Effects of Graphene on Yield, Grain Quality, 2-AP Biosynthesis and Antioxidant Systems of Fragrant Rice

Abstract

The application of nanotechnology in agricultural neighborhoods is rapidly developing with the aim of promoting growth and enhancing crop tolerance to environmental stresses. However, there are fewer reports on the application of graphene nanoparticles in practical production, especially in fragrant rice. In early-season and late-season pot experiments conducted in 2022, the effects of graphene on the yield, grain quality, 2-acetyl-1-pyrroline (2-AP) and antioxidant systems of two fragrant rice cultivars (19× and Meixiangzhan) were examined at concentrations of 9 g/hm², 18 g/hm² and 27 g/hm². The results showed that graphene T1 treatment at 9 g/hm² significantly increased the activity of PDH and P5CS, promoted the synthesis of proline and P5C and significantly increased the 2-AP content of the grains of the two fragrant rice cultivars by 10.33–39.88% and 22.05–65.76%, respectively, in both growing seasons. Meanwhile, the lower concentration of T1 treatment (9 g/hm²) increased the grains per panicle and 1000-grain weight, enhancing the grain yield of both fragrant rice cultivars. The T1 treatment (9 g/hm²) had significant effects on the appearance and nutritional quality of both fragrant rice cultivars. It increased the head rice rate and protein content of the grains while also increasing the amylose content of 19× and reducing the chalkiness degree of 19×. Conversely, the T1 treatment reduced the amylose content and increased the chalkiness degree of Meixiangzhan. In addition, the low concentration of T1 treatment significantly increased the POD and SOD activities, increased the content of photosynthetic pigments and decreased the content of MDA in the leaves. However, 18 g/hm² and 27 g/hm² had slightly negative effects on yield, grain quality and fragrance biosynthesis in both fragrant rice cultivars. Furthermore, the results of structural equation modeling showed that antioxidant enzymes had a significant, positive effect on the grain’s 2-AP content and 2-AP synthesis-related enzyme activity, and photosynthetic pigments had a significant, positive effect on yield and grain appearance quality, while rice appearance quality and nutritional quality had significant, positive effects on yield. Overall, this study showed that suitable concentrations of graphene have good potential for use in fragrant rice production, but additional attention should be paid to the concentration of graphene application.

1. Introduction

Rice is a type of crop known for its long history of consumption, and it is a staple food for most people in the Asian region. Fragrant rice is a rice cultivar with a special aroma that gives it a higher value than conventional rice cultivars, so its demand has increased [1,2]. Consequently, numerous researchers have taken an interest in studying fragrant rice in recent years [3,4].

Research has indicated that the primary component responsible for fragrant volatiles in fragrant rice cultivars is 2-acetyl-1-pyrroline (2-AP) [5,6]. Typically, 2-AP is present in all growing parts of fragrant rice except the root system [7,8]. Currently, the biosynthetic pathway of 2-AP in aromatic rice involves two mechanisms. One pathway involves proline, ornithine and glutamate being transformed into pyrrolin-5-carboxylic acid (P5C), which is then converted into pyrroline by proline dehydrogenase (PDH), pyrrolin-5-carboxylic acid synthase (P5CS) and ornithine aminotransferase (OAT) [9,10]. In the other pathway, betaine aldehyde dehydrogenase (BADH) is inactivated, resulting in the lack of regulation of the transformation of γ-aminobutyraldehyde into γ-aminobutyric acid; instead, it is converted into pyrroline, which ultimately leads to the formation of 2-AP [11]. Previously, work by Mo et al. [1] found that silica fertilization increased 2-AP content in the grains and improved rice quality. Drought and salt conditions have also been reported to promote 2-AP synthesis [12]. Moreover, foliar spraying of chelated selenium improved rice quality in fragrant rice and promoted 2-AP synthesis by moderating related enzymes and precursors [13]; it has also been shown that exogenous copper application can increase 2-AP in the grains by increasing the content of precursors, such as proline and P5C [14]. Therefore, it is feasible that the 2-AP content of fragrant rice can be increased by suitable agronomic measures.

Advances in nanotechnology have boosted the creation of diverse carbon nanomaterials. These materials have demonstrated positive effects on plant growth and development [15], eliminating heavy metals from the soil [16], enhancing the effectiveness of pesticides and insecticides [17] and maintaining the slow-release effect of fertilizers [18,19]. Graphene is a new type of nano-carbon broadly used in the materials, energy and environmental fields due to its unique surface morphology, large specific surface area and excellent physicochemical properties, while graphene also has many applications in agriculture. Graphene has been reported to ameliorate saline–alkaline stress injury in alfalfa [20]. Moreover, a study showed that graphene oxide invoked the expression of tomato root development-related genes (SlExt1 and LeCTR1) and increased root growth hormone content [21]. Similarly, another study demonstrated that graphene oxide can increase drought tolerance in soybean by increasing the content of salicylic acid, jasmonic acid and abscisic acid, as well as promoting the expression of drought tolerance genes [22]. However, high concentrations of graphene may have adverse effects on crops, limiting plant growth and biomass, which was observed in cabbage, tomato and spinach [23]. Additionally, it was shown that 100–200 mg/L of graphene significantly inhibited root length, stem length, root fresh weight and aboveground fresh weight of rice seedlings [24].

