Optimizing Selenium Delivery in Grapevines: Foliar vs. Rhizosphere Fertilization Effects on Photosynthetic Efficiency, Fruit Metabolites, and VOCs of ‘Muscat Hamburg’ Grape (Vitis vinifera L.)

Abstract

This study examined the effects of selenium (Se) fertilization, applied via foliar and rhizosphere methods, on the physiological and biochemical characteristics of ‘Muscat Hamburg’ grapes. Sodium selenite (Na₂SeO₃) treatments were administered at three concentrations (50, 100, and 150 ppm) during critical phenological stages. The results showed that Se at 50 ppm effectively increased the chlorophyll content and enhanced chlorophyll fluorescence parameters. Se significantly elevated total soluble solid content and reduced titratable acidity, thereby increasing the TSS/TA ratio. Foliar fertilization with 50 ppm Se enhanced cluster size without affecting berry dimensions, whereas rhizosphere fertilization increased both with increasing Se concentrations, albeit with negative impacts on berry size at higher concentrations. Se increased flavonoid content in grape peels, with rhizosphere fertilization exerting more pronounced effects. Se—via rhizosphere fertilization at 100 and 150 ppm—significantly influenced VOCs derived from fatty acid and isoprene metabolic pathways. Mantel’s test confirmed that foliar fertilization significantly increased chlorophyll content and fluorescence indices, while rhizosphere fertilization had more marked effects on flavonoid content, berry and cluster size, and VOCs, particularly through fatty acid metabolism. These findings suggest that Se can enhance grape quality, but optimal concentrations and fertilization methods must be carefully determined to avoid adverse effects.

1. Introduction

Global climate change poses one of the most urgent challenges to agriculture, significantly impacting crop production systems worldwide. Grapes, a vital horticultural commodity, are particularly susceptible to the shifting environmental conditions. Extreme weather events, altered precipitation patterns, and rising temperatures threaten grape yield and quality [1]. Consequently, devising strategies to mitigate these losses has become imperative for agricultural production.

Previous studies have demonstrated the positive effects of selenium (Se) on plant growth and development, affecting pollen viability, fruit production and quality, and heavy metal uptake and translocation [2,3,4,5]. Optimal Se dosages were shown to increase explant weight and boost total Se content in in vitro cultivated olives [6]. Additionally, Se mitigated the generation of excessive reactive oxygen species (ROS) in plants exposed to various environmental stresses [7,8]. Se protects wheat seedlings against salt stress-mediated oxidative damage by up-regulating antioxidant and osmolyte metabolism [9]. At low concentrations, Se promotes the synthesis of chlorophyll precursors, leading to increased photosynthesis and crop yield [10]. Studies have also demonstrated that Se positively influences amino acid biosynthesis and nitrogen accumulation in plants, contributing to enhanced metabolic processes and overall plant health [11].

Currently, two principal application modalities for Se exist: foliar spraying and rhizosphere application. Foliar spraying facilitates rapid absorption and translocation of Se to photosynthetically active chloroplasts, circumventing potential soil fixation and complexation processes [12,13]. In contrast, root application offers a more gradual yet systemic supply of Se, enabling assimilation into the plant’s root xylem and phloem transport systems, ensuring a stable and continuous influx of the element throughout the plant’s tissues [14]. However, each method presents distinct limitations influenced by environmental and physiological factors.

Recent studies have focused on augmenting Se levels in grape berries using various additives [15,16,17]. Low concentrations of Se have been shown to confer beneficial impacts on various plants, including citrus, blueberry, peach, and pear, as it stimulates their growth and bolsters their resilience against environmental stresses [10,18]. Conversely, elevated Se concentrations can be deleterious, acting as prooxidants and inducing damage. Intriguingly, Se addition has been documented to enhance volatile aroma compounds in plants such as Brassica oleracea, Artemisia annua, and Cardamine violifolia [19,20,21]. These volatile compounds predominantly originate from amino acid metabolism, fatty acid metabolism, and isoprene metabolism pathways. Although Liu et al. [22] reported the favorable effect of a 25 mg∙L^𢄡 Se solution on preserving the flavor of red earth grapes, the precise mechanisms underlying Se’s influence on different metabolic pathways remain elusive. Elucidating the distinct mechanisms of selenium uptake through foliar versus rhizosphere application, while refining transport pathway optimization, is essential for maximizing selenium bioavailability in grapes under practical farming conditions.

This study systematically examined the effects of foliar and rhizosphere fertilization techniques, utilizing a range of Se concentrations, on multiple physiological and biochemical characteristics of ’Muscat Hamburg’ grapes (Vitis vinifera L.). These characteristics encompassed photosynthetic pigments, grape appearance, flavor quality, nutritional components, and volatile compounds. We delved into the correlations among these diverse parameters to gain a comprehensive understanding. Specifically, our objectives were to identify the optimal Se concentration for both foliar and rhizosphere fertilization, and to compare the differing impacts of both fertilization methods on leaf photosynthetic functionality and fruit quality. By doing so, we aimed to provide profound insights into the utilization of Se for the enhancement of grape cultivation practices and postharvest quality, ultimately yielding potential benefits for the fruit industry.

