FW (Merlot) at 30 mM allantoin, corresponding to increases of 53% and 17%, respectively. Merlot consistently maintained higher absolute anthocyanin levels across all treatments in both tissues.

Fig. 3.

Effects of allantoin treatments on leaf phenolic compounds, berry phenolic compounds, leaf anthocyanin, and berry anthocyanin contents in Merlot and Sultana grape cultivars. Data are presented as mean ± standard error. Different letters (a, b, c, d, e) above bars indicate statistically significant differences according to Duncan’s multiple range test (p < 0.05, n = 3).

Effects of allantoin on vegetative growth and photosynthetic pigments

In our results, foliar application of allantoin markedly influenced leaf morphological parameters and pigment composition in both Vitis vinifera cultivars, Sultana and Merlot, demonstrating a clear concentration-dependent enhancement across all measured traits. Increasing allantoin concentrations from 0 to 30 mM led to significant and progressive improvements in leaf length, leaf width, carotenoid, and total chlorophyll contents (p < 0.05). In both cultivars, leaf length increased steadily with allantoin application. The control plants exhibited the lowest values (4.3 cm in Sultana and 3.8 cm in Merlot), while maximum values were recorded at 30 mM allantoin, reaching 10.8 cm and 8.7 cm, respectively. Compared with the untreated control, these increases correspond to approximately 145% in Sultana and 120% in Merlot. Similarly, leaf width followed a comparable trend, with the highest values of 6.1 cm and 5.6 cm in Sultana and Merlot at 30 mM, compared to 3.0 cm and 2.9 cm in the control. Pigment contents also responded significantly to allantoin treatments. Carotenoid levels increased from 5.0 to 7.5 mg g⁻¹ FW in Sultana and from 2.0 to 7.2 mg g⁻¹ FW in Merlot, showing enhanced pigment biosynthesis in both cultivars with increasing allantoin concentration. Likewise, total chlorophyll content rose markedly with allantoin application. At 30 mM, total chlorophyll reached 52.8 mg g⁻¹ FW in Sultana and 40.5 mg g⁻¹ FW in Merlot, compared with 32.0 and 21.4 mg g⁻¹ FW in control plants (Fig. 4).

Fig. 4.

Effects of allantoin treatments on leaf length, leaf width, carotenoids, and total chlorophyll contents in Merlot and Sultana grape cultivars. Data are presented as mean ± standard error. Different letters (a, b, c, d, e) above bars indicate statistically significant differences according to Duncan’s multiple range test (p < 0.05, n = 3).

Effects of allantoin on stress tolerance indicators

Foliar allantoin application induced significant changes in chlorophyll a, chlorophyll b, proline, and malondialdehyde (MDA) contents of Vitis vinifera cultivars Sultana and Merlot, showing clear concentration-dependent responses (p < 0.05). The enhancement in chlorophyll pigments was accompanied by an increase in proline accumulation and a marked reduction in lipid peroxidation levels as allantoin concentration increased. Chlorophyll a content increased gradually in both cultivars, with control plants showing the lowest levels (18.5 mg g⁻¹ FW in Sultana and 14.2 mg g⁻¹ FW in Merlot), while the highest values were recorded at 30 mM allantoin (29.5 and 25.4 mg g⁻¹ FW, respectively). Similarly, chlorophyll b followed the same trend, rising from 11.2 to 7.6 mg g⁻¹ FW in the controls to 21.8 and 16.9 mg g⁻¹ FW at 30 mM in Sultana and Merlot, respectively. Proline content, an indicator of osmotic adjustment and stress protection, also increased with allantoin treatment. The lowest values were found in untreated plants (15.2 µmol g⁻¹ FW in Sultana and 14.7 µmol g⁻¹ FW in Merlot), whereas the maximum accumulation occurred at 30 mM allantoin (21.8 and 20.9 µmol g⁻¹ FW, respectively). In contrast, MDA content, a marker of lipid peroxidation, decreased significantly with increasing allantoin concentration. Control plants had the highest MDA values (2.1 µmol g⁻¹ FW in Sultana and 2.0 µmol g⁻¹ FW in Merlot), while the lowest values were detected at 30 mM (1.5 and 1.3 µmol g⁻¹ FW, respectively). (Fig. 5).

