Exogenously Applied Sodium Nitroprusside Alleviated Cadmium Toxicity in Different Aromatic Rice Cultivars by Improving Nitric Oxide Accumulation and Modulating Oxidative Metabolism

Abstract

Exogenous application of sodium nitroprusside (SNP) has previously been reported to trigger plant tolerance against a variety of environmental stresses. The present study was planned to investigate the possible role/s of exogenously applied SNP (50 or 100 μM) in alleviating cadmium (Cd)-induced effects on physio-biochemical processes, yield attributes, and grain quality traits of three fragrant rice cultivars, viz., Meixiangzhan-2 (MXZ), Guixiangzhan (GXZ), and Xiangyaxiangzhan (XYXZ) under 50 mg Cd kg⁻¹ of soil. The results revealed that foliar spray of SNP (50 or 100 μM) on Cd-stressed rice plants reduced oxidative stress (lower hydrogen peroxide (H₂O₂), malondialdehyde (MDA), and electrolyte leakage (EL)) and improved the photosynthetic apparatus through higher chlorophyll contents, gas exchange attributes, and intact chloroplast configurations, and reduced Cd concentration in the leaves and grains of aromatic rice cultivars. The reduced levels of cellular ROS, MDA, and EL were related to the endogenous NO-mediated improvement in the activity of anti-oxidative enzymes and those involved during the ascorbate–glutathione cycle. However, among the different SNP levels, the foliar spraying of 50 μM of SNP was recorded to be the best treatment for fragrant rice growth, which increased grain yield by 42.06%, 46.03%, and 31.21%, and the quality trait of 2-acetyl-1-pyrroline (2-AP) content by 43.12%, 55.84%, and 35.72% in MXZ, GXZ, and XYXZ respectively, suggesting that GXZ is more responsive to SNP than MXZ and XYXZ fragrant rice cultivars. Collectively, our results deduced that cultivating the GXZ fragrant rice cultivar along with foliar application of 50 μM of SNP could sustain the grain yield and quality features of aromatic rice cultivation in heavy metal (especially Cd)-polluted soils.

1. Introduction

Industrialization and the broad use of farming techniques have caused a sudden rise in the amount of potentially harmful substances that have a negative impact on crop production around the globe [1]. The major heavy metals, including cadmium (Cd), aluminum (Al), mercury (Hg), chromium (Cr), and lead (Pb), are well reported to induce phytotoxicity, which affects plant metabolism and defense systems [2]. Due to cadmium’s high mobility, solubility, and easy soil–plant transfer, it seriously threatens all life on earth [3]. In the metal-affected rhizosphere, Cd is readily absorbed by plant roots and subsequently travels up the food chain to humans, where it can harm their neurological, reproductive, and immune systems [4].

Cd building up over the threshold limit provokes many phytotoxic consequences including poor seed germination, stunted growth, and reduced defensive mechanisms [2]. Usually, the photosynthetic system is more vulnerable under Cd stress. Chlorophyll (Chl) is essential for photosynthesis, and any decline in its biosynthesis under Cd toxicity hinders the process of photosynthesis [5]. Cd inhibits Chl biosynthesis through the upregulation of chlorophyllase and the substitution of Mg²⁺ with Cd²⁺ ions [6]. Likewise, quantum yields of photosystem I (PSI) and photosystem II (PSII) are the principal targets of Cd toxicity, which may affect the electron transport rate (ETR) owing to attachment to sensitive regions of the photosynthetic apparatus [7]. Cd stress causes a reduction in the cellular NADPH production, which disrupts the flow of electrons from PSII to PSI in chloroplasts and from complex I to IV in mitochondria [8]. These electrons react with molecular oxygen, which causes oxidative stress through reactive oxygen species (ROS) aggravation [9]. ROS produced during stress becomes the reason for cellular oxidative damage and genotoxicity [8]. The Cd-induced toxicity symptoms include a reduction in plant growth (biomass and elongation) and deteriorated crop yield and quality owing to the disruptions in various physio-biochemical processes, such as photosynthesis, photochemical efficiency, mitochondrial electron transport, and cellular functioning [10,11].

In recent times, one of the possible strategies to increase plant adaptability to stressful environments has been the use of signaling molecules. When it comes to biological messengers, NO stands out as a key player in plant tissues. It has a significant impact on the physio-biochemical and metabolic processes and the growth characteristics of plants under stressful environments [2]. NO, an endogenous plant bioactive signaling molecule, is involved in a variety of biotic and abiotic stress-related processes in plants, including fruit ripening, pollen tube growth, seed germination, photosynthesis, root growth, and interactions with other plant hormones [12,13]. The direct chemical interactions of NO with superoxide (O₂·⁻) or lipid peroxyl radicals (LOO^⋅) are of particular interest because they illustrate a molecular basis for some of the apparently antagonistic outcomes of similar chemical reactions. The reaction with O₂·⁻ is rapid and facile (k = 6.7 × 10⁹ M/s), resulting in the formation of peroxynitrite (ONOO⁻) [14]. Similarly, a termination reaction occurs between NO and LOO^· (k = 2 × 10⁹ M/s) [15] and the biological effects of these two reactions are generally viewed as being opposite, with the formation of ONOO⁻ usually associated with a pro-oxidant response and scavenging of LOO^⋅ being an antioxidant response [16]. Additionally, NO regulates several tolerance mechanisms in plants under harsh environments and regulates plants’ antioxidative defenses to counteract ROS, and prevents superfluous ROS production [17,18]. Many studies reported that adding exogenous NO via SNP improved plant tolerance to oxidative stresses caused by drought [19], aluminum toxicity [20], nickel stress [21], and salinity [22]. Foliar application of SNP has recently been proposed as a viable strategy for heavy metal detoxification in plants [18,23]. NO has emerged as a potential novel molecule in terms of extending post-harvest longevity while preserving fruit quality attributes including nutritional contents, flavor, color, and texture [24]. However, no studies have been conducted to understand how NO controls a range of Cd tolerance mechanisms, yield attributes, grain aroma, growth performance, and nutritional quality factors in aromatic rice.