Previous studies have revealed that several agronomic practices can influence the yield, accumulation of fragrance and antioxidant system of fragrant rice [3,25]. In addition, the majority of current research concerning the effects of graphene on crop growth has been conducted through hydroponic experiments, yet the impact of graphene on fragrance formation and growth of fragrant rice remains unexplored. Therefore, we conducted an investigation and analyzed the effects of graphene on indicators of growth and the antioxidant system of fragrant rice in potted plants, simulating a field environment. The specific aims of this study were: (1) to explore the impact of graphene on the yield, grain quality and fragrance formation; and (2) to analyze the effects of antioxidant responses on the yield, grain quality and accumulation of 2-AP in fragrant rice under graphene treatments.

2. Materials and Methods

2.1. Plant Material and Experimental Details

To explore the effects of graphene on the growth and aroma synthesis of fragrant rice, we selected 19× and Meixiangzhan as the experimental materials. The trial was conducted at an experimental farm at South China Agricultural University in 2022, with the early season trial conducted from March to July 2022 and the late season trial conducted from July to November 2022. The two fragrant rice cultivars are widely planted in southern China, and the experimental material was provided by the School of Agriculture at South China Agricultural University. The seeds were soaked in water for 24 h before sowing. They were sown on 10 March in the early season and on 14 July in the late season. The seedlings were grown to about 15 cm in moist conditions and transplanted into plastic pots (30 cm × 25 cm), each containing 12.5 kg of rice soil. We harvested them on 10 July for the early season and 27 October for the late season.

Graphene is available in powder form with a particle size of 0.68–3.4 nm, provided by Guangzhou Longteng Energy Equipment Company (Guangzhou, China). It was mixed with fertilizer before sowing and then applied to the soil one time. The three treatments were: T1: 9 g/hm²; T2: 18 g/hm²; and T3: 27 g/hm²; and the normal fertilizer application without graphene was the control CK. For each rice treatment, a total of 12 pots were treated with 3.52 g of urea (46% N), 4.32 g of calcium superphosphate (12% P₂O₅) and 1.08 g of potassium chloride (K₂O) per pot. During the early and late season growth and development, a water layer of 2–4 cm was maintained, while other standards followed the fragrant rice cultivation protocols.

2.2. Determination of Yield and Yield-Related Attributes

At maturity, four pots were randomly selected from each treatment, dried in the sun (grain moisture content of about 13%) and then threshed by hand to obtain the yield per pot, expressed in g/pot. The panicle number per hill was determined by calculating the average number of spikes from 10 rice plants of consistent growth condition in each treatment. From four representative rice plants under each treatment, we calculated the number of grains per panicle, the 1000-grain weight and the filled grains rate.

2.3. Determination of Grain Quality

Rice quality was determined according to the method of Li et al. [26]. Each treatment was taken from the warehouse with 400 g of rice, first dehulled in a huller, then processed in a finishing machine (Taizhou, China) and finally weighted and screened to obtain the head rice rate. The chalkiness of fragrant rice was assessed using an SDE-A light box (Guangzhou, China). The determination of the grain protein and amylose content was performed by an Infratec 1241 grain analyzer (FOSS-TECATOR). The head rice rate was determined based on the method provided by Mo et al. [1] and calculated using the following formula:

Head rice rate (%) = Weight of the head rice/Weight of the grain sample

2.4. Determination of 2-AP Content in Grains

The 2-AP content was determined according to the method of Mo et al. [8]. Grains (at full heading) were ground with liquid nitrogen, and then approximately 2.0 g of powdered sample was weighed, mixed with 10 mL of dichloromethane and held in an ultrasonic cleaner at 40 °C for 4 h. Afterward, an appropriate amount of anhydrous sodium sulfite was added to the extract to absorb water, and the extracts were analyzed using GCMS-QP 2010 Plus (Shimadzu Corporation, Kyoto, Japan). The 2-AP content was expressed in µg kg⁻¹ fresh weight (FW).

2.5. Determination of Parameters Related to 2-AP Accumulation

The content of proline, P5C and pyrroline in leaves at full heading stage was determined according to the method of Li et al. [26]. In addition, the activities of PDH and P5CS were determined by improving on the method of Ren et al. [14]. The proline contents were expressed as ug g⁻¹ FW; the P5C contents were expressed as µmol g⁻¹ FW; the pyrroline content was expressed as mmol g⁻¹ FW; and the PDH and P5CS activities were expressed as µmol g⁻¹ FW and U g⁻¹ h⁻¹ FW.

2.6. Determination of Parameters Related to Antioxidant Reactions

Leaves were collected from each treatment at the full heading stage and removed from the ultra-low temperature refrigerator at −80 °C for the measuring. The activities of POD, SOD, CAT and MDA were determined according to the method of Gui et al. [3]. The POO, SOD and CAT activities were expressed as U g⁻¹ min⁻¹ FW, U g⁻¹ FW and mmol min⁻¹ g⁻¹ FW, respectively, and the MDA content was expressed as µmol g⁻¹ FW.