2. Materials and Methods

2.1. Materials

The experiment was conducted in the greenhouse of the Binhai Chadian Grape Science and Technology Park, located in Tianjin, China (latitude 39°17′56″ N, longitude 117°18′49″ E) during the 2022 growing season. This region benefits from a sunshine duration of 2998.9 h and a frost-free period spanning 210 d, with an average annual temperature of 13.0 °C, annual solar radiation of 129.5 MJ·cm⁻², and an annual average rainfall of 566.0 mm. During the entire experimental period, environmental conditions in the greenhouse were regulated using fans, shading nets, and heating equipment, maintaining daytime temperatures below 30 °C and nighttime temperatures above 15 °C, with relative humidity controlled within 40%~70%. The vineyard soil had a sandy clay texture with an average salt content of 1.4% and a pH of 7.78. Nine-year-old ‘Muscat Hamburg’ grapevines were selected as experimental materials, with a planting density of 2200 vines per hectare (1.2 × 3.0 m between vines and rows, respectively). Standard vineyard management practices, including pruning, spraying, irrigation, soil tillage, cluster thinning, leaf removal, and cluster tip cutting, were performed.

2.2. Experimental Design

Selenium solutions were administered through both foliar spraying and rhizosphere fertilization, with three concentrations of sodium selenite (Na₂SeO₃) established for each method: 50 ppm, 100 ppm, and 150 ppm. A control group (0 ppm) was included as CK treatment, utilizing distilled water. These treatments were applied in three separate installments of 2.5 L each, timed to correspond with key developmental stages of ‘Hamburg Muscat’ grapes: fruit set (26 May 2022), fruit expansion (26 June 2022), and fruit color change (26 July 2022). It is important to note that the two fertilizer application methods were independent and did not interact. The study comprised eight distinct treatments, each replicated across six plants, resulting in a total of 48 grapevines. The experimental design adhered to a randomized block group layout, with a spacing of 1.2~1.3 m between individual plants to minimize potential fertilization impacts on adjacent grapevines.

2.3. Photosynthetic Pigments Content and Fluorescence Analysis

Photosynthetic pigment levels were measured in mature leaves near the fruit cluster at three developmental stages: fruit swelling (25 June 2022, BBCH73), fruit color transformation (25 July 2022, BBCH81), and fruit ripening (25 August 2022, BBCH89). The contents of photosynthetic pigments, namely chlorophyll a, chlorophyll b, and carotenoid, were determined from these samples using a spectrophotometer method.

The chlorophyll fluorescence induction kinetics curve (OJIP) and its associated parameters were measured utilizing a Handy PEA rapid fluorescence analyzer (Handy PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK). Measurements were conducted on the 5th to 7th functional leaves between 22~25 August 2022, with each treatment measured on three leaves, repeated three times. Prior to the measurements, the leaves were adapted to darkness for 20 m using leaf clips provided by Hansatech Instruments Ltd., ensuring the complete oxidation of the photosynthetic system. The fluorescence transients were induced by exposing the leaves to a 1 s incident light pulse with a maximum intensity of 3000 μmol (photon) ·m⁻²·s⁻¹ over a 4 mm diameter leaf area, using a portable fluorometer (Handy PEA). Based on the OJIP fluorescence transient curve, the JIP test was evaluated in accordance with the theory of energy flow across biomembranes [23,24].

2.4. Fruit Appearance and Biochemical Properties

Cluster samples were collected at harvest maturity and brought to the laboratory. On 26 August, 30 healthy grape berries that had attained full industrial ripeness were randomly chosen from diverse locations within the clusters of the identical treatment. For physical properties, berry width and length were measured with a caliper, cluster width and length with a ruler, and berry and cluster weight with a precise balance (ES20K-1D; China). Total soluble solids (TSSs) in the berry were measured with a refractometer (PAL-1; ATAGO, Itabashi City, Tokyo, Japan) and the total titratable acidity (TA) was determined by the sodium hydroxide (NaOH) titration method. The TSS/TA ratio was calculated by dividing the TSS and TA values [25].

Ten berries were carefully harvested from each treatment group. The peel and pulp fractions were manually dissected using sterile forceps under aseptic conditions. Both tissue components were then lyophilized and pulverized using a cryogenic grinder, respectively, with liquid nitrogen to ensure complete homogenization. Following the protocol detailed by Tohidi et al. (2017) [26], the total phenolic content (TPC) was assessed using the Folin–Ciocalteu method. For total flavonoid determination, the aluminum chloride spectrophotometric method was employed as previously reported [27]. Anthocyanins were extracted utilizing a methanol solution containing 0.1% HCl [28].