Fragrant rice (Oryza sativa L.) is distinct from unscented rice by its distinguishing pleasant aroma, distinctive flavor, and taste [25]. Among the various fragrance-contributing volatile compounds, 2-acetyl-1-pyrroline (2-AP) is a strong volatile compound that produces a characteristic aromatic flavor and taste in aromatic rice grains [26]. Recent studies have revealed that stressful conditions, whether heavy metals or plastic pollutants (nanoplastics), along with adversely affecting the grain quality and yield attributes of aromatic rice also repress plant growth and physio-biochemical processes [27,28]. Similarly, we have comprehensively elucidated that the fragrance-conducive features of aromatic rice grains, including the precursors and enzymes involved in the 2-AP biosynthetic pathway, are highly vulnerable to heavy metals [11]. In this context, the present experiment examined the potential of NO to strengthen fragrant rice’s natural defenses, including the effects on the photosynthetic apparatus, osmolyte accumulation, the antioxidant defense system, and grain quality traits and yield attributes of different fragrant rice cultivars under heavy metal (especially Cd)-polluted soils.

2. Materials and Methods

2.1. Experimental Site, Plant Husbandry, and Growth Environments

A pot experiment was conducted in a rain-protected net house in open-air conditions at the Experimental Research Farm (23°15′ N, 113°21′ E), Agricultural College, South China Agricultural University (SCAU), Guangzhou, China. The mean temperature ranged from 23 °C to 29 °C during the growing season, with an average relative humidity of 75–85%. Each experimental pot contained 10 kg of soil, collected from paddy fields that had been cultivated for years, with fundamental chemical properties listed in Supplementary Table S1. Pots were filled with air-dried soil around 30 days before nursery transplanting and soil was contaminated with Cd by dissolving CdCl₂·2.5H₂O in deionized water and applying it at different concentrations, i.e., 0 and 50 mg kg⁻¹ of soil. These concentrations were uniformly applied to the respective pots while mixed thoroughly at the same time. Pots were submerged with a water layer (3–4 cm) above the soil surface to maintain fully anaerobic and puddle-like conditions within the pots. After 10 days of transplanting, foliar spraying of SNP (50 or 100 µM) as Na₂[Fe(CN)₅NO] was initiated on the abaxial and adaxial sides of plant leaves with 20-day intervals (a total of 4 sprays).

Three fragrant rice cultivars, Meixiangzhan-2 (MXZ), Guixiangzhan (GXZ), and Xiangyaxiangzhan (XYXZ), were obtained from the Agricultural College of SCAU, Guangzhou, China. These fragrant rice cultivars have an almost identical growth cycle and are regionally renowned for their distinctive aroma and are widely cultivated on a commercial scale by local farmers in South China. The soil-filled pots were transplanted with uniform and stable rice seedlings (25-day-old) with 3–4 seedlings per hill and 5 hills per pot by following a cross mark (+) type of planting pattern (four seedlings a side and the fifth in the middle of the pot). All the pots were supplied with 2.25 g N (Urea), 3.33 g P (phosphorus pentoxide), and 1.35 g K (potassium oxide) with a starting dose of 70% and 30% at the tillering stage. There were four replications in our experiment. The fresh plant samples were selected at random for physio-biochemical analyses at the panicle heading stage. However, in order to quantify the buildup of above-ground biomass, the plants were harvested at maturity and grain quality traits and yield attributes were measured.

2.2. Estimating the Photosynthetic Efficacy, Pigment Contents, and Transmission Electron Microscopy

For the gas exchange measurement, one fully expanded mature leaf from the top was used per plant and five plants were randomly selected per treatment and the results were averaged. Net photosynthesis rate (Pn), stomatal conductance (Gs), transpiration rate (E), and intercellular CO₂ concentration (Ci) in the rice plant leaves were measured by using a LI-6800 Portable Photosynthetic System on the same day at around 10:00 a. m. with an average temperature (25–30 °C) and relative humidity (75–80%). The photosynthetic pigments (Chl a, b, CAR) were measured in the same leaves as those used for photosynthetic attributes and fresh leaf tissues were extracted with 95% alcohol and a spectrophotometer was used to measure absorbance at 665, 649, and 470 nm [29].

Fresh leaf samples were randomly collected from different plants of fragrant rice cultivars for TEM analysis. Small sections of leaves 1–3 mm in length, were fixed in 4% glutaraldehyde (v/v) in 0.2 mol/L PBS (sodium phosphate buffer, pH 7.2) for 6–8 h and post-fixed in 1% OsO4 [Osmium (VIII) oxide] for 1 h, then in 0.2 mol/L PBS (pH 7.2) for 1–2 h. Dehydration was performed in a graded ethanol series (50%, 60%, 70%, 80%, 90%, 95%, and 100%) followed by acetone, and then samples were filtrated and embedded in Spurr’s resin. Ultra-thin sections (80 nm) were prepared and mounted on copper grids for viewing under a transmission electron microscope (TALOS L120C, Comin Biotechnology Co., Ltd., Shanghai, China).