2.7. Determination of Pigment Contents

The content of chlorophyll a, chlorophyll b and carotenoids was estimated according to the method of Cheng et al. [25]. The extract was obtained with 95% ethanol solution for 24 h, followed by absorbance readings at 665, 649 and 470 nm using an enzyme marker. The pigment content was expressed as mg g⁻¹ FW.

2.8. Statistical Analysis

In this study, we utilized Statistic software, version 8.0 (Analytical Software, Tallahassee, FL, USA) to collect and analyze the data. To compare multiple variables, we used the least significant difference (LSD) test with a significance level of p < 0.05. Graphs were drawn using OriginPro 2021 software. In addition, we generated structural equation models using the nlme (version 3.1–162), lme4 (version 1.1–32), piecewiseSEM (version 2.1.2) and QuantPsyc (version 1.6) packages of R software, version 4.2.1, adopting the method of Tian et al. [27].

3. Results

3.1. Effect of Graphene Application on Yield and Yield-Related Attributes in Rice

Season (S), cultivar (C), treatment (T), S × T and S × C × T significantly affected the yield of both fragrant rice cultivars (

Table 1. Effect of graphene application on yield and yield-related attributes of fragrant rice in early and late seasons.

Season	Cultivar	Treatment	Panicle Number per Hill	Grains per Panicle	Filled Grains Rate (%)	1000-Grain Weight (g)	Yield (g/pot)
Early season	19×	CK	7.17 $\pm$ $0.75 \mathrm{a}$	101.17 $\pm$ $2.92$ ab	75.46 $\pm$ $1.22 \mathrm{b}$	20.91 $\pm$ $0.25 \mathrm{b}$	61.16 $\pm$ $1.42 \mathrm{ab}$
		T1	7.33 $\pm$ $1.03 \mathrm{a}$	107.10 $\pm$ $4.97$ a	77.03 $\pm$ $3.09 \mathrm{b}$	22.34 $\pm$ $0.60$ a	62.94 $\pm$ $2.73 \mathrm{a}$
		T2	7.50 $\pm$ $1.05 \mathrm{a}$	103.77 $\pm$ $5.30$ ab	85.08 $\pm$ $2.97\mathrm{a}$	21.34 $\pm$ $0.80$ ab	58.75 $\pm$ $1.56 \mathrm{b}$
		T3	7.00 $\pm$ $0.89 \mathrm{a}$	95.98 $\pm$ $5.60$ b	80.09 $\pm$ $3.18 \mathrm{ab}$	20.67 $\pm$ $0.61 \mathrm{b}$	59.25 $\pm$ $1.69 \mathrm{ab}$
	Meixiangzhan	CK	9.00 $\pm$ $1.10 \mathrm{a}$	83.12 $\pm$ $3.81$ ab	89.10 ± 1.78 ab	19.84 $\pm$ $0.24 \mathrm{b}$	48.60 $\pm$ $2.08 \mathrm{b}$
		T1	9.33 $\pm$ $1.63 \mathrm{a}$	90.46 $\pm$ $3.17$ a	90.47 ± 1.19 a	21.08 $\pm$ $0.74 \mathrm{a}$	53.76 $\pm$ $0.26 \mathrm{a}$
		T2	8.83 $\pm$ $0.98 \mathrm{a}$	76.66 b $\pm$ $2.75$ bc	86.46 ± 2.45 ab	20.41 $\pm$ $0.24$ ab	50.72 $\pm$ $3.22 \mathrm{ab}$
		T3	9.17 $\pm$ $1.17 \mathrm{a}$	73.43 $\pm$ $3.84$ c	84.62 ± 1.28 c	19.76 $\pm$ $0.22$ b	47.88 $\pm$ $1.52 \mathrm{b}$
Late season	19×	CK	6.83 $\pm$ $0.75 \mathrm{a}$	104.84 $\pm$ $2.51 \mathrm{b}$	62.67 $\pm$ $1.36$ a	20.64 $\pm$ $0.10$ ab	39.57 $\pm$ $2.21$ b
		T1	7.50 $\pm$ $1.05 \mathrm{a}$	121.42 $\pm$ $1.60 \mathrm{a}$	65.52 $\pm$ $1.53$ a	20.93 $\pm$ $0.32$ ab	48.66$\pm$ $3.23$ a
		T2	6.67 $\pm$ $0.52 \mathrm{a}$	105.01 $\pm$ $3.30 \mathrm{b}$	64.26 $\pm$ $2.44$ a	21.08 $\pm$ $0.27$ a	45.19 $\pm$ $3.05$ ab
		T3	6.50 $\pm$ $1.05 \mathrm{a}$	102.18 $\pm$ $2.24 \mathrm{b}$	62.58 $\pm$ $1.84$ a	20.26 $\pm$ $0.36$ b	41.11 $\pm$ $1.79$ b
	Meixiangzhan	CK	7.00 $\pm$ $0.89 \mathrm{a}$	76.85 $\pm$ $3.08$ ab	73.36 ± 1.19 b	17.21 $\pm$ $0.09$ b	34.13 $\pm$ $1.89$ ab
		T1	7.67 $\pm$ $0.82 \mathrm{a}$	81.77 $\pm$ $4.79$ a	77.44 ± 1.45 a	17.81 $\pm$ $0.36$ a	37.42 $\pm$ $1.37$ a
		T2	7.50 $\pm$ $0.84 \mathrm{a}$	78.44 $\pm$ $2.67$ a	73.90 ± 1.46 b	17.75 $\pm$ $0.28$ a	35.12 $\pm$ $1.56$ ab
		T3	7.33 $\pm$ $1.21 \mathrm{a}$	69.25 $\pm$ $5.71$ b	70.82 ± 2.17 b	17.05 $\pm$ $0.14$ b	32.63 $\pm$ $1.07$ b
ANOVE	S		*	ns	**	**	**
	C		**	**	**	**	**
	T		**	**	**	**	**
	S × C		**	*	ns	**	ns
	S × T		ns	ns	ns	ns	*
	C × T		Ns	ns	**	ns	ns
	S × C × T		Ns	*	**	ns	**