2.5. Volatile Organic Compounds Analyses

A total of 50 g berries were delicately gathered from diverse clusters within the same grapevine, replicated three times per treatment group. These grapes were promptly stored in a freezer to preserve their original characteristics. Hand-peeled fruit samples were pulverized in liquid nitrogen, centrifuged, and filtered to obtain clear juice for gas chromatography–mass spectrometry (GC-MS) analysis. The GC-MS system (Agilent 7890B-5977A) was equipped with an HP-MS chromatographic column (Agilent Technologies, Santa Clara, CA, USA). The chromatographic conditions were meticulously controlled as follows [29,30]: (1) The sample inlet temperature was set at 250 °C; (2) helium, serving as the carrier gas, was circulated at a constant flow rate of 1 mL·min⁻¹; (3) the GC inlet was set in the splitless mode to ensure optimal sample injection; (4) the column temperature was programmed to initially heat to 35 °C and hold for 2 min. It was then increased to 200 °C at a rate of 4 °C·min⁻¹ and held for 5 min. Afterward, the temperature was raised by 30 °C·min⁻¹ for another 5 min until it reached 250 °C. (5) For mass spectrometry, the ion source temperature was set at 230 °C, while the quadrupole temperature was maintained at 250 °C; (6) the ionization mode used was electron ionization (EI) with an ionization energy of 70 eV. The mass spectrometric data range was set from 30 to 300 m·z⁻¹.

The sample vial was equilibrated at 45 °C for 5 min. The solid phase microextraction fiber (SPME, 50/30 μm DVB/CAR/PDMS, Supelco, Bellefonte, PA, USA) was utilized to extract volatile compounds from the sample at 45 °C for 50 min. After extraction, the fiber was inserted into the gas chromatograph (Agilent 7890B GC, Agilent Technologies) injection port. The fiber was desorbed in the splitless mode at 250 °C for 2 min. The GC system was equipped with a 5977A mass-selective detector (Agilent Technologies) to facilitate compound identification. For compound identification, the NIST library along with the mass spectrum and retention times of chromatographic standards (Sigma-Aldrich, St. Louis, MO, USA) were utilized.

2.6. Statistical Analysis

The software SPSS 24 (IBM, Chicago, IL, USA) was used for statistical analyses. All data were normalized and subjected to a homogeneity test before one-way analysis of variance and Duncan’s multiple range test (p < 0.05). The illustration was produced through Origin Pro 2021 (Origin Lab Inc., Northampton, MA, USA) and the ggplot2 package within the R programming environment. OPLS-DA analysis was employed to investigate volatile organic compounds (www.metaboanalyst.ca, accessed on 24 November 2024), and VIP scores were plotted for the two datasets exhibiting statistically significant differences.

3. Results

3.1. Leaf Photosynthetic Pigments

Under foliar fertilization, Se concentrations below 50 ppm increased chlorophyll a, chlorophyll b, and chlorophyll a+b levels, while carotenoid levels decreased (

Table 1. Effect of different concentrations of Se by foliar (A) and rhizosphere (B) fertilization on photosynthetic pigment content of ‘Muscat Hamburg’ grapes in different periods (mean ± SE). Different letters indicate significant differences (p < 0.05) across Se concentrations in the same period.