2.3. Estimation of ROS Level, Lipid Peroxidation, Electrolyte Leakage, and Proline Content

To determine hydrogen peroxide (H₂O₂) levels in fragrant rice plants, fresh leaves were homogenized with 0.1% of trichloroacetic acid (TCA) and centrifuged for 20 min at 12,000× g. The reaction mixture contained supernatant, 100 mM potassium phosphate buffer (pH 6.8), and 1 M potassium iodide [30]. The H₂O₂ levels were quantified by a UV–VIS 2550 spectrophotometer operating at 390 nm and following the calibration curve (Supplementary Table S3). To measure malondialdehyde (MDA) contents, fresh plant samples were homogenized in 0.1% (w/v) cold TCA, and the homogenate was centrifuged at 12,000× g for 20 min at 4 °C. The reaction mixture contained 0.5 mL of supernatant and 2.5 mL of 0.5% thiobarbituric acid (TBA) solution (dissolved in 20% TCA). The reaction mixture was boiled for 30 min, and then rapidly cooled and centrifuged for 5 min at 12,000× g. The difference between the absorbance values at 532 and 600 nm with an extinction coefficient of 155 mM cm was applied to calculate the MDA contents [31]. To determine electrolyte leakage (EL), fresh leaf samples (0.3 g) were washed with distilled water and placed in water-containing closed vials (10 mL, deionized) and incubated at 25 °C for 6 h and then electrical conductivity (EC) (EC1) was recorded with an EC meter (SX-650, Sansin, Guangzhou, China). Then, samples were incubated at 90 °C for 2 h and cooled down to 25 °C to record second EC (EC2). The EL of the leaf samples was calculated as (EL (%) = EC1/EC2 × 100) in triplicate and averaged [30]. The proline contents were estimated according to Bates et al. [32] by using ninhydrin. The reaction mixture was extracted with 5 mL toluene and the absorbance of the red chromophore in the toluene fraction was measured at 520 nm. The amount of proline was estimated using a calibration curve (Supplementary Table S4) and expressed as μg g⁻¹ FW.

2.4. Estimating the Cd Concentrations in Roots, Leaves, and Grains

To measure Cd concentration, fragrant rice plant roots, leaves, and grains were sampled, oven-dried and pulverized, and then digested using an MLS1200 microwave oven (Milestone, FKV, Varese, Italy) in a di-acidic combination of HClO₄:HNO₃ (1:5). The Cd concentrations were estimated using an AA6300C Atomic Absorption Spectrophotometer, Nippon Instruments Corporation, Osaka, Japan, [33].

2.5. Estimation of NO Content

Griess reagent was used to measure the NO contents [19]. Shortly, plant samples were mixed in 50 mM acetic acid buffer (pH 3.6) and zinc acetate (4%). The supernatant was reacted with activated carbon (0.1 g) and, subsequently, reacted with a mixture of sulfanilamide (1%) and HCl (3 M) and then incubated for 10 min (37 °C) under darkness. The absorbance was triplicated at 540 nm using a spectrophotometer (Shimadzu, Kyoto, Japan).

Endogenous NO production was detected using diaminoflourescein-FM diacetate (DAF-FM DA) and confocal microscopy (Life Technologies, Carlsbad, CA, USA) [19]. The freshly cut plant samples were treated with 10 µM DAF-FM DA in 20 mM HEPES buffer (pH 7.8) for 30 min at 37 °C and then rinsed with 0.2 M sodium phosphate buffer (7.8 pH) for 20 min. The images were captured by a laser confocal scanning microscope (LSM-510) with a 515 nm emission and 495 nm excitation.

2.6. Estimating the Antioxidant Enzymes

The fresh plant samples were normalized in 50 mM phosphate-buffered saline (PBS) (pH 7.8) for antioxidant determinations. The supernatant was collected by centrifuging for 10 min at 12,000× g and the superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, and ascorbate peroxidase (APX) activity were estimated [31]. The commercial detection kits “GSH-2-W”, “GR-2-W”, “DHAR-2-W”, “MDHAR-2-W”, and “ASA-1-W” were used to measure glutathione (GSH), glutathione reductase (GR), dehydroascorbate reductase (DHAR), manodehydroascorbate reductase (MDHAR), and ascorbic acid (AsA) levels, respectively, bought from (http://www.cominbio.com, accessed on 15 March 2024) Comin Biotechnology Co. Ltd., Shanghai, China.

2.7. Estimating the Grain Yield Attributes and Quality Traits of Fragrant Rice

Grain yield and contributing attributes were determined at the maturity stage. The grains were manually threshed and sun-dried to around 12–14% moisture content. The productive tillers per pot were counted at the reproductive stage of the plants. To obtain the 1000-grain weight (g), 1000 grains were weighed, while grain yield pot⁻¹ was calculated as the total weight of paddy collected from each pot. The harvested grains were taken from a stored seed lot to determine rice quality attributes. A rice huller (Jingsu, China) was used to obtain brown rice and brown rice rate (BRR) was calculated as follows: (brown rice weight/paddy weight) × 100. The brown rice was milled with a Jingmi testing rice miller (Zhejiang, China) and milling rate was recorded as follows: (milled rice weight/original weight) × 100. Grain chalkiness degree was assessed with the help of an SDE-A light box (Guangzhou, China).

2.8. Quantification of 2-Acetyl-1-Pyrroline (2AP)

Around 5 g of the fresh grains were weighed and ground for 2AP measurement. The 2AP content in the extraction solution was determined after drying over sodium sulfate and filtering by using the synchronization distillation and extraction method (SDE) combined with a GCMS-QP 2010 Plus (Shimadzu Corporation, Kyoto, Japan). The conditions of the GCMS-QP 2010 Plus were as follows: the gas chromatograph was equipped with an RTX-5MS (Shimadzu, Kyoto, Japan) silica capillary column (30 m × 0.32 mm × 0.25 μm) and high-purity helium gas (99.999%, Guangzhou Gases Co., LTD, Guangzhou, China) at a flow rate of 2.0 mL min⁻¹. The temperature of the GC oven was 40 °C, increased to 65 °C and held at 65 °C for 1 min, and then further increased to 220 °C and held at 220 °C for 10 min. The ion source temperature was 200 °C. Under these conditions, the retention time of 2-AP was 7.5 min [26]. The 2-AP contents were determined using the calibration curves (Supplementary Data S1 and Table S5) and expressed as ng g⁻¹ fresh weight (FW).