Fig. 5.

Effects of allantoin treatments on chlorophyll a, chlorophyll b, proline, and malondialdehyde contents in Merlot and Sultana grape cultivars. Data are presented as mean ± standard error. Different letters (a, b, c, d, e) above bars indicate statistically significant differences according to Duncan’s multiple range test (p < 0.05, n = 3).

Correlation analysis among traits

The correlation analysis revealed distinct associations among morphological, physiological, and biochemical traits of Merlot and Sultana grape cultivars treated with allantoin (Fig. 6). Strong positive correlations were observed between berry length and berry weight (r = 0.94). A very high correlation was also found between pH and berry weight (r = 0.82) and between pH and berry length (r = 0.80). In contrast, berry weight exhibited strong negative correlations with TSS (r = − 0.78), leaf width (r = − 0.55), and leaf length (r = − 0.47). Similarly, berry length was negatively correlated with TSS (r = − 0.81), leaf width (r = − 0.52), and leaf length (r = − 0.45). Panicle weight showed only weak associations with other traits, with the highest positive correlation recorded with leaf length (r = 0.35). Overall, chlorophyll displayed relatively weak correlations with the studied parameters, the highest being with berry weight (r = 0.25).

Fig. 6.

Correlation heatmap of morphological, physiological, and biochemical traits of Merlot and Sultana grape cultivars treated with allantoin.

Discussion

Cultivar-specific metabolic responses to allantoin application

The differential responses observed between Sultana and Merlot cultivars to allantoin application reflect fundamental differences in their genetic backgrounds and metabolic regulation. Sultana, as a white seedless cultivar primarily developed for table grape and raisin production, demonstrated superior responsiveness in reproductive parameters (panicle weight, berry size) and vegetative growth metrics (leaf dimensions, total chlorophyll). This enhanced response may be attributed to Sultana’s breeding history emphasizing vigorous vegetative growth and high productivity, which could predispose this cultivar to greater phenotypic plasticity in response to nitrogen-rich bio stimulants. In contrast, Merlot, a wine grape cultivar selected for quality traits rather than yield maximization, exhibited more pronounced enhancement in secondary metabolite accumulation (phenolics, anthocyanins) and stress tolerance indicators (proline, MDA). This pattern aligns with the known metabolic trade-offs between primary growth and secondary metabolism, where wine grape cultivars typically allocate more resources toward quality-determining compounds²³. The molecular basis for these cultivar-specific responses likely involves differential regulation of nitrogen assimilation pathways, transcription factors controlling anthocyanin biosynthesis (such as MYB and bHLH family members), and stress-responsive gene networks. Allantoin, through its role in ureide metabolism, provides readily available nitrogen that can be rapidly assimilated into amino acids and proteins, thereby influencing numerous downstream metabolic pathways²³. The observed correlation between morphological enhancements and biochemical changes suggests coordinated developmental programming rather than isolated physiological responses, highlighting the systemic nature of allantoin’s effects on grapevine physiology.