(A)
Period	Se Concentration /ppm	Chlorophyll a /mg·g−1FW	Chlorophyll b /mg·g−1FW	Total Chlorophyll /mg·g−1FW	Chlorophyll a/b	Carotenoid /mg·g−1FW
BBCH73	0	3.15 ± 0.04 b	7.70 ± 0.24 a	10.85 ± 0.27 a	0.41 ± 0.01 b	2.36 ± 0.21 ab
	50	3.49 ± 0.14 a	7.91 ± 0.07 a	11.40 ± 0.21 a	0.44 ± 0.01 a	2.15 ± 0.09 b
	100	2.36 ± 0.03 c	5.83 ± 0.20 b	8.18 ± 0.23 b	0.41 ± 0.01 b	2.88 ± 0.64 a
	150	2.36 ± 0.13 c	5.71 ± 0.30 b	8.07 ± 0.42 b	0.41 ± 0.01 b	2.77 ± 0.18 ab
BBCH81	0	3.65 ± 0.06 b	8.76 ± 0.15 b	12.41 ± 0.19 b	0.42 ± 0.02 a	3.31 ± 0.24 a
	50	3.92 ± 0.09 a	9.25 ± 0.14 a	13.17 ± 0.23 a	0.42 ± 0.01 a	2.97 ± 0.26 b
	100	3.75 ± 0.05 ab	8.86 ± 0.12 b	12.61 ± 0.17 b	0.42 ± 0.01 a	3.87 ± 0.12 a
	150	3.22 ± 0.04 b	7.71 ± 0.08 b	10.93 ± 0.12 b	0.42 ± 0.02 a	3.50 ± 0.08 a
BBCH89	0	2.34 ± 0.15 a	5.56 ± 0.26 a	7.90 ± 0.40 a	0.42 ± 0.02 a	2.70 ± 0.50 a
	50	2.39 ± 0.13 a	5.89 ± 0.25 a	8.28 ± 0.38 a	0.41 ± 0.01 a	3.20 ± 0.30 a
	100	1.86 ± 0.11 b	4.50 ± 0.11 b	6.36 ± 0.22 b	0.41 ± 0.01 a	2.98 ± 0.51 a
	150	1.72 ± 0.09 b	4.10 ± 0.30 b	5.82 ± 0.39 b	0.42 ± 0.02 a	2.88 ± 0.21 a
(B)
Period	Se Concentration /ppm	Chlorophyll a /mg·g−1FW	Chlorophyll b /mg·g−1FW	Total Chlorophyll /mg·g−1FW	Chlorophyll a/b	Carotenoid /mg·g−1FW
BBCH73	0(CK)	2.46 ± 0.08 b	5.86 ± 0.16 b	8.31 ± 0.24 b	0.42 ±0.01 ab	3.40 ± 0.13 b
	50	2.78 ± 0.15 a	6.52 ± 0.06 a	9.29 ± 0.17 a	0.43 ± 0.02 a	3.15 ± 0.18 b
	100	2.74 ± 0.01 a	6.86 ± 0.09 a	9.60 ± 0.10 a	0.40 ± 0.01 b	3.77 ± 0.13 a
	150	2.34 ± 0.11 b	5.58 ± 0.31 b	7.92 ± 0.42 b	0.42 ± 0.01 ab	2.64 ± 0.27 c
BBCH81	0(CK)	3.60 ± 0.13 ab	8.60 ± 0.19 b	12.19 ± 0.32 b	0.42 ± 0.01 a	4.08 ± 0.32 a
	50	3.86 ± 0.07 a	9.20 ± 0.18 a	13.06 ± 0.14 a	0.42 ± 0.01 a	4.19 ± 0.24 a
	100	3.57 ± 0.19 b	8.60 ± 0.32 b	12.16 ± 0.51 b	0.41 ± 0.02 a	3.27 ± 0.28 b
	150	3.45 ± 0.16 b	8.34 ± 0.37 b	11.79 ± 0.53 b	0.41 ± 0.02 a	4.34 ± 0.13 a
BBCH89	0(CK)	2.38 ± 0.05 a	5.76 ± 0.19 a	8.14 ± 0.23 a	0.41 ± 0.01 a	8.31 ± 0.27 ab
	50	2.53 ± 0.05 a	6.00 ± 0.49 a	8.53 ± 0.48 a	0.42 ± 0.04 a	8.53 ± 0.48 a
	100	2.11 ± 0.16 b	5.07 ± 0.23 b	7.18 ± 0.15 b	0.42 ± 0.05 a	7.18 ± 0.11 b
	150	1.94 ± 0.13 b	4.54 ± 0.37 b	6.48 ± 0.46 c	0.43 ± 0.03 a	6.48 ± 0.46 b

A). No significant difference was observed in the chlorophyll a/b ratio. When compared to the CK, the chlorophyll a, chlorophyll b, and chlorophyll a+b levels increased significantly by 7.3%, 5.6%, and 6.1%, respectively, at a Se concentration of 50 ppm during the BBCH81 stage. At Se concentrations exceeding 100 ppm via foliar fertilization, the chlorophyll a, chlorophyll b, and chlorophyll a+b levels decreased significantly compared to 50 ppm. During the BBCH73 and BBCH81 stages, carotenoid levels decreased by 8.9% and 10.3%, respectively, at 50 ppm Se compared to the CK.

For rhizosphere fertilization, chlorophyll a achieved maximum levels at a Se concentration of 50 ppm, experiencing a 13.0% increase during the BBCH73 stage (Table 1B). Chlorophyll b and chlorophyll a+b levels peaked at 100 ppm during BBCH73, and 50 ppm during BBCH81 and BBCH89. Chlorophyll a/b ratios remained consistent across all treatments. In comparison to the CK, the carotenoid levels significantly decreased by 22.4% at 150 ppm during BBCH73, 19.8% at 100 ppm during BBCH81, and 22.0% at 150 ppm during BBCH89.