2.9. Statistical Analysis

The analysis of variance (ANOVA) for the studied parameters of individual aromatic rice cultivars were performed and the least significant difference test (LSD) was performed for treatment comparison at p < 0.05 using the ‘Statistix 8.1′ (Analytical Software, Tallahassee, FL, USA). SigmaPlot 12.0 (Systat Software Inc., San Jose, CA, USA) was used for graphical representation.

3. Results

3.1. Impacts of SNP Treatments on Cadmium Concentration, Nitric Oxide Content, and Plant Growth

Cd toxicity significantly diminished the plant dry biomass of all three aromatic rice cultivars as compared to non-Cd-stressed rice plants either with or without SNP treatments (

Table 1. Effects of sodium nitroprusside on plant dry weight, nitric oxide contents, and cadmium conc. in aromatic rice cultivars under Cd stress.

Rice Cultivars	Treatments		Total Plant DW (g)	NO Content in Roots (nmol g−1 FW)	NO Content in Leaves (nmol g−1 FW)	Cd Conc. in Root (mg kg−1 DW)	Cd Conc. in Leaves (mg kg−1 DW)	Cd Conc. in Grains (mg kg−1 DW)
MXZ	SNP0	Cd0	3.21 ± 0.18 bc	8.34 ± 0.56 b	15.69 ± 1.03 b	–	–	–
		Cd50	2.17 ± 0.10 d	5.16 ± 0.23 c	9.65 ± 0.55 c	27.06 ± 1.16 a	19.97 ± 1.27 a	1.21 ± 0.10 a
	SNP50	Cd0	4.69 ± 0.35 a	15.42 ± 1.26 a	28.84 ± 1.52 a	–	–	–
		Cd50	2.98 ± 0.18 c	8.14 ± 0.21 b	15.53 ± 1.03 b	26.87 ± 0.69 a	14.86 ± 0.41 b	0.59 ± 0.05 b
	SNP100	Cd0	3.79 ± 0.22 b	13.49 ± 0.86 a	26.24 ± 1.45 a	–	–	–
		Cd50	2.65 ± 0.17 cd	6.83 ± 0.15 bc	13.53 ± 0.66 b	25.62 ± 1.22 a	15.21 ± 0.49 b	0.68 ± 0.04 b
GXZ	SNP0	Cd0	3.70 ± 0.42 c	8.78 ± 0.51 c	16.99 ± 0.94 c	–	–	–
		Cd50	2.64 ± 0.10 d	5.70 ± 0.47 d	11.26 ± 0.80 d	27.94 ± 1.31 a	18.42 ± 0.92 a	0.98 ± 0.07 a
	SNP50	Cd0	5.66 ± 0.38 a	17.25 ± 1.24 a	31.92 ± 1.60 a	–	–	–
		Cd50	3.71 ± 0.19 c	9.64 ± 0.45 c	19.62 ± 0.93 c	25.08 ± 0.91 a	12.93 ± 0.95 b	0.35 ± 0.02 c
	SNP100	Cd0	4.86 ± 0.19 b	12.44 ± 0.90 b	24.56 ± 1.80 b	–	–	–
		Cd50	3.51 ± 0.10 c	8.67 ± 0.64 c	17.04 ± 1.31 c	26.91 ± 1.12 a	14.40 ± 0.46 b	0.48 ± 0.04 b
XYXZ	SNP0	Cd0	2.81 ± 0.13 b	8.28 ± 0.66 bc	14.19 ± 0.80 b	–	–	–
		Cd50	1.88 ± 0.07 c	4.84 ± 0.32 e	8.08 ± 0.42 d	28.86 ± 1.27 a	20.55 ± 0.82 a	1.47 ± 0.11 a
	SNP50	Cd0	4.36 ± 0.22 a	13.67 ± 0.78 a	24.44 ± 1.64 a	–	–	–
		Cd50	2.53 ± 0.17 b	7.20 ± 0.57 cd	12.47 ± 0.55 bc	25.20 ± 1.05 a	16.82 ± 1.17 b	0.94 ± 0.07 b
	SNP100	Cd0	3.77 ± 0.30 a	9.43 ± 0.70 b	21.41 ± 1.74 a	–	–	–
		Cd50	2.36 ± 0.21 bc	6.51 ± 0.10 de	10.61 ± 1.12 cd	26.93 ± 1.10 a	17.54 ± 0.66 b	0.84 ± 0.04 b

Note: Cd in the form of CdCl₂·2.5H₂O was treated in the soil as 0 and 50 mg/kg, while foliar SNP treatments were in the form of Na₂[Fe(CN)₅NO] at three levels (0, 50, and 100 µM). The numerical values reflect means from four independent replicates (±S.E.) with different treatments. Data presented in individual columns indicated with dissimilar alphabets (a, b, c, etc.) differ significantly within a cultivar by LSD test at p < 0.05. MXZ, Meixiangzhan-2; GXZ, Guixiangzhan; and XYXZ, Xiangyaxiangzhan.