Values followed by different lowercase letters within a column represent significant differences among the treatments at p < 0.05. S: Season; C: Cultivar; T: Treatment; S × C: Interaction between season and cultivar; S × T: Interaction between season and treatment; C × T: Interaction between cul-tivar and treatment; S × C × T: Interaction among season, cultivar, and treatment, * and ** represent a significant difference at p < 0.05 and p < 0.01, respectively; ns represents a non-significant differ-ence (LSD).

). The yield of both fragrant rice cultivars was highest with the T1 treatment in both seasons, with significant increases of 10.62% and 22.98% in the early season for Meixiangzhan and 19× in the late season, respectively, compared with CK. However, with the increase in graphene concentration, the yield of both fragrant rice cultivars subsequently decreased. For 19×, the graphene treatment had no significant effect on panicle number per hill but had significant effects on grains per panicle, filled grains rate and 1000-grain weight, with the T1 treatment significantly increasing 6.36% of 1000-grain weight in the early season and the T2 treatment significantly increasing the filled grains rate by 12.74%; meanwhile, the T1 treatment significantly increased by 15.82% the grains per panicle in the late season. For Meixiangzhan, the early season T1 treatment significantly increased the 1000-grain weight by 6.26%. The late season T1 treatment significantly increased the filled grains rate by 5.56% of and the 1000-grain weight by 3.47%. Notably, the high concentration of graphene T3 treatment significantly reduced the number of grains per panicle and the filled grains rat in the early season of Meixiangzhan by 9.89% and 5.03%, respectively.

3.2. Effect of Graphene Application on Grain Quality

Cultivar (C) and treatment (T) both significantly affected the head rice rate, chalkiness degree and amylose content (

Table 2. Effect of graphene application on the quality of fragrant rice in early and late seasons.

Season	Cultivar	Treatment	Head Rice Rate (%)	Chalkiness Degree (%)	Protein Content (%)	Amylose Content (%)
Early season	19×	CK	40.07 ± 1.06 b	14.68 ± 1.99 a	6.70 ± 0.08 b	16.60 ± 0.10 b
		T1	42.05 ± 0.55 a	9.34 ± 0.87 b	7.10 ± 0.08 a	17.10 ± 0.26 a
		T2	41.52 ± 0.99 ab	9.86 ± 0.59 b	6.75 ± 0.17 b	16.53 ± 0.23 b
		T3	41.10 ± 0.49 ab	10.46 ± 1.38 b	6.93 ± 0.21 ab	16.40 ± 0.20 b
	Meixiangzhan	CK	31.76 ± 0.45 c	22.50 ± 1.23 bc	7.03 ± 0.05 c	16.53 ± 0.47 a
		T1	33.71 ± 0.76 b	20.22 ± 1.56 c	7.10 ± 0.05 bc	16.27 ± 0.38 a
		T2	34.10 ± 0.31 b	23.14 ± 1.80 ab	7.30 ± 0.08 a	16.10 ± 0.26 a
		T3	35.17 ± 0.50 a	25.27 ± 0.60 a	7.23 ± 0.10 ab	15.93 ± 0.15 a
Late season	19×	CK	54.18 ± 0.57 b	3.71 ± 0.56 a	6.73 ± 0.05 c	18.57 ± 0.23 ab
		T1	56.67 ± 0.75 a	2.21 ± 0.24 b	6.75 ± 0.06 bc	18.80 ± 0.10 a
		T2	55.49 ± 0.55 ab	2.87 ± 0.44 ab	7.03 ± 0.05 a	18.30 ± 0.10 b
		T3	54.17 ± 1.04 b	3.52 ± 0.74 a	6.85 ± 0.10 b	18.50 ± 0.10 b
	Meixiangzhan	CK	62.26 ± 0.69 b	9.10 ± 0.08 c	7.10 ± 0.08 a	18.47 ± 0.15 a
		T1	63.52 ± 0.50 a	10.30 ± 1.00 c	7.13 ± 0.10 a	17.80 ± 0.20 b
		T2	64.23 ± 0.87 a	12.17 ± 0.74 b	7.15 ± 0.13 a	17.93 ± 0.21 b
		T3	63.94 ± 0.40 a	15.00 ± 1.22 a	7.20 ± 0.14 a	17.90 ± 0.10 b
ANOVE	S		**	**	ns	**
	C		**	**	**	**
	T		**	**	**	**
	S × C		**	**	ns	ns
	S × T		ns	**	ns	ns
	C × T		**	**	ns	**
	S × C × T		ns	ns	**	ns