3.2. Leaf Chlorophyll Fluorescence

Se application via foliar and rhizosphere fertilization at various concentrations resulted in consistent OJIP curve patterns (Figure 1A,B). The fluorescence intensity at the I point and P point (maximum fluorescence) were higher with 50 ppm Se via foliar fertilization compared to other treatments, whereas a significant increase was observed at 150 ppm via rhizosphere fertilization. The values of S_m, N, and S_m/(tF_m) decreased at 50 ppm via foliar fertilization (Figure 1C,D). Compared to the CK, significant differences were observed in ETo/RC, ABS/CSo, DIo/CSo, TRo/CSo, ETo/CSo, ABS/RC, DIo/RC, and TRo/RC. The 50 ppm treatment prolonged the “t for Fm” in ‘Muscat Hamburg’ grapes under rhizosphere fertilization. The differences in Fv/Fm among the treatments were relatively small, whereas the differences in dVG/dto and dV/dto were larger. Under 50 ppm Se, the Sm value increased, while N and S_m/(tF_m) values decreased. Both PI_abs and PI_total significantly increased under the 50 ppm treatment via both fertilization.

3.3. Fruit Appearance Traits

Grape berry appearance traits were measured as changes in the length, width, and weight of ‘Muscat Hamburg’ grape berries, as well as cluster length and width (

Table 2. Effect of different concentrations of Se by foliar (A) and rhizosphere (B) fertilization on fruit appearance traits of ‘Hamburg’ grape (mean ± SE). Different letters indicate significant differences (p < 0.05) across Se concentrations.

(A)
Se Concentration /ppm	Berry Length /mm	Berry Width /mm	Berry Weight /g	Cluster Length /cm	Cluster Width /cm	Cluster Weight /g
0 (CK)	19.95 ± 0.77 a	18.77 ± 0.55 ab	4.93 ± 0.53 ab	22.35 ± 0.58 c	14.80 ± 0.32 c	634.32 ± 15.03 b
50	19.96 ± 0.20 a	19.31 ± 0.75 a	5.49 ± 0.34 a	24.93 ± 0.37 a	19.27 ± 0.63 a	721.09 ± 24.80 a
100	18.33 ± 0.73 b	18.70 ± 0.58 ab	4.70 ± 0.64 bc	23.33 ± 0.52 b	16.33 ± 0.52 b	626.08 ± 26.95 b
150	18.76 ± 0.58 b	18.04 ± 0.50 b	4.24 ± 0.28 c	19.63 ± 0.36 d	13.17 ± 0.67 d	617.62 ± 19.70 b
(B)
Se Concentration /ppm	Berry Length /mm	Berry Width /mm	Berry Weight /g	Cluster Length /cm	Cluster Width /cm	Cluster Weight /g
0 (CK)	18.64 ± 0.50 b	17.67 ± 0.85 c	4.94 ± 0.42 a	22.35 ± 0.58 b	14.83 ± 0.26 d	529.88 ± 8.11 d
50	18.97 ± 0.53 b	18.34 ± 0.59 bc	5.05 ± 0.43 a	24.50 ± 0.63 a	16.33 ± 0.55 c	628.98 ± 21.95 c
100	19.95 ± 0.77 a	19.31 ± 0.75 a	5.49 ± 0.34 a	24.01 ± 0.89 a	20.00 ± 0.14 a	876.47 ± 29.32 a
150	19.72 ± 0.43 a	18.67 ± 0.76 ab	4.30 ± 0.70 b	24.33 ± 0.68 a	19.13 ± 0.58 b	727.24 ± 30.24 b

). Compared to the CK via foliar fertilization, 50 ppm Se increased berry length and width by 0.11%, with no significant difference. At 100 ppm and 150 ppm Se, berry length decreased by 8.84% and 6.34%, respectively, with significant differences (Table 2A). Berry width at 150 ppm decreased by 4.05% without significance. Berry weight decreased by 16.27% at 150 ppm, a statistically significant change. Cluster length and width significantly increased under the 50 ppm and 100 ppm treatments, while 150 ppm led to a significant reduction. Cluster weight increased by 13.68% at 50 ppm compared to the control.

Compared to CK via rhizosphere fertilization, the length and width of ‘Muscat Hamburg’ grape berries increased under the 50 ppm treatment with no significance, whereas the berry length and width significantly increased significantly under the 100 ppm and 150 ppm treatments, respectively (Table 2B). Berry weight increased by 2.22% and 11.13% at 50 ppm and 100 ppm, respectively, though not significantly. At 150 ppm, berry weight significantly decreased by 14.88%. Cluster length and width significantly increased with increasing Se levels, and cluster weight increased by 18.70%, 65.41%, and 37.25% at 50 ppm, 100 ppm, and 150 ppm, respectively.

3.4. Fruit Flavor Qualities

In comparison to the CK, the total soluble solids (TSSs) of the berry significantly increased by 9.84% and 9.63% at 50 ppm and 100 ppm Se, respectively, via foliar fertilization with Se. Under rhizosphere fertilization, the TSSs increased by 19.38% at 100 ppm and 14.67% at 150 ppm (Figure 2A,B). Titratable acidity (TA) decreased by 20.91% at 50 ppm via foliar fertilization and 15.43% at 100 ppm via rhizosphere fertilization compared to the CK, respectively (Figure 2C,D). The TSS/TA ratio initially rose and subsequently declined with increasing Se concentration, peaking at 50 ppm for foliar fertilization and 100 ppm for rhizosphere fertilization, representing significant increases of 38.83% and 41.72%, respectively (Figure 2E,F).