Integrated mechanisms of allantoin-mediated growth enhancement

The enhancement of reproductive and vegetative growth parameters observed following allantoin application likely involves multiple integrated physiological pathways operating at cellular, tissue, and whole-plant levels. The most striking result to emerge from our data is that allantoin foliar application significantly enhanced panicle weight, with cultivar-specific responses playing a key role. The greater relative increase in Sultana may be due to its lower baseline panicle weight or higher sensitivity to foliar nitrogen sources. Similar cultivar-dependent effects of foliar and biostimulant applications on yield-related traits have been reported previously²⁴. These findings emphasize the importance of cultivar-specific and tailored allantoin foliar treatments in improving grape yield. Our findings demonstrate that berry weight enhancement followed similar patterns, with both the grapevine’s genetic background and external allantoin application serving as key factors influencing berry development. The significant increase in berry weight may be due to allantoin’s role in enhancing nitrogen metabolism, osmotic regulation, and stress tolerance, all of which support fruit growth and development¹². The greater response in Sultana indicates that cultivars with lower initial berry weights might benefit more from allantoin supplementation, possibly due to their higher physiological plasticity or increased responsiveness to nitrogenous compounds^8,12. Previous research has shown that exogenous application of biostimulants like allantoin can lead to cultivar-dependent improvements in reproductive growth parameters, including berry size and weight²⁵. At the metabolic level, allantoin serves as an efficient nitrogen source that can be rapidly catabolized through sequential action of allantoinase and allantoicase enzymes, releasing four ammonium ions per molecule for assimilation into amino acids and proteins during critical developmental windows^8,11,12. This enhanced nitrogen availability during fruit set and early berry development stages directly supports the high metabolic demands associated with cell division and expansion processes. Based on our findings, on the other hand, foliar application of allantoin significantly increased TSS content in the grapevine cultivars. The highest TSS was observed in vines treated with 20 mM allantoin, while untreated control vines exhibited the lowest TSS values. The enhancement of TSS following allantoin treatment may be attributable to allantoin’s role in improving nitrogen metabolism, stress tolerance, and overall plant physiological performance, factors known to influence carbohydrate accumulation and sugar metabolism in grape berries^26,27. Previous research has shown that the impact of foliar biostimulants on grape quality parameters is often cultivar-dependent, with some cultivars responding more markedly to exogenous applications²⁸. The observed improvement in TSS is consistent with general findings that foliar supplementation can enhance fruit quality in grapevines²⁹. The study found that foliar application of allantoin had a highly significant effect on berry length in the Sultana grape cultivar. The observed enhancement in berry length may be attributed to allantoin’s role in promoting cell elongation and enhancing tolerance to environmental stresses^30,31. These physiological processes are critical for fruit development and have been linked to improvements in both berry size and overall yield in various crops following the application of biostimulants³². Studies have shown that biostimulants and growth regulators can positively impact berry morphology by improving nutrient uptake and hormonal regulation, leading to improved fruit quality traits³³.

Beyond its nutritional role, allantoin functions as a signaling molecule that activates abscisic acid (ABA) and jasmonic acid (JA) signaling pathways, which regulate cell expansion, stress responses, and secondary metabolite biosynthesis³⁴. It should be emphasized that the present study did not directly measure phytohormone levels (ABA, JA, IAA) or key enzyme activities (e.g., phenylalanine ammonia-lyase [PAL], UDP-glucose: flavonoid 3-O-glucosyltransferase [UFGT], allantoinase) involved in these pathways. Therefore, the mechanistic interpretations regarding ABA/JA signaling and nitrogen remobilization pathways represent working hypotheses based on established biochemical frameworks from previous research³⁴ rather than direct experimental evidence from our study. Future investigations employing targeted phytohormone profiling (e.g., LC-MS/MS quantification of ABA, JA, and auxin), enzyme activity assays (allantoinase, PAL, UFGT), and gene expression analysis (qRT-PCR of anthocyanin biosynthesis genes such as VvMYBPA1, VvUFGT, and nitrogen metabolism genes including VvNRT, VvGS) would be essential to validate these proposed molecular mechanisms and establish causative relationships between allantoin application and the observed physiological responses. Recent molecular studies indicate that allantoin can modulate the expression of genes involved in nitrogen metabolism (nitrate reductase, glutamine synthetase), photosynthetic carbon fixation (Rubisco, PEPC), and antioxidant defense systems (superoxide dismutase, catalase, ascorbate peroxidase). The observed increases in berry dimensions, panicle weight, and leaf growth in our study represent the cumulative outcomes of these interconnected processes, where improved nitrogen status supports cell division and expansion, while enhanced stress tolerance maintains optimal growth conditions throughout berry development. The mechanistic basis for allantoin-mediated growth enhancement likely involves multiple integrated pathways. First, allantoin serves as an efficient nitrogen source that can be rapidly catabolized to release ammonium ions through the sequential action of allantoinase and allantoicase enzymes, providing nitrogen for amino acid biosynthesis during critical developmental windows. Second, allantoin has been demonstrated to function as a signaling molecule that activates abscisic acid (ABA) and jasmonic acid (JA) signaling pathways, which regulate cell expansion, stress responses, and secondary metabolite biosynthesis³⁴. Third, recent molecular studies indicate that allantoin can modulate the expression of genes involved in nitrogen metabolism (nitrate reductase, glutamine synthetase), photosynthetic carbon fixation (Rubisco, PEPC), and antioxidant defense systems (superoxide dismutase, catalase, ascorbate peroxidase). The observed increases in berry length and panicle dimensions in our study are likely the cumulative result of these interconnected physiological processes, where improved nitrogen status supports cell division and expansion, while enhanced stress tolerance maintains optimal growth conditions throughout berry development. However, it must be acknowledged that the present study remains primarily descriptive in nature, focusing on phenotypic and biochemical endpoints rather than elucidating the underlying molecular mechanisms. Future investigations employing transcriptomic, proteomic, and metabolomic approaches would be valuable to comprehensively characterize the molecular cascades initiated by allantoin application. Specifically, time-course analysis of gene expression patterns for key enzymes in nitrogen metabolism (e.g., ureide transporters, allantoinase, ureidoglycolate), anthocyanin biosynthetic pathway genes (PAL, CHS, DFR, UFGT, MYB transcription factors), and antioxidant defense components would provide mechanistic insights into the observed physiological responses. Additionally, investigating the potential interactions between allantoin-derived nitrogen and endogenous phytohormone dynamics (particularly ABA, JA, and auxin) would help explain the coordinated enhancement of both vegetative and reproductive traits observed in our study.