). However, in Cd-stressed conditions, exogenous application of SNP with 50 µM and 100 µM on aromatic rice cultivars increased plant dry weight by 37.29% and 22.16% in MXZ, 40.22% and 32.86% in GXZ, and 34.89% and 25.61% in XYXZ, respectively, compared to no SNP (Table 1). Correspondingly, Cd stress lowered the NO contents, while SNP spray enriched the NO levels both in the shoot and root tissues of all three aromatic rice cultivars as follows, GXZ > MXZ > XYXZ, suggesting that the GXZ cultivar is more receptive to SNP application than other two aromatic rice cultivars. Under Cd-stressed conditions, foliar application of SNP with 50 µM and 100 µM on aromatic rice cultivars increased NO content in shoots by 60.88% and 40.14% in MXZ, 74.17% and 51.27% in GXZ, and 54.46% and 31.34% in XYXZ, respectively, while in roots by 57.74% and 32.43% in MXZ, 69.18% and 52.09% in GXZ, and 48.65% and 34.52% in XYXZ, respectively, compared to no SNP (Table 1). Moreover, the results were confirmed by taking visual pictures with a DAF-FM DA confocal microscope (Figure 1). The Cd concentration was higher under Cd treatments (Table 1) and roots retained higher quantities of Cd than shoots. Conversely, SNP treatment significantly decreased the Cd concentrations in all aromatic rice cultivars. Under Cd-stressed conditions, foliar application of SNP with 50 µM and 100 µM decreased Cd in shoots by 26.95% and 19.79% in MXZ, 29.16% and 24.48% in GXZ, and 24.49% and 14.65% in XYXZ, and in roots by 21.10% and 18.06% in MXZ, 24.23% and 21.99% in GXZ, and 15.92% and 8.96% in XYXZ, while in grains by 58.98% and 44.10% in MXZ, 64.13% and 51.07% in GXZ, and 35.83% and 42.99% in XYXZ, respectively, compared to no SNP (Table 1).

3.2. Effects of SNP Treatments on Photosynthetic Pigments

Cadmium toxicity decreased chlorophyll a, chlorophyll b, and total chlorophyll in all three aromatic rice cultivars MXZ, GXZ, and XYXZ, as compared with respective control plants. However, under Cd-stressed conditions, foliar application of SNP with 50 µM and 100 µM on aromatic rice cultivars increased total Chl content by 50.37% and 35.76% in MXZ, 55.98% and 41.34% in GXZ, and 42.67% and 30.99% in XYXZ, respectively, compared with no SNP application (Figure 2). The exogenous application of 50 µM SNP was more successful than the 100 µM treatment in preventing Chl loss under Cd stress (Figure 2C). In contrast to Chl a and b, the ratio of Chl a/b reduced with 50 µM and 100 µM SNP treatments by 4.29% and 3.08% in MXZ, 6.28% and 5.80% in GXZ, and 6.21% and 4.78% in XYXZ, respectively, compared to no SNP under Cd stress (Figure 2D). CAR contents decreased with Cd stress in all aromatic rice cultivars while foliar treatment of 50 µM and 100 µM SNP to Cd-stressed aromatic rice plants increased CAR by 38.68% and 31.74% in MXZ, 43.77% and 35.66% in GXZ and 33.80% and 39.58% in XYXZ, respectively, relative to no SNP application (Figure 2E).

3.3. SNP Application Improved Photosynthetic Apparatus

Cadmium toxicity decreased the efficacy of photosynthetic attributes (P_n, E, C_i, and G_s) in aromatic rice cultivars in the following order: GXZ < MXZ < XYXZ. However, foliar spray of 50 µM and 100 µM SNP enhanced net photosynthetic efficiency (P_n) by 43.73% and 46.45% in MXZ, 54.96% and 47.36% in GXZ, and 37.07% and 30.23% in XYXZ, respectively, as compared to no SNP application under Cd stress (Figure 3). Notably, both 50 and 100 µM SNP treatments enhanced photosynthetic attributes under Cd-stressed aromatic rice plants but the 50 µM treatment showed more prominent effects than the 100 µM SNP treatment, suggesting that the 50 µM SNP treatment was more efficient than the 100 µM SNP treatment in aromatic rice plants. Since photosynthetic efficiency largely depends upon the chloroplast integrity in green plants, SNP treatments effects were evaluated on the chloroplast ultrastructural configurations under Cd toxicity. In this experiment, Cd toxicity had a significantly negative impact on the integrity and ultrastructural configuration of the chloroplasts (Figure 4A–C). On the other hand, it was found that the SNP-treated plants’ chloroplasts were more uniform, well-organized, regularly oval shaped, and gathered more starch grains (Figure 4D–F). Moreover, under Cd stress, plant cells were observed with ruptured cell walls, scattered organelles, and fewer chloroplasts, while SNP treatment reversed the Cd-induced inhibitory effects in all aromatic rice cultivars (Figure 4).

3.4. Impact of Foliar SNP Treatments on Membrane Integrity

In all aromatic rice cultivars, cadmium stress significantly increased proline, hydrogen peroxide (H₂O₂), malondaldehyde (MDA), and electrolyte leakage (EL) either with or without SNP treatments. Foliar treatment of 50 µM and 100 µM SNP further accumulated proline by 67.55% and 44.75% in MXZ, 75.17% and 55.13% in GXZ, and 63.03% and 54.89% in XYXZ respectively, relative to no SNP treatment under Cd stress (Figure 5A). In contrast to proline content, H₂O₂, MDA, and EL were accumulated in the order of XYXZ > MXZ > GXZ under Cd stress, indicating that, in comparison to other cultivars, GXZ is relatively more resistant to Cd toxicity (Figure 5B–D). However, foliar application of SNP (50 µM) reduced H₂O₂ content (38.22%, 41.31%, and 34.82%), MDA (26.92%, 32.43%, and 27.66%), and EL (24.27%, 29.49%, and 22.12%) in MXZ, GXZ, and XYXZ, respectively, relative to no SNP treatment under Cd stress. Foliar application of 50 µM SNP proved to be more effective in terms of reducing EL, MDA, and H₂O₂ contents than 100 µM SNP application (Figure 5).