3.5. Berry Antioxidant Substances

At 100 ppm Se, the flavonoid concentration in the peel increased significantly by 16.81% via foliar fertilization and 26.34% via rhizosphere fertilization, reaching its peak (Figure 3). In the pulp, the flavonoid concentration remained relatively stable with foliar Se fertilization, while decreasing significantly by 28.80%, 20.92%, and 24.55% with increasing concentrations via rhizosphere fertilization. At 150 ppm Se, the total phenol in the peel reached its lowest level, experiencing significant decreases of 4.04% via foliar fertilization and 6.05% via rhizosphere fertilization. The total phenol content in the pulp was minimally affected by foliar Se fertilization but increased by 4.22% at 100 ppm via rhizosphere fertilization compared to the CK. Anthocyanin content in the peel decreased with increasing Se concentration via foliar fertilization, with a 38.35% reduction at 150 ppm compared to the CK. Conversely, rhizosphere fertilization increased peel anthocyanin content by 30.02%, 30.35%, and 86.67% at different concentrations.

3.6. Volatile Organic Compounds

A total of 56 volatile organic compounds were quantitatively identified in the berries of ‘Muscat Hamburg’ grapes, with 5 originating from the amino acid metabolic pathway, 41 from the fatty acid metabolic pathway, and 10 from the isoprene metabolic pathway (Figure 4A). Se treatment with 50 ppm via foliar fertilization significantly elevated VOCs derived from the fatty acid and isoprene metabolic pathways in the berries. However, no significant impact was observed on the VOCs derived from the amino acid metabolic pathway via foliar fertilization. Compared to the CK, the total concentrations of VOCs derived from the amino acid, fatty acid, and isoprene metabolic pathways increased significantly under the 100 ppm and 150 ppm treatments via rhizosphere fertilization (Figure 4B).

OPLS-DA analysis was carried out using VOCs detected in ‘Muscat Hamburg’ grapes as the variables to further elucidate the influence of Se treatment. The VIP scores plot (Figure 4C,D) revealed that Se treatment significantly affected 15 volatile compounds (VIP > 1). Specifically, 50 ppm treatment via foliar fertilization significantly increased 1-pentanol, 3-octanol, butyl butyrate, isoamyl caproate, ethyl lactate, beta-pinene, N-hexanal, and isobutyl acetate. However, 100 ppm via rhizosphere fertilization promoted the accumulation of 15 compounds, including isoamyl caproate, phenylacetaldehyde, citronellyl acetate, 2-methyl-1-pentanol, isobutyl alcohol, caprate, geraniol, N-hexanal, butyric acid ethyl ester, isobutyl acetate, 2-nonanone, ethyl lactate, nonanoate, beta-pinene, and citronellol. Notably, isoamyl caproate, ethyl lactate, N-hexanal, isobutyl acetate, and beta-pinene increased in both 50 ppm treatment via foliar fertilization and 100 ppm via rhizosphere fertilization.

3.7. Correlation Between Fertilization Method and Grape Indices

Mantel’s test analysis (Figure 5) indicated that foliar application of Se significantly increased chlorophyll a, chlorophyll b, and total chlorophyll content (all p < 0.05), with notable effects on chlorophyll fluorescence. Foliar fertilization had no significant effect on TSSs (p = 0.210) but significantly impacted TA (p = 0.002), resulting in a significant change in the TSS/TA ratio (p = 0.009). Foliar fertilization had no significant effect on the flavonoid content in both the peel and pulp of the berry (p = 0.579 and 0.454, respectively), and a limited influence on phenolic compounds (p = 0.805 and 0.632, respectively). Foliar fertilization exhibited a certain positive influence on the length and width of the grape clusters, but its impact on fruit length, width, and weight was relatively weak (p > 0.05). Rhizosphere fertilization significantly increased the flavonoid content in both the pulp and peel (both p < 0.001) and had a more pronounced effect on the size of grape clusters and fruits. Additionally, rhizosphere fertilization significantly improved grape aroma, particularly by promoting VOCs produced through fatty acid metabolism pathways.

4. Discussion

Selenium, recognized as a beneficial element for plant growth and development, regulates key metabolic pathways and enhances stress tolerance mechanisms in plants [31]. This study systematically compared the impacts of foliar and rhizosphere fertilization on the fruit and berry qualities of grapes, combined with the photosynthetic physiology of the leaves. Notably, the effects of Se application varied significantly across measured parameters, demonstrating clear concentration-dependent and application method correlations.

