Hydrogen-Rich Water Pretreatment Alleviates the Phytotoxicity of Bispyribac-Sodium to Rice by Increasing the Activity of Antioxidant Enzymes and Enhancing Herbicide Degradation

Abstract

Bispyribac-sodium (BS) is an herbicide often used to control weeds in rice fields. Hydrogen-rich water (HRW) has recently been recommended for alleviating adverse effects, but whether and how HRW alleviates the injury to rice from exposure to BS is still largely unknown. In this study, a greenhouse hydroponic experiment showed that BS alone could substantially inhibit the plant height and fresh weight of both indica and japonica rice seedlings. For indica rice, its pretreatment with HRW at 75% saturation could markedly alleviate the impact on its size but not so with either 50% or 100% HRW. For japonica rice, all the concentrations of HRW used in this study (50%, 75%, and 100% HRW) were capable of reversing the plant size reductions. Further results revealed that the HRW supplement could increase the activity of antioxidative enzymes, including that of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD), which assist in the effective removal of ROS (reactive oxygen species). Subsequent results demonstrated a weaker inhibition of the acetolactate synthase (ALS) enzyme within five days by BS in rice seedlings pretreated with HRW than those receiving the BS treatment alone, and that the HRW pretreatment can hasten the rate at which BS is degraded in rice. Taken together, these findings strongly suggest that pretreatment with HRW may offer a promising and effective strategy to improve the ability of rice to tolerate BS.

1. Introduction

Weeds compete with rice (Oryza sativa L.) plants for water, nutrients, space, and light, seriously interfering with the growth and development of cultivated rice, which inevitably reduces the grain yield and quality [1,2]. With advances in agricultural methods, a variety of weeding methods have emerged, such as manual, flame, mulching, mechanical, and chemical weeding. Yet, chemical weed control remains the most convenient and economical method being practiced [3]. Bispyribac-sodium (sodium 2,6-bis(4,6-dimethoxypyrimidin-2-yloxy) benzoate, BS) is a pyrimidinyl carboxy herbicide with a wide spectrum and high effect. It has mainly been used to control grass and broadleaf weeds, such as Echinochloa spp., knotgrass (Paspalum distichum), arrowleafed monochoria (Monochoria vaginalis), and threeleaf arrowhead (Sagittaria trifolia), at the 2–4 leaf stage [4,5]. BS is an acetolactate synthase (ALS) inhibitor. ALS is essential for the biosynthesis of branched-chain amino acids (i.e., valine, leucine, and isoleucine) in plants. ALS inhibitors can be quickly absorbed by the stems and leaves, and inhibiting ALS activity in plants after application, which ultimately causes the sensitive population die [6,7].

The evolution of herbicide-resistant weeds has greatly increased the use of BS, and phytotoxic damage to rice caused by the greater application of BS has occurred [6,8,9]. Excessive use of BS not only increases its accumulation in the food chain but can also lead to toxic effects in humans [10]. Some research has reported that BS might also affect the germination of seeds, inhibit the growth of stems and tillers, and reduce the chlorophyll content of barnyardgrass (E. crus-galli) [11]. Further studies have shown that japonica rice cultivars are more sensitive to BS than are indica rice cultivars, although the sensitivity of the ALS enzyme is similar between them; hence, rice sensitivity to BS may be related to non-targeted tolerance [8,12]. For example, Saika et al. (2014) found that a cytochrome P450 gene CYP72A31 confers tolerance to BS in indica rice cultivars, but this gene was inactivated by a frameshift mutation in japonica rice cultivars [13].

Hydrogen is the most abundant chemical element, constituting approximately 75% of the mass of the universe. Its molecular form, hydrogen gas (H₂), is colorless, odorless, rare in Earth’s atmosphere, and is gaining recognition as a novel energy source [14]. Evidently, H₂ is a potent antioxidative and anti-inflammatory agent with promising potential for medical applications [15,16]. However, because H₂ is flammable, explosive, and dangerous, the much safer hydrogen-rich water (HRW) is often used to replace hydrogen. Hydrogen-rich water is a saturated hydrogen-rich solution prepared by dissolving pure hydrogen in an aqueous solution. One study reported that HRW could alleviate oxidative stress damage induced in the rat brain by focal ischemia and reperfusion by reducing the abundance of hydroxyl radicals (^•HO) and peroxynitrite (ONOO^–) [17]. In humans, the consumption of HRW by adults effectively alleviated the symptoms of diabetes [18], mitigated cisplatin-induced nephrotoxicity [19], and prevented the risk occurrence of chronic allograft nephropathy after undergoing renal transplantation [20]. The evidence accumulated by these studies suggests that H₂ could protect various cells, tissues, and organs against oxidative injury [15,21,22]. In plants, experiments have shown that H₂ could promote growth vigor and seed germination in certain crops (soybean, barley, canola, spring wheat and mung bean) [23,24], promote the synthesis of anthocyanin in radish sprouts [25], and delay the post-harvest ripening and senescence of kiwifruit [26]. Additionally, the exogenous application of HRW could improve the tolerance of plants to certain heavy metals, such as Cd, Al and Hg [27,28,29]. Recently, there has been burgeoning interest in its ability to improve the tolerance of plants to various abiotic stress factors, such as salt [30,31], drought [32], strong light [33], high temperature [34], and freezing [32].