Photosynthetic enhancement and carbohydrate metabolism

The interaction between grape cultivar and foliar application of allantoin significantly influenced both panicle and leaf lengths. These findings underscore the effectiveness of allantoin as a biostimulant in improving vegetative and reproductive growth traits in grapevines. Allantoin is known for its roles in enhancing nitrogen assimilation, promoting cellular expansion, and mitigating stress, all of which can contribute to increased organ size and overall plant vigor^27,29. The differential response observed between cultivars suggests genotypic variation in sensitivity to exogenous allantoin; such cultivar-specific effects have been widely reported in grapevine studies examining foliar treatments and growth regulators³⁵. Similar improvements in shoot and leaf growth following foliar application of biostimulants have been previously documented, highlighting that enhanced vegetative growth can have downstream positive effects on reproductive structures such as panicles^30,36. The findings revealed that foliar application of allantoin enhanced chlorophyll content in grapevine leaves, which aligns with previous studies demonstrating allantoin’s role in improving pigment biosynthesis^8,27. Improved SPAD values are closely associated with enhanced photosynthetic capacity and improved plant nutritional status, both of which can positively influence grapevine vigor and productivity³⁷. The differential response between cultivars highlights the influence of genetic background on the effectiveness of foliar biostimulants^32,38. Our results demonstrated that the interaction between grape cultivar and foliar allantoin application significantly influenced the contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids. These findings are consistent with the established roles of allantoin in promoting pigment biosynthesis and antioxidant defense in plants^15,39. Allantoin acts as a signaling molecule and metabolic enhancer, improving nitrogen assimilation and stress tolerance, processes fundamental to chlorophyll formation and carotenoid accumulation¹³. The substantial increases in both chlorophylls and carotenoids suggest enhanced photosynthetic efficiency and protective capacity against oxidative stress following allantoin application. The increased carbohydrate content after allantoin application indicates an improvement in the photosynthetic ability and metabolic activity of grapevine leaves¹³. Both Sultana and Merlot showed significant increases in carbohydrates, although varietal differences may reflect different physiological or metabolic responses to allantoin³². These cultivar-specific interactions align with previous findings that biostimulant effectiveness can vary depending on grapevine genotype and environmental conditions¹³. The results support the potential of allantoin foliar sprays to enhance carbohydrate accumulation, which is vital for grapevine growth, fruit set, and overall yield²⁶. Allantoin has been recognized as a metabolic stimulant that boosts various biochemical processes in plants, potentially resulting in greater synthesis of simple sugars in the fruit²⁶. By supporting stronger root growth and enhancing nutrient absorption, allantoin can promote the accumulation of carbohydrates and sugars critical for fruit development¹². These physiological improvements effectively increase the carbohydrate content of fruits. Experimental studies have shown that applying allantoin to fruit crops raises sugar levels and improves fruit flavor¹². Research investigating allantoin’s influence on fruit quality has revealed that it not only raises sugar concentrations but also enhances the sensory characteristics of fruits⁴⁰.