The role of Se lies in increasing photosynthetic pigments by delaying the tissue senescence and raising the leaf chlorophyll content [32]. Filek et al. [33] found that the addition of Se allowed the restoration of the chloroplast ultrastructure, reorganization of the structure of the thylakoids and stroma, and led to an increase in the chloroplast size, fatty acid unsaturation, and fluidity of the cell membrane. In this experiment, the concentration of 50 ppm Se through both fertilization methods led to an increase in the chlorophyll a, chlorophyll b, and chlorophyll a+b (Table 1). A similar pattern has been observed in studies involving in Vitis vinifera [16], Phaseolus aureus [34], and Brassica napus [35]. The underlying mechanism may involve Se-mediated reactivation of membrane-bound enzymes (particularly those containing cysteine thiol groups) and the restoration of chloroplast metabolite transport systems. The improvement of cell membrane integrity in response to Se may reduce electrolyte leakage in plants exposed to environmental stresses [36,37]. However, during the same period, inconsistencies in chlorophyll content variations were observed, attributing to differential Se utilization mechanisms under distinct fertilization methods. Selenium ions can be directly assimilated through leaf stomata and cuticular pathways by foliar application, subsequently undergoing rapid translocation to mesophyll cells via symplastic transport [12]. In contrast, selenium absorption through roots involves sequential processes including rhizosphere uptake, longitudinal xylem transportation, and systemic redistribution—a more protracted translocation pathway that may influence selenium bioavailability and subsequent physiological responses in tissues [13].

Se application enhances photosynthetic efficiency by increasing excitation energy allocation for electron transport and ATP synthesis for CO₂ assimilation, while reducing the plant NPQ, improving heat dissipation, and preventing photodamage [18]. The analysis of OJIP transients demonstrated that Se improved PSII function by enhancing excitation energy trapping (TR0/CS) and electron transport (ET0/CS) per excited cross-section of leaves [38]. The progressive increase in fluorescence signals from the basal level (F0) to the maximum level (Fm), with characteristic intermediate J and I phases, confirmed photosynthetic activity across all treatments [39,40]. The suppression of all reference points of the OJIP transient (0.02, 0.3, 2, 30, and 300 ms) with 50 ppm Se likely indicates a greater allocation of energy to photochemical quenching, given that there was no difference in terms of heat dissipation (DI0/RC) for these treatments (Figure 1A,B). The performance indexes (PI_ABS and PI_Total), which integrate multiple functional and structural parameters [41], showed higher values under the 50 ppm Se treatment, indicating an improved energy conservation capacity of the photosynthetic apparatus [40]. Specifically, elevated PI_ABS values demonstrated Se’s role in maintaining the energy conservation potential for intersystem electron acceptor reduction [24,42]. However, at 50 ppm Se, differential responses were observed between application methods: foliar application reduced S_m and S_m/tF_m (Figure 1C,D), indicating lower energy dissipation, while rhizosphere application prolonged tF_m, suggesting delayed PSII reaction center closure. These findings highlight distinct light utilization optimization strategies between fertilization methods.

The research focus on selenium (Se) treatments has evolved from berry and cluster morphology to their effects on phytochemical alterations. Sucu and Yagci [43] demonstrated a positive correlation between increasing Se concentrations and berry/cluster weights, although cultivar-specific variations were evident. For ‘Muscat Hamburg’ grapes, it was observed that foliar application of Se at a concentration of 50 ppm led to an increase in both berry weight and cluster weight, whereas a significant increase was only evident with rhizosphere application at 100 ppm (Table 2). This differential response suggests that foliar-applied Se might have a more direct and rapid impact on sink strength (cluster and berry development) than rhizosphere. Meanwhile, the TSSs were noted to be increased, with Se contents increasing by foliar and rhizosphere fertilization (Figure 2A,B). These findings align with Zhu et al. [44], who reported similar trends in the ‘Hutai No.08’, ‘Crimson Seedless’, ‘Red Barbara’, and ‘Summer Black’ varieties. The accumulation of dry matter is attributed to an enhanced photosynthetic rate, where optimal selenium concentrations optimize photosynthetic performance by elevating stomatal conductance, boosting chlorophyll biosynthesis, and regulating the tree water status [45,46]. The observed accumulation of TSSs may be attributed to the Se-mediated upregulation of carbohydrate metabolism, a process potentially driven by redox potential modulation [47]. In addition, the application of Se decreased organic acid levels and increased the TAA/TA ratio in berries (Figure 2E,F), as confirmed in kumquat fruit [48]. Foliar fertilization required a lower Se concentration than root fertilization to achieve the desired acid ratio, suggesting that foliar application of Se may be a more sustainable and cost-effective approach for modulating fruit quality parameters.