3.5. Foliar Application of SNP-Enhanced Antioxidant Enzyme Activity under Cd Toxicity

In comparison to cultivars without Cd stress, the activity of antioxidant enzymes (SOD, POD, CAT, APX, and GR) were considerably high under Cd stress in all aromatic rice cultivars. Moreover, foliar spray of SNP (50 µM) further enhanced SOD activity by 37.43%, 53.32%, and 29.54%, POD by 56.96%, 65.34%, and 48.19%, CAT by 34.45%, 35.62%, and 31.99%, APX by 79.73%, 85.40%, and 62.21%, and GR by 48.89%, 51.10%, and 44.58%, in MXZ, GXZ, and XYXZ, respectively, relative to no SNP treatments under Cd stress (Figure 6). In this study, foliar SNP treatment with 50 µM proved to be more efficient than 100 µM in all aromatic rice cultivars.

3.6. Foliar Application of SNP Regulated AsA-GSH Cycle

Cd stress reduced AsA content and dehydroascorbate reductase (DHAR) and manodehydroascorbate reductase (MDHAR) activities in all aromatic rice cultivars relative to without Cd stress. However, SNP treatment (50 µM) increased AsA content by 62.98%, 68.81%, and 52.92%, DHAR activity by 48.69%, 53.26%, and 40.36%, and MDHAR by 49.38%, 50.73%, and 47.17% in MXZ, GXZ, and XYXZ, respectively, relative to no SNP treatment under Cd stress (Figure 7). In contrast, GSH content increased under Cd toxicity relative to non-Cd-stressed plants. Furthermore, foliar treatments of 50 µM and 100 µM SNP further enhanced the GSH contents by 67.12% and 44.75% in MXZ; 81.97% and 51.48% in GXZ, and 56.28% and 39.15% in XYXZ, respectively, relative to no SNP treatment under Cd toxicity (Figure 7D).

3.7. Influence of SNP Treatments on Aromatic Rice Yield and Grain Attributes

Cd toxicity degraded the grain quality characters as well as significantly decreased grain yield and yield attributes. Conversely, SNP treatments mitigated Cd-induced adversity in rice plants and except from 1000-grain weight, significantly improved all the grain yield attributes (

Table 2. Effects of SNP treatments on yield traits and grain quality attributes of three aromatic rice cultivars under Cd toxicity.

			Yield Traits					Grain Quality Attributes
Rice Cultivars	Treatment		Productive Tiller per Pot	Grains per Panicle	1000-Grains Weight	Grain Yield per Pot		Browning Rate (%)	Milling Rate (%)	Chalkiness Degree (%)	2AP Content (ng g−1 FW)
MXZ	SNP0	Cd0	25.10±1.27 b	137.66 ± 2.10 bc	18.49 ± 0.47 ab	53.35 ± 1.51 b		70.08 ± 3.58 a	56.52 ± 1.74 bc	13.96 ± 0.30 bc	136.70 ± 6.17 bc
		Cd50	19.93 ± 0.61 c	119.76 ± 5.62 c	17.40 ± 0.99 b	40.42 ± 2.86 c		67.53 ± 1.69 a	51.32 ± 2.35 c	18.91 ± 1.35 a	98.18 ± 7.11 c
	SNP50	Cd0	36.63 ± 2.47 a	175.04 ± 11.37 a	20.95 ± 0.92 a	80.79 ± 5.53 a		76.43 ± 1.84 a	65.77 ± 2.56 a	10.92 ± 0.86 d	276.00 ± 25.26 a
		Cd50	28.44 ± 1.41 b	159.34 ± 8.06 ab	18.88 ± 0.89 ab	57.42 ± 2.45 b		71.35 ± 2.19 a	54.91 ± 1.82 bc	14.87 ± 0.64 b	140.53 ± 8.82 b
	SNP100	Cd0	34.85 ± 1.29 a	159.68 ± 9.11 ab	19.04 ± 0.67 ab	71.85 ± 5.54 a		73.35 ± 3.78 a	62.96 ± 5.73 ab	12.10 ± 0.19 cd	259.26 ± 10.96 a
		Cd50	25.85 ± 1.80 b	149.94 ± 5.04 b	18.57 ± 1.61 ab	52.87 ± 2.14 b		71.29 ± 4.30 a	53.33 ± 1.53 c	16.26 ± 0.86 b	148.27 ± 9.17 b
GXZ	SNP0	Cd0	30.75 ± 2.00 b	140.33 ± 8.85 c	19.15 ± 0.65 b	64.36 ± 4.16 b		81.52 ± 1.61 ab	66.39 ± 2.05 a	11.55 ± 0.20 bc	158.47 ± 11.30 bc
		Cd50	21.24 ± 0.76 c	120.99 ± 4.16 c	17.98 ± 0.89 b	46.08 ± 2.76 c		77.28 ± 2.90 b	57.28 ± 2.17 b	15.31 ± 0.44 a	115.32 ± 4.30 c
	SNP50	Cd0	43.80 ± 3.12 a	195.44 ± 10.82 a	21.94 ± 1.28 a	99.23 ± 6.75 a		90.06 ± 1.68 a	69.14 ± 1.47 a	9.58 ± 0.53 d	302.23 ± 29.26 a
		Cd50	31.57 ± 0.98 b	166.81 ± 7.64 b	19.90 ± 0.14 ab	67.30 ± 1.17 b		83.35 ± 1.93 ab	64.79 ± 2.73 ab	12.64 ± 0.41 b	179.72 ± 14.47 b
	SNP100	Cd0	38.09 ± 2.11 a	182.69 ± 11.96 ab	20.76 ± 0.98 ab	90.72 ± 6.22 a		86.65 ± 3.29 ab	68.82 ± 4.17 a	10.58 ± 0.58 cd	283.60 ± 21.02 a
		Cd50	30.04 ± 1.92 b	174.54 ± 4.64 ab	18.28 ± 1.18 b	63.51 ± 4.68 b		82.95 ± 6.90 ab	66.09 ± 1.82 a	14.42 ± 0.35 a	158.10 ± 8.91 bc
XYXZ	SNP0	Cd0	22.68 ± 1.18 bc	128.20 ± 2.49 bc	18.19 ± 0.85 bc	47.61 ± 3.76 b		64.10 ± 3.28 abc	52.05 ± 1.34 c	19.75 ± 0.64 c	117.31 ± 8.59 b
		Cd50	17.63 ± 1.29 c	111.54 ± 5.23 c	15.99 ± 0.70 c	35.51 ± 2.68 c		59.23 ± 1.48 c	49.11 ± 1.99 c	23.90 ± 0.88 a	80.10 ± 6.38 c
	SNP50	Cd0	35.08 ± 2.67 a	162.53 ± 9.84 a	20.63 ± 1.07 a	77.42 ± 5.76 a		69.81 ± 2.22 ab	61.34 ± 2.32 ab	12.33 ± 0.55 d	245.17 ± 23.62 a
		Cd50	23.88 ± 1.67 b	145.68 ± 7.37 ab	16.90 ± 0.43 c	46.59 ± 3.86 bc		64.12 ± 3.54 abc	55.52 ± 1.24 bc	22.42 ± 0.74 ab	108.72 ± 8.72 bc
	SNP100	Cd0	31.74 ± 2.71 a	144.26 ± 6.96 ab	20.34 ± 0.77 ab	78.17 ± 4.04 a		71.96 ± 5.48 a	64.04 ± 5.01 a	11.64 ± 0.47 d	212.55 ± 6.99 a
		Cd50	22.15 ± 1.19 bc	136.41 ± 2.55 b	16.40 ± 0.89 c	44.68 ± 0.88 bc		61.32 ± 1.80 bc	56.98 ± 2.15 abc	21.64 ± 1.03 bc	101.36 ± 3.70 bc