Secondary metabolite enhancement and quality improvement

Our analysis revealed that foliar application of allantoin significantly enhanced the phenolic profile of grapevines, affecting both vegetative and reproductive tissues. Phenolic compounds are crucial antioxidants, contributing to plant defense mechanisms against both abiotic and biotic stresses, and are important determinants of fruit quality and health-promoting properties in grapes and derived products^41,42. The substantial increase in fruit phenolics following allantoin treatment highlights its potential utility as a biostimulant for improving grape antioxidant potential and market value. The more pronounced increases in Merlot may reflect genotype-specific differences in metabolic responsiveness to allantoin, in line with earlier reports highlighting the variable effect of biostimulants and nutrient management strategies among grape cultivars^13,32. The results demonstrated a significant interaction between grape cultivar and foliar allantoin application concerning both leaf and fruit anthocyanin contents. These findings highlight the potent influence of foliar allantoin application on anthocyanin biosynthesis in grapevines. Anthocyanins are vital secondary metabolites, primarily responsible for the red, purple, and blue pigmentation in grapes, and are potent antioxidants crucial for fruit quality, appeal, and human health benefits⁴³. The significant increases observed, particularly in fruit anthocyanins, underscore allantoin’s potential to enhance the commercial and nutritional value of grape produce. The cultivar-specific responses, with Merlot showing a strong increase in both leaf and fruit anthocyanins and Sultana exhibiting a remarkable boost primarily in fruit anthocyanins, are consistent with the understanding that genetic background plays a pivotal role in determining a plant’s metabolic response to external stimuli⁴³. Allantoin, as a purine derivative, is known to participate in nitrogen metabolism and signaling pathways, which can indirectly influence the synthesis of secondary metabolites like anthocyanins, potentially by modulating enzymatic activities involved in the flavonoid pathway or by alleviating stress conditions⁴⁴. The enhancement of these secondary metabolites likely results from improved carbon-nitrogen balance, activation of phenylpropanoid pathway regulatory genes, and ABA-mediated transcriptional regulation of anthocyanin biosynthetic genes, as previously demonstrated in grape berry ripening studies. Our findings indicate that allantoin can markedly improve protein metabolism in grapevines, although the magnitude of the response varies by cultivar. The increase in total soluble protein content is likely linked to allantoin’s known roles in promoting nitrogen uptake and assimilation, which are fundamental for protein biosynthesis in plants³⁹. Allantoin has been implicated in modulating signaling pathways and protective mechanisms under environmental stress conditions, which may further contribute to increased protein accumulation. Varying allantoin concentrations can directly influence the amino acid profile and specific protein composition in grape berries, thereby enhancing the nutritional quality of the fruit¹³.