Se enhances plant antioxidant capacity by upregulating key enzymatic activities, including peroxidase (POD) and superoxide dismutase (SOD), while simultaneously inhibiting malondialdehyde (MDA) accumulation [49]. This mechanism effectively scavenges the overproduction of reactive oxygen species (ROS) induced by abiotic stress, thereby mitigating oxidative damage [50]. In grapevine systems, polyphenols serve as potent antioxidants that work in concert with other cellular defense mechanisms to preserve redox homeostasis during stressful conditions [27]. In this study, at appropriate levels of Se via foliar and rhizosphere fertilization, the content of flavonoid compounds increased, and the total phenol decreased in the peel. In the pulp, the flavonoids decreased, and total phenol increased with rhizosphere fertilization, whereas no significance was observed with foliar fertilization (Figure 3). In the study by Karimi [51], the total phenol content of vine leaves treated with 20 mg·L⁻¹ Se decreased, and the highest total flavonoid content was observed at concentration of 5 and 10 mg·L⁻¹. This is attributed to the fact that Se exhibits oxidant properties at high levels [52]. As the Se concentration increased, the anthocyanin content in the peel of ‘Muscat Hamburg’ grapes treated via foliar fertilization dropped to its lowest level, while that of ‘Muscat Hamburg’ berries receiving rhizosphere fertilization rose to its highest. This discrepancy may stem from the rapid absorption of Se when sprayed on the leaves, leading to the oxidative breakdown of anthocyanins [31]. Rhizosphere Se enhanced anthocyanins, possibly through improved nutrient availability and antioxidant synergy [53].

Se plays a pivotal role in promoting the synthesis of aroma components in fruits. In this study, Se treatment at 50 ppm via foliar fertilization significantly increased VOCs from the fatty acid and isoprene pathways, while 100 ppm and 150 ppm treatments via rhizosphere fertilization increased VOCs from all three pathways (Figure 4B). The production of volatile compounds including alcohols, aldehydes, ketones, acids, and esters in fruits may be related to the activity of enzymes like lipoxygenase and peroxidase. Liu et al. [19] showed that Se treatment increased the expression of two LOX genes in broccoli heads. LOX is a cold non-heme iron oxygenase that catalyzes the conversion of unsaturated fatty acids into various volatile lipids. Additionally, Se appears to stimulate the biosynthesis of specific carbohydrates, namely citronellol, linalool, geraniol, α-terpineol, and nerol, which are the primary components responsible for the delightful Muscat flavor [54] (Figure 4C,D). This indicates that Se treatment possesses the potential to enhance the flavor of ‘Muscat Hamburg’ grapes, with a more pronounced effect observed in rhizosphere fertilization (Figure 5). This may be attributed to the fact that rhizosphere application of Se results in a lower proportion of the oxidized forms of essential oils compared to foliar application [20,55]. The overlap in upregulated compounds (e.g., beta-pinene, isoamyl caproate) between foliar and rhizosphere treatments suggests conserved metabolic targets, though pathway dominance varies with the application method. This provides a new direction for future research.

5. Conclusions

The present study elucidates the differential impacts of Se fertilization methods (foliar vs. rhizosphere) and concentrations on grape physiology, fruit quality, and metabolism. Optimal Se application significantly enhanced photosynthetic efficiency, with foliar fertilization at 50 ppm increasing chlorophyll a, b, and total chlorophyll levels, while higher concentrations (>100 ppm) exhibited inhibitory effects. Se also improved chlorophyll fluorescence parameters, indicating enhanced energy capture and electron transport within the photosynthetic system. Se treatment significantly enhanced TSSs and reduced the TA of grapes, with the most pronounced effects observed at 50 ppm via foliar fertilization and 100 ppm via rhizosphere fertilization. Se via foliar fertilization reduced anthocyanins in peels due to oxidative stress, while rhizosphere fertilization synergistically boosted anthocyanins and phenolics. Se at 50 ppm via foliar fertilization enhanced berry weight and cluster traits, whereas rhizosphere application at 100 ppm maximized VOCs, likely through the upregulation of fatty acid and isoprene metabolic pathways. Mantel’s test further emphasized the distinct and sometimes complementary roles of foliar and rhizosphere fertilization. Foliar fertilization was more effective in modulating photosynthetic parameters and had a moderate influence on fruit size and flavor components. Rhizosphere fertilization, on the other hand, had a stronger impact on fruit antioxidant content, overall size, and aroma. These findings indicate that Se as a growth enhancer and stress mitigator is critically dependent on both concentration and delivery route. However, this study was conducted in a greenhouse environment with controlled and stable temperature, lighting, and humidity conditions, which differ significantly from outdoor settings. Despite our recommendation for its use, a higher degree of caution is warranted when applying these findings to outdoor cultivation. In future research, not only should the synergistic effects of combined foliar and rhizosphere applications be explored for their potential to enhance grapevine performance, but also the impact of Se application on the absorption and enrichment of other macro- and microelements should be thoroughly considered.