Values followed by different lowercase letters within a column represent significant differences among the treatments at p < 0.05. S: Season; C: Cultivar; T: Treatment; S × C: Interaction between season and cultivar; S × T: Interaction between season and treatment; C × T: Interaction between cultivar and treatment; S × C × T: Interaction among season, cultivar, and treatment, ** represents a significant difference at p < 0.01; ns represents a non-significant difference (LSD).

). For 19×, the T1 treatment significantly increased the head rice rate by 4.94% and 4.60% in the early and late seasons, respectively. As for nutritional quality, the T1 treatment also significantly increased the protein content by 5.97% and amylose content by 3.01% in the early season, whereas the T2 and T3 treatments significantly increased the protein content by 4.38% and 1.78% in the late season, respectively. However, all graphene treatments significantly reduced chalkiness degree by 19× in the early season, but only the T1 treatment significantly reduced the chalkiness degree by 40.43% in the late season. For Meixiangzhan, graphene treatments significantly increased the head rice rate in both early and late seasons. In terms of nutritional quality, the T2 and T3 treatments significantly increased the protein content by 3.84% and 2.77% in the early season, respectively. Notably, the graphene treatments reduced the amylose content by 1.59–3.61% in the early season while decreasing the amylose content by 2.91–3.63% in the late season, reaching a significant level. Meanwhile, the T3 treatment significantly increased the chalkiness degree in the early season by 12.31%, while the T2 and T3 treatments significantly increased the chalkiness by 33.74% and 64.87%, respectively.

3.3. Effect of Graphene Application on 2-AP Content in Grains

Season (S), cultivar (C), treatment (T), S × T and C × T significantly influenced the 2-AP content in grains (

Table 3. Analysis of variance (ANOVA) of the investigated rice parameters.

	S	C	T	S × C	S × T	C × T	S × C × T
2-AP	**	*	**	ns	**	**	ns
Proline	*	ns	**	ns	ns	*	**
P5C	ns	**	**	ns	ns	ns	ns
Pyrroline	**	ns	**	*	*	ns	ns
PDH	ns	**	**	ns	**	**	ns
P5CS	ns	ns	**	ns	*	*	**
POD	ns	ns	**	ns	ns	ns	**
SOD	**	ns	**	ns	**	**	**
CAT	*	*	*	**	**	ns	*
MDA	**	ns	**	ns	ns	**	ns
Chlorophyll a	**	**	**	ns	**	**	**
Chlorophyll b	**	ns	**	**	**	**	**
Total chlorophyll	**	*	**	ns	**	**	**
Carotenoid	**	ns	**	**	**	**	**

* and ** represent a significant difference at p < 0.05 and p < 0.01, respectively; ns represents a non-significant difference (LSD). S, season; C, cultivar; T, treatment.

). The trend of 2-AP content in the grains of the two fragrant rice cultivar at the full heading stage was the same in the early and late seasons, showing an increasing and then decreasing trend with the increase in graphene treatment concentration (Figure 1A,B). For 19×, the T1 treatment significantly increased the 2-AP content by 10.33% and 39.88% in both seasons. For Meixiangzhan, all graphene treatments significantly increased the 2-AP content in the early season, but only T1 and T2 treatments significantly increased the 2-AP content in the late season, and T1 treatment had the highest 2-AP content of 240.98 µg kg⁻¹ and 231.37 µg kg⁻¹ in the early and late seasons, respectively.

3.4. Effect of Graphene Application on Proline, P5C, and Pyrroline Content

Graphene treatment (T) significantly affected proline, P5C and pyrroline content in full heading stage leaves (Table 3). For 19×, the T1 treatment significantly increased proline content by 17.67% and pyrroline content by 71.61% in the early season, while only the T1 treatment significantly increased the pyrroline content of 19× by 51.96% in the late season. In contrast, for Meixiangzhan, the T1 treatment significantly increased the proline content by 20.92%, P5C content by 23.48% and pyrroline content by 35.25% in the early season (Figure 2A,C,E). In the late season, the T1 and T2 treatments significantly increased the proline content of Meixiangzhan by 57.54% and 43.85%, along with T1 treatments significantly increasing the P5C content by 52.55% and the pyrroline content by 44.19% (Figure 2B,D,F). Notably, high graphene concentrations of the T2 and T3 treatments significantly reduced the proline content of 19× by 19.58% and 23.29% (Figure 2B), respectively, in parallel with T3 treatments significantly reducing the P5C content by 19.50% (Figure 2D).