Stress tolerance and antioxidant system modulation

It is important to clarify that our experimental design evaluated allantoin efficacy under naturally occurring semi-arid field conditions rather than as a controlled stress-imposition study comparing stressed versus non-stressed treatments. Semi-arid viticulture represents the baseline growing condition for major grape-producing regions globally, where chronic moderate water limitation, high vapor pressure deficit, and elevated evapotranspiration constitute inherent environmental constraints rather than acute experimental stresses. The control treatment (0 mM allantoin) in our study represents standard commercial practice without biostimulant supplementation under these prevailing conditions. The measurement of stress indicators (MDA and proline) in our study is justified by the fact that grapevines grown under semi-arid field conditions experience chronic moderate stress characterized by water limitation, high vapor pressure deficit, and elevated temperatures, even in the absence of experimentally imposed acute stress treatments. MDA and proline are not exclusively “stress markers” appearing only under severe stress, but rather represent fundamental components of cellular metabolism and redox homeostasis that operate across a continuum of environmental conditions. The study revealed a significant interaction between grape cultivar and foliar application of allantoin on proline content. Proline is a key osmoprotectant and stress indicator in plants, playing crucial roles in cellular osmotic adjustment, scavenging of reactive oxygen species, and stabilizing proteins and membranes, particularly under abiotic stress conditions⁴⁵. Proline accumulation serves multiple functions beyond stress tolerance, including osmotic adjustment, stabilization of proteins and membranes, and acting as a compatible solute and antioxidant⁴⁵. The baseline proline levels observed in control plants (15.2 µmol g⁻¹ FW in Sultana and 14.7 µmol g⁻¹ FW in Merlot) are substantially elevated compared to values typically reported for well-watered grapevines (typically 5–8 µmol g⁻¹ FW), confirming the presence of baseline environmental stress at our study site. The results demonstrated a statistically significant interaction between grape cultivar and foliar application of varying allantoin concentrations on malondialdehyde (MDA) content. MDA is a well-established marker of lipid peroxidation and oxidative stress in plant tissues⁴⁶. Elevated MDA levels are indicative of enhanced cellular membrane damage due to the accumulation of reactive oxygen species, often under adverse environmental conditions. The observed decreases in MDA following allantoin application suggest that allantoin confers improved oxidative stress tolerance, likely through the enhancement of the plant’s antioxidant defense systems^28,44. The coordinated reduction in MDA levels and increase in proline content following allantoin treatment indicate improved cellular homeostasis and oxidative stress management capacity under semi-arid conditions. The mechanistic relationship between enhanced proline content and improved fruit yield following allantoin treatment likely involves multiple interconnected pathways. First, proline’s role in maintaining cellular turgor and membrane integrity under water-limited conditions supports sustained cell expansion during critical berry development phases, directly influencing final berry size and weight. Second, proline acts as a molecular chaperone protecting photosynthetic apparatus and metabolic enzymes from oxidative damage, thereby maintaining the carbon assimilation capacity necessary to support reproductive sink strength. Third, proline serves as a readily mobilizable nitrogen and carbon reserve that can be catabolized during periods of high metabolic demand associated with fruit development, providing precursors for protein synthesis and secondary metabolite biosynthesis. However, it should be noted that the observed correlation between proline accumulation and enhanced yield does not necessarily imply direct causation, and the increases in fruit yield likely result from the integrated effects of improved nitrogen nutrition, enhanced antioxidant capacity.

Endogenous allantoin dynamics and future mechanistic investigations

An important limitation of the present study is the absence of endogenous allantoin quantification in response to both semi-arid conditions and exogenous allantoin application. Under stress conditions, endogenous allantoin accumulation is typically enhanced through downregulation of allantoinase activity, which converts allantoin to allantoate in the purine catabolism pathway^39,44. In Arabidopsis, allantoin accumulation mediated by allantoinase downregulation confers stress tolerance, with allantoin acting as both a nitrogen reserve and signaling molecule (Takagi et al., 2016). Similarly, in rice under drought stress, allantoin accumulation correlates with enhanced stress tolerance through activation of ABA-dependent pathways³¹. In our study, the baseline semi-arid conditions likely induce moderate endogenous allantoin accumulation in control plants, while exogenous allantoin supplementation provides additional substrate that can be either catabolized for nitrogen assimilation or function as a signaling molecule to activate stress-responsive pathways. Future investigations should quantify endogenous allantoin levels in grapevine tissues under semi-arid conditions with and without exogenous application, measure allantoinase and allantoicase enzyme activities, and characterize expression patterns of genes encoding ureide transporters and catabolic enzymes to comprehensively elucidate the metabolic fate and signaling functions of applied allantoin.