Note: Cd in the form of CdCl₂·2.5H₂O was treated in the soil as 0 and 50 mg/kg, while foliar SNP treatments were in the form of Na₂[Fe(CN)₅NO] at three levels (0, 50, and 100 µM). The numerical values reflect means from four independent replicates (±S.E.) with different treatments. Data presented in individual columns indicated with dissimilar alphabets (a, b, c, etc.) differ significantly within a cultivar by LSD test at p < 0.05. MXZ, Meixiangzhan-2; GXZ, Guixiangzhan; and XYXZ, Xiangyaxiangzhan; 2AP, 2-acetyle-1-pyrolline.

). Foliar treatments of 50 µM and 100 µM SNP improved grain yield by 42.06% and 30.79% in MXZ, by 46.03% and 37.81% in GXZ, and by 31.21% and 25.82% in XYXZ, respectively, relative to no SNP treatment under Cd stress (Table 2). Similarly, the degree of chalkiness, browning rate, milling rate, and 2AP concentrations in fragrant rice grains were all negatively impacted by Cd toxicity (Table 2). The Cd stress decreased the 2AP contents in the grains of all fragrant rice cultivars while SNP application restored the 2AP concentration in seeds. It was observed that GXZ was the least affected fragrant rice cultivar while being more responsive to SNP application than the other two cultivars, suggesting that GXZ is the most resistant relative to MXZ and XYXZ. However, foliar treatments of 50 µM and 100 µM SNP improved 2AP content by 43.12% and 51.02% in MXZ, 55.84% and 37.09% in GXZ, and 35.72% and 26.53% in XYXZ, respectively, relative to no SNP treatment under Cd stress (Table 2).

4. Discussion

In agricultural plants, all heavy metals, especially Cd toxicity, impair a variety of physio-biochemical processes, the antioxidant defense system, the photosynthetic apparatus, and plant metabolism. This leads to deteriorated grain quality and significant yield losses [34,35]. Hence, implementing Cd-toxicity-mitigating methods in plant growth, and improving yield and quality relapses through better plant metabolism, remain difficult objectives for plant scientists. Recent studies revealed that NO functions as a signaling molecule and helps plants withstand biotic and abiotic stresses [3,8]. Therefore, the present study demonstrated the advantageous impacts of foliar application of NO donor SNP on different aspects of plant metabolism, including the antioxidant defense system, photosynthetic apparatus, yield, and quality attributes of three fragrant rice cultivars exposed to Cd stress.

In the present experiment, photosynthetic pigments and photosynthetic attributes were reduced under Cd stress (Figure 2 and Figure 3), which agrees with previous findings that Cd stress decreased photosynthetic efficacy and chlorophyll levels [4,8]. The possible reasons might be that Cd toxicity disorganized chloroplast ultrastructural configurations and induced higher production of ROS and this coincides with previous findings that Cd toxicity disrupted photosynthesis efficacy by affecting the stability and structural configuration of pigment–protein complexes [36]. On the other hand, foliar SNP treatments reversed the Cd-induced adversities in photosynthetic pigments, photosynthetic efficiency, and chloroplast ultrastructure configurations (Figure 2, Figure 3 and Figure 4). The possible reason might be the ability of NO to decrease oxidative damage and preserve the ultrastructural configuration and membranes of chloroplasts [2,37] as well as reduce ROS production (Figure 5). Our findings agree with earlier reports that foliar application of NO maintained the structural and functional integrity of stomata and photosynthetic attributes [22] and protected chloroplast membranes and ultrastructural configurations [38,39].

Plants accumulate compatible solutes/osmolytes, particularly proline, to perform a variety of functions, including the protection of numerous enzymes, cellular membrane stability, acting as an osmoticum to maintain turgor, and ROS scavenging activities [40]. Proline contents were significantly higher in aromatic rice plants under Cd toxicity and these results also corroborate with other heavy metals and crops like Cr in Brassica juncea [41] and Ni in Glycine max [42].