3.5. Effect of Graphene Application on Activity of PDH and P5CS

Cultivar (C), treatment (T), S × T and C × T significantly affected PDH activity in leaves (Table 3). The PDH activity of both 19× and Meixiangzhan in both the early and late seasons showed a trend of increasing and then decreasing with increasing graphene concentration. For 19×, the T2 treatment significantly increased the PDH activity of 19× in the early season by 31.43% (Figure 3A). In the late season, the T1 and T2 treatments significantly increased the PDH activity of 19× by 30.48% and 38.90%, respectively, reaching the highest with the T2 treatment (Figure 3B). However, the high concentration treatment significantly decreased the PDH activity, with the T3 treatment significantly diminishing the PDH activity in leaves by 43.54% in the late season (Figure 3B). For Meixiangzhan, the T1 treatment significantly increased PDH activity in the early and late seasons by 21.69% and 52.35% (Figure 3A,B), respectively, but high concentrations of graphene T2 and T3 treatments significantly reduced the PDH activity by 9.11% and 30.23% in the late season (Figure 3B).

Treatments (T), S × T, C × T and S × C × T significantly affected the P5CS activity in leaves (Table 3). For 19×, in the early season, graphene treatments significantly increased the P5CS activity in leaves by 8.20–20.59% (Figure 3C), but in the late season, only the T1 treatment increased the P5CS activity in leaves by 26.66%. In contrast, the high graphene concentration of T3 treatment significantly diminished the P5CS activity compared to CK, with a significant decrease of 24.70% (Figure 3D). For Meixiangzhan, the T1 treatment significantly increased the P5CS activity in early season leaves by 9.87% (Figure 3C), whereas T1 and T2 treatments significantly increased by 41.77% and 29.62% the P5CS activity in the late season, respectively (Figure 3D).

3.6. Effect on Antioxidant Reaction Parameters

Treatment (T) and S × C × T significantly affected the activity of POD (Table 3). Application of graphene treatment increased the POD activity of both fragrant rice cultivars in both seasons to different degrees, but the POD activity showed a trend of increasing and then decreasing with increasing graphene concentrations. For 19×, the highest activity was observed in the early season T1 treatment with a significant increase of 54.63% compared to the controls, and the maximum increase in POD activity was 83.79% in the late season T2 treatment. Conversely, for Meixiangzhan, the elevation of POD activity for each treatment did not increase as greatly as in 19×, with only a significant increase of 45.99% with the early season T2 treatment and 34.38% with the late season T1 treatment (Figure 4A,B).

Season (S), treatment (T), S × T, C × T and S × C × T significantly affected the activity of SOD (Table 3). In the early season, 19× showed a trend of increasing and then decreasing SOD activity with increasing graphene concentrations, and the T1 treatment significantly increased SOD activity by 18.12%; in contrast, the SOD activity of Meixiangzhan showed an increase with the increasing graphene application concentrations, reaching significance with all treatments. In the late season, the SOD activity of both 19× and Meixiangzhan showed a trend of increasing and then decreasing with the applied graphene concentrations. Remarkably, the high concentration of graphene T3 treatment significantly decreased the SOD activity of 19× by 9.62% (Figure 4C,D).

Season (S), cultivar (C), treatment(T), S × C, S × T and S × C × T significantly affected the activity of CAT (Table 3). Graphene increased the activity of CAT in full heading stage leaves relatively slightly. In the case of 19×, the highest activity of CAT was observed in the early season under T2 treatment, while the late season showed the highest activity under T1 treatment. For Meixiangzhan, the T2 and T3 treatments significantly increased the activity of CAT in the early season by 5.19% and 5.97%, respectively, while only the T1 treatment significantly increased the activity of CAT in the late season by 6.46% (Figure 4E,F).

Season (S), treatment (T) and C × T significantly affected the content of MDA (Table 3). For 19×, the graphene treatments reduced MDA content in leaves by 17.38–31.49% and 10.51–21.29% in two seasons, respectively, but the effect was weakened with increasing graphene concentrations. The T1 treatment was the most effective in reducing MDA content in both seasons and significantly reduced the MDA content in leaves. For Meixiangzhan, MDA in leaves accumulated with increasing graphene concentrations in both seasons, and the T1 treatment had some effect in reducing MDA content in leaves compared to CK in the late season, but it did not have a significant effect. In particular, the elevated T3 treatment significantly increased MDA content in leaves in both seasons, by 44.25% and 39.30%, respectively (Figure 4G,H).