Applied implications and future research directions

From an applied viticulture perspective, our findings demonstrate that allantoin foliar application represents a practical biostimulant strategy for enhancing grapevine productivity and fruit quality under semi-arid conditions. The optimal concentration appears to be cultivar-dependent, with 20–30 mM providing maximum benefits for most parameters. The application timing employed in this study (fruit set and early berry development stages) coincides with critical periods of resource allocation and can be readily integrated into existing vineyard spray programs. The cost-effectiveness of allantoin application, combined with its compatibility with integrated pest management practices and organic viticulture systems, positions this approach as a sustainable alternative or complement to conventional nitrogen fertilization strategies. Nevertheless, the absence of positive control treatments (conventional nitrogen sources such as urea or commercial biostimulant products) in our experimental design limits direct performance benchmarking and economic comparison. Future field trials should include parallel treatments with standard foliar nitrogen fertilizers and commercially available biostimulants to establish allantoin’s relative efficacy, determine cost-benefit ratios, and identify specific application scenarios where allantoin may offer advantages over conventional approaches. Also, several research gaps remain that warrant future investigation. First, field trials across multiple growing seasons and diverse environmental conditions are essential to validate these controlled-study findings and assess the consistency of treatment effects under variable climatic scenarios. Second, economic analysis comparing allantoin application costs against yield and quality improvements would inform commercial adoption decisions. Third, investigation of potential synergistic effects between allantoin and other biostimulants (e.g., seaweed extracts, humic substances, beneficial microorganisms) could lead to optimized biostimulant formulations. Fourth, studies examining residual effects of allantoin on subsequent growing seasons and long-term vine health would provide insights into the sustainability of repeated applications. Finally, extending this research to other commercially important cultivars and different grapevine growing regions would establish the broader applicability of allantoin-based biostimulation strategies in global viticulture.

Conclusions

This study demonstrates that foliar application of allantoin (10–30 mM) significantly enhances grapevine productivity and berry quality in Vitis vinifera L. cultivars Sultana and Merlot grown under semi-arid field conditions. Allantoin-treated vines exhibited marked improvements in reproductive parameters (panicle weight increases up to 151% in Sultana, 110% in Merlot), berry quality indicators (TSS, phenolic content, anthocyanin accumulation), photosynthetic capacity (chlorophyll content increases of 40–65%), and stress tolerance markers (reduced MDA, enhanced proline accumulation). Importantly, cultivar-specific response patterns were identified: Sultana demonstrated superior responsiveness in vegetative and reproductive growth parameters, while Merlot showed more pronounced enhancement in secondary metabolite accumulation and antioxidant capacity. These differential responses highlight the necessity for cultivar-specific optimization of allantoin application protocols in commercial viticulture, with optimal concentrations appearing to be 20–30 mM applied at fruit set and early berry development stages. However, this study remains primarily descriptive, characterizing phenotypic and biochemical endpoints without elucidating underlying molecular mechanisms. Future research priorities should include: (1) molecular-level investigations (transcriptomic, proteomic analyses) to characterize gene expression patterns and metabolic pathway dynamics mediating allantoin’s effects; (2) quantification of endogenous allantoin levels and ureide metabolism enzyme activities under semi-arid conditions; (3) multi-season field validation across diverse environmental conditions and viticultural regions; (4) evaluation of potential synergistic effects with other biostimulants; and (5) economic cost-benefit analyses to inform commercial adoption decisions. In conclusion, while this study provides clear evidence for beneficial effects of allantoin foliar application on grapevine performance under semi-arid conditions and offers preliminary insights for developing targeted biostimulant strategies, significant mechanistic and applied research remains necessary to fully optimize the utilization of allantoin in sustainable viticulture systems.

Author contributions

Conceptualization, data curation, formal analysis and methodology, project administration, writing draft—review and editing, A. B., S.M., G.E., H.S.H., and O.K. All authors have read and agreed to the published version of the manuscript.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethical approval

All procedures were conducted following the relevant institutional, national, and international guidelines and legislations.

Plant material authentication and deposition

Plant material identification was conducted by Dr Ali Bahmani (Department of Horticultural Science, University of Maragheh). Voucher specimens have been deposited in the Herbarium of the Faculty of Agriculture, University of Maragheh, Iran, with voucher numbers UM-VV-2023-001 (Sultana) and UM-VV-2023-002 (Merlot), where they are publicly accessible. All plant materials were collected from the university-owned research vineyard with institutional permission. This research complies with IUCN guidelines and CITES regulations; Vitis vinifera L. is a domesticated species not listed as threatened or endangered.