The oxidative damage of Cd toxicity was seen in all three aromatic rice cultivars, as manifested by elevated levels of H₂O₂, MDA, and EL (Figure 5). In previous studies, it was also reported that heavy metals, such as Ni in rice [21] and Cr in Brassica juncea [41], elevated H₂O₂, EL, and MDA contents. Nevertheless, foliar SNP treatments, in this study, significantly reduced H₂O₂ production, and limited lipid peroxidation (Figure 5). The possible reason is that improved enzymatic antioxidant activities in SNP-treated aromatic rice plants decreased ROS production and increased membrane stability (Figure 6), and similar findings were reported in Vigna angularis [13] and Lycopersicon esculentum [43].

SOD dismutates superoxide radicals generated in the cells, thereby preventing the formation of toxic hydroxyl, and the resultant H₂O₂ is either neutralized by APX or CAT during the AsA-GSH cycle [8,44]. The Cd toxicity exacerbated oxidative damage and increased antioxidant enzymes activities (Figure 6). Higher antioxidant enzyme activity has also been reported under various heavy metals like Ni in Oryza sativa [21], Pb in Lycopersicon esculentum [43], and Cd in Vigna angularis [13]. However, foliar spray of SNP further increased their activities while reducing oxidative damages. The reason might be that NO prevents O₂·⁻-mediated cytotoxic effects by converting O₂·⁻ to ONOO⁻ [45], thus rendering plants tolerance to oxidative stress. Moreover, antioxidant enzymes such as SOD, POD, APX, CAT, and GR scavenge ROS and stimulate the plant defense mechanism to shield from oxidative damage [46]. Our results are consistent with past research showing that NO treatment enhances plants’ resistance to free radicals in the presence of heavy metals, drought, cold, and heat stresses [21,23].

The non-enzymatic antioxidants AsA and GSH function as a redox buffer in the presence of heavy metal toxicity [47]. The SNP-induced higher AsA and GSH contents coincide with reduced H₂O₂, MDA, and EL levels, indicating that endogenous NO protected the cellular components from damage. It has also been discovered that applying exogenous NO to plants increases their tolerance to stress and activates the AsA-GSH cycle [20]. AsA, created from DHA with the help of GSH through the AsA-GSH cycle, functions as a substrate during the detoxification of H₂O₂ [43]. During H₂O₂ to H₂O conversion, APX generates MDHA and DHA. Similarly, AsA synthesis depends on MDHA and DHA, which in turn are mediated by MDHAR and DHAR, respectively. According to [48], transgenic tobacco showed less oxidative damage with overexpressed DHAR. In the current study, NO-induced stress alleviation might be due to maintaining a higher GSH concentration because GSH helps during the synthesis of phytochelatins which chelate toxic metals [3]. Our results also confirm earlier studies that endogenous NO accelerated plant responses to stressful conditions through higher DHAR and GR activities and triggered several downstream signaling events to maintain higher AsA and GSH concentrations [20,49].

In rice, productive tillers, sterility percentage, 1000-grain weight, and number of grains per panicle are the determinants of grain yield, while milling recovery, chalkiness rate, and 2AP content are the key quality estimates of fragrant rice [11,26]. In the current experiment, the presence of Cd stress resulted in a decline in both yield-contributing traits and rice quality attributes. The most probable explanation is greater oxidative damage (Figure 5) and dwindled photosynthetic characteristics (Figure 2, Figure 3 and Figure 4), as the precursors and enzymes involved during the 2AP synthesis pathway are extremely susceptible to stressed conditions [11]. Previous research [38,39] indicated that endogenous NO production promoted net photosynthesis, modulated the ultrastructural configurations of chloroplasts both in vivo and in vitro, and mediated photosynthesis in the presence of stressed conditions. Similarly, in the current experiment, NO donor SNP treatments increased the activity of antioxidant enzymes, decreased MDA and H₂O₂ levels, and well maintained the photosynthetic apparatus and antioxidant defense system. Taken together, the current study infers that among three fragrant rice cultivars, GXZ is more resistant to Cd and more responsive to SNP application, as evidenced by the higher grain yield and improved quality traits which correspond with the enhanced photosynthetic machinery and strengthened enzymatic and non-enzymatic antioxidant defense system under Cd stress compared to other fragrant rice cultivars. Future studies, however, might look at how NO donor SNP treatments affect the precursors and intermediates involved during the 2AP synthesis pathway in aromatic rice cultivated in heavy metal-contaminated soil.

5. Conclusions

The current study’s findings indicate that different fragrant rice cultivars were severely and negatively affected by Cd stress in terms of plant growth, physio-biochemical processes, and grain yield and quality parameters. The Cd toxicity boosted H₂O₂ generation and enhanced electrolyte leakage as compared to the control. However, SNP treatments effectively mitigated the impediments caused by Cd toxicity on the metabolism, grain production, and quality features of fragrant rice plants. This can be explained by a combination of factors such as reduced absorption of Cd, improved photosynthetic machinery, consistent configurations of chloroplasts, and increased ROS neutralization. Our research revealed that between the two exogenously applied SNP treatments, the 50 µM of SNP treatment was the best treatment for the fragrant rice plants for improving the plant growth, grain quality, and yield attributes under Cd stress. Moreover, the GXZ fragrant rice cultivar showed the highest resistance under Cd stress and was more responsive to SNP application than XYXZ and MXZ cultivars. We deduced that cultivating the GXZ fragrant rice cultivar along with foliar application of 50 µM of SNP could sustain the grain yield and quality features for fragrant rice cultivation under heavy metal (especially Cd)-polluted soils.