3.7. Effect of Graphene Application on the Photosynthetic Pigments Contents

Season (S), treatment (T), S × T, C × T and S × C × T significantly affected the content of photosynthetic pigments in leaves (Table 3). Concerning 19×, the T1 treatment significantly enhanced the content by 37.99% of chlorophyll a, by 38.04% of chlorophyll b, by 24.08% of carotenoids and by 34.77% of total chlorophyll in the early season, compared to CK (Figure 5A,C,E,G), whereas high concentrations of graphene T2 and T3 treatments significantly decreased the content of pigments. Only the T1 treatment significantly increased chlorophyll a by 28.41% and carotenoids by 14.77% in 19× in the late season (Figure 5B,D,F,H). However, for Meixiangzhan, all graphene treatments significantly increased chlorophyll a, chlorophyll b, carotenoids and total chlorophyll in the early season leaves (Figure 5A,C,E,G), but only the T1 treatment significantly increased the content of these pigments in the late season (Figure 5B,D,F,H).

3.8. Correlation Analysis

As can be seen from the heatmap (Figure 6), yield was significantly positively correlated at p ≤ 0.05 with grains per panicle, filled grains rate, 1000-grain weight, the activity of PDH and CAT and the content of 2-AP, proline, P5C, MDA, chlorophyll a, chlorophyll b, total chlorophyll and carotenoid at the p ≤ 0.05 level while significantly negatively correlated with the activity of SOD, content of pyrroline, head rice rate, protein and amylose. The content of 2-AP was significantly positively correlated with yield, grains per panicle, panicle number per hill, filled grains rate, 1000-grain weight, the activity of PDH, P5CS, POD and CAT and the content of proline, P5C, chlorophyll a, chlorophyll b, total chlorophyll and carotenoid at the p ≤ 0.05 level, while significantly negatively correlated with the activity of SOD, head rice rate and amylose.

3.9. Path Analysis

Moreover, for further clarification of the effects of photosynthetic pigments, antioxidant enzymes and parameters related to 2-AP synthesis regarding yield, grain quality and 2-AP content, a structural equation model was constructed (Figure 7). The results of the structural equation fit were Fisher’s C = 15.73, p-value = 0.61, AIC = 99.73 and BIC = 159.96, indicating a good model fit (* p < 0.05, ** p < 0.01, *** p < 0.001). Photosynthetic pigments had a significant, direct, positive influence on yield and grain appearance quality, and antioxidant enzymes had a significant, positive influence on 2-AP content and 2-AP synthesis-related enzymes. In contrast, MDA had a significant, positive influence on the nutritional quality of grain rice but a negative influence on yield, 2-AP synthesis-related enzymes and 2-AP synthesis-related substance. At the same time, it is worth mentioning that the appearance and nutritional quality of the grain had a negative influence on the 2-AP content.

4. Discussion

4.1. Effects of Different Graphene Treatments on the Rice Yield

Numerous studies have investigated the interactions between graphene nanomaterials and plants, with varying effects on plant growth depending on the graphene material’s physicochemical properties. For example, Younes et al. [28] found that spraying graphene nanoflakes significantly increased eggplant and pepper yields, while Park et al. [29] observed that moderate concentrations of graphene oxide promoted watermelon girth and increased sugar content. Our study found similar trends in yield variation between two cultivars. Graphene applied at appropriate concentrations led to increased yields. However, the promotion effect decreased at higher concentrations and even reduced the yield. Comprehensively, the T1 treatment exhibited the best yield-increasing effect, primarily due to significant increases in 1000-grain weight, grains per panicle and the filled grains rate (Table 1), which are key yield components positively associated with yield [14]. However, the T3 treatment decreased the yield of both cultivars, most likely by reducing the grains per panicle and 1000-grain weight. We hypothesize that excess graphene may have affected the fertility process during the grain filling period of fragrant rice; hence, the yield was reduced with high concentration treatments. Furthermore, previous studies have shown that high concentrations of graphene may inhibit crop growth. Research by Zhang et al. [30] reported that exposing rice seeding to 100 mg/L and 250 mg/L of graphene oxide inhibited above-ground growth and reduced above-ground and root biomass.

4.2. Effects of Different Graphene Treatments on the Grain Quality

Previous studies have demonstrated the notable influence of nanomaterials on crop quality. For instance, Cheng et al. [31] reported improved quality, flavonoids and amino acid content in cherry radish by foliar spraying of selenium nanoparticles. Similarly, a study by Gao et al. [32] found that 30 mg/L of graphene effectively relieved the negative effects of cadmium on lettuce growth and increased soluble sugar, protein and vitamin C content in lettuce leaves. Our results showed that the application of graphene treatments increased the head rice rate in two fragrant rice cultivars. Treatment with low-concentration T1 graphene reduced the rice chalkiness degree, whereas treatment with high-concentration T2 and T3 graphene increased it (Table 2). Generally, the quality of grain appearance strongly influences the consumer’s perception of product quality [33], implying that high-concentration graphene may have negative marketing implications for rice. Moreover, Yang et al. [34] also discovered that the utilization of ZnO nanoparticles could raise the brown rice percentage and zinc content in brown rice. Our evaluation of the nutritional quality of grain samples found that graphene treatment increased the protein content in grains of both fragrant rice cultivars. Hence, these results implied that appropriate concentrations of graphene may have enormous potential to improve grain quality.

4.3. Effects of Different Graphene Treatments on 2-AP Content in Grains

The biosynthesis of 2-AP is limited by several factors [8,14,35], and despite the role of genetic factors in determining it, the 2-AP content in the grain is still susceptible to external environmental influences during the growth cycle [36]. Today, agricultural management mechanisms, harvest timing, irrigation systems and growth regulators have been well established [37,38]. The synthesis of 2-AP primarily relies on the conversion of proline into pyrroline by proline dehydrogenase (PDH) and pyrroline-5-carboxylate decarboxylase (P5CS) [2,14,39]. The present study found that applying suitable concentrations of graphene (T1 and T2 treatment) increased the 2-AP content in grains (Figure 1), which was primarily attributed to the elevation of the content of precursor substances for 2-AP synthesis. Our study showed that variations in precursor substances and related key enzyme activities (PDH, P5CS) were highly correlated with 2-AP content in grains, as shown by the significant, positive correlation observed in the heatmap (Figure 6). These results are consistent with the study of Yang et al. [37], who revealed the significant and positive correlations among the content of 2-AP, the activity of PDH and P5CS. Notably, the heatmap results showed significant, positive correlations between antioxidant enzymes POD, CAT and photosynthetic pigments and 2-AP content (Figure 6), which aligns with the research by Gui et al. [3].

4.4. Effects of Different Graphene Treatments on the Antioxidant Response of Fragrant Rice Leaves

Antioxidant enzymes, such as CAT, POD, SOD and APX, are critical defense mechanisms for plants. Previous research reported by Zhao et al. [40] showed that nanoparticles can stimulate the plant defense system by triggering stress responses and enhancing tolerance to growth and environmental stresses. In particular, graphene has been shown to affect the antioxidant system in plants. Zhao et al. [22] reported that appropriate graphene oxide significantly increased the activity of SOD, CAT, POD and APX and enhanced drought tolerance in soybean. In our study, we investigated the effects of graphene treatments on the antioxidant system and photosynthetic pigments of 19× and Meixiangzhan. We found that T1 treatment significantly increased the activity of POD in 19× and Meixiangzhan, as well as CAT activity. The improvement of the antioxidant system increased the plants’ resistance to biotic stresses [41]. Moreover, based on the structural equation, we propose that the activity of antioxidant enzymes has a significant effect on 2-AP content (Figure 7). It is likely that graphene stimulates the plant defense system and enhances the scavenging of free radicals and ROS, thus increasing the activity of 2-AP synthesis enzymes. Therefore, we speculate that applying appropriate concentrations of graphene has the effect of improving growth, 2-AP accumulation and resistance in fragrant rice.

In addition, nanoparticles affect the alteration of photosynthetic pigments in plants. Chlorophyll, as an important cofactor of photosystem I and photosystem II, plays an important role in photosynthesis [42]. Lu et al. [43] showed that graphene promotes the electron transfer efficiency of photosystem II in vesicles and enhances photosynthetic phosphorylation in chloroplasts. In this experiment, we observed that T1 treatment significantly increased the content of chlorophyll a, chlorophyll b, total chlorophyll and carotenoids in both cultivars, consistent with previous findings [28]. However, high concentrations of graphene decrease the photosynthetic pigment content in leaves, which may be related to the toxic effect of high concentrations of nanoparticles.

Moreover, our experiment revealed that T3 treatment decreased the activity of antioxidant enzymes in leaves while increasing the MDA content in leaves. Our results agreed with the research by Zhang et al. [44], who found a similar effect of high concentrations of graphene on plant chlorophyll content and MDA. Additionally, correlation analysis showed that the activities of POD and SOD were negatively correlated with the content of MDA. The excessive accumulation of MDA content in plant cells can cause membrane lipid peroxidation [45], which has a negative impact on normal plant growth. At the same time, excessive nanoparticles can accumulate in the plant root system and impede nutrient transport [46]. Further investigations have revealed that nanomaterials potentially influence the internal structure of plant cells and modulate gene expression [47]. In the context of increasing populations and limited natural resources, agricultural production faces numerous challenges, and the utilization of nanotechnology to enhance crop resilience is an efficient and sustainable strategy. Nevertheless, the possible safety risks associated with nanoparticles must be considered in future research.

5. Conclusions

Two seasons of experiments showed that appropriate concentrations of graphene (9 g/hm²) enhanced the activity of antioxidant enzymes (POD, SOD and CAT) and the photosynthetic pigment content of fragrant rice to promote growth and improved grain quality. In addition, appropriate graphene concentration (9 g/hm²) up-regulated 2-AP biosynthesis-related precursor substances (proline, P5C and pyrroline) to improve 2-AP content in the grains. However, higher concentrations of graphene (18 g/hm² and 27 g/hm²) may cause disruption of the protective enzyme system and increase MDA content in the leaves, adversely affecting yield accumulation and grain quality in fragrant rice. Consequently, applying appropriate concentrations of graphene can optimize the antioxidant system of fragrant rice and thus generate positive effects on its growth and 2-AP biosynthesis.