Crop plants are often exposed to one or more herbicide stresses (as well as abiotic ones). Despite long knowing that H₂ is an important bio-regulator, relatively few studies have focused on H₂ biology in crop plants under herbicide stress. The exogenous herbicide stress often induces the production of reactive oxygen species (ROS), which can cause membrane lipid peroxidation and protein denaturation, among other forms of damage. In order to protect against ROS-induced damage, plants have evolved a series of detoxification systems, namely the antioxidant enzymatic systems of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD). Studies have shown that HRW can alleviate ROS pressure by increasing the activity of these enzymes, suggesting that it may also be effective at relieving stress caused herbicide exposure [27,28,32,33,34]. Therefore, whether and how HRW affects the sensitivity of rice to BS should be studied because of the universality of BS phytotoxicity to rice and the accessibility of HRW.

A certain yield loss caused by herbicide injury to crops is inevitable but hard to predict. In particular, the impact of BS upon rice plants warrants more attention. As a novel antioxidant and signaling molecule, HRW has great application potential in scavenging for ROS in animals and plants. The study brings HRW to bear upon the mitigation of herbicidal phytotoxicity as an attempt. In this paper, “9311” (O. sativa spp. Indica) and “Nipponbare“ (O. Sativa spp. Japonica) rice were selected as experimental materials. The changes in root length, biomass and plant height of rice seedlings after treatment with HRW and BS were measured by bioassay. The changes in indexes related to the antioxidant defense system and the activity of ALS in japonica and indica rice were studied. The residue of BS in the leaves of both kinds of rice was determined by liquid phase and liquid mass analysis. This work expands our understanding of the mechanisms by which H₂ ameliorates exogenous stress in plants.

2. Materials and Methods

2.1. Chemicals

The 20% Bispyribac-sodium (wettable powder) used in this work was kindly supplied by the Jiangsu Institute of Ecomones Co., Ltd. (Changzhou, China). Chemical reagents such as thiamine pyrophosphate (TPP), polyvinylpyrrolidone (PVP), and dithiothreitol (DTT) were purchased from Sigma (St. Louis, MO, USA). Ethylenediaminetetraacetic acid (EDTA) and nitroblue tetrazolium (NBT) were purchased from Bio Basic Inc. (Toronto, ON, Canada). Analytical grade ethyl acetate and chromatographic grade methanol were bought from the Shanghai Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of HRW

Purified H₂ gas (99.99%, v/v) generated by an H₂-producing apparatus (SCH-300, Saikesaisi Hydrogen Energy Co., Ltd., Jinan, China) was bubbled into 1000 mL of half-strength Hoagland’s solution (pH 6.0) at a rate of 150 mL min⁻¹ for 30 min [35]. Then, the saturated 100% stock solution was immediately diluted to the required concentration (50%, 75%, 100% (v/v)). Under our experimental conditions, the H₂ concentration in the freshly prepared HRW was analyzed by gas chromatography (Shimadzu Corporation, Kyoto, Japan) and found to be 0.22 mM; this was maintained at a relatively constant level at 25 °C for at least 12 h.

2.3. Plants, Growth Conditions, and Treatments

The seeds of “9311” (O. sativa spp. Indica) and “Nipponbare” (O. Sativa spp. Japonica) rice cultivars—kindly supplied by Institute of Food Crops, Jiangsu Academy of Agricultural Sciences (Nanjing, China)—were surface-sterilized with 10% hydrogen peroxide for 10 min, rinsed extensively in distilled water, and germinated at 25 °C under darkness. Seeds were evenly placed in 12 cm-diameter glass Petri dishes lined with two pieces of Whatman No. 1 filter paper (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) moistened with distilled water. All dishes were placed inside incubators at 25 °C with 0 h light/24 h darkness. Every 30 uniform seedlings were then chosen and transferred to one plastic chamber (height: 12 cm) and cultured in a nutrient medium (half-strength Hoagland’s solution). These seedlings were grown in an illuminating incubator (12 h of light at an intensity of 151 µmol m⁻² s⁻¹, 32 °C ± 1 °C; 12 h in the dark, at 20 °C ± 1 °C). When the seedlings had reached the 2–3-leaf stage, they were incubated in nutrient solution containing one of three concentrations of HRW (50%, 75%, and 100%) alone for 24 h (solutions were renewed every 12 h to maintain constant concentrations) or sprayed with the indicated concentration of BS alone (30 or 45 g a.i./hm²), or done so in combination. For each combination treatment, rice seedlings were firstly treated with the indicated concentration for 24 h; after that, the seedlings were sprayed with BS at 30 or 45 g a.i./hm². To do this, seedlings were treated using a laboratory sprayer equipped with a flat-fan nozzle to deliver 280 L/hm² at 230 kPa spray pressure. The untreated seedlings (cultured on the nutrient solution, receiving neither BS nor HRW) served as the control group. The examination of growth response variables was carried out on three replicates of 10 plants each. Five days after applying the treatments, the measurements of root length, plant height, and fresh weight of entire seedling were obtained using a ruler (0.1 cm) and an electronic balance ME203 (Mettler Toledo, Zurich, Switzerland) (accuracy: 0.001 g). For the residue analysis of BS, at least 30 plants per treatment were sampled, then immediately frozen in liquid nitrogen, and stored at –80 °C until their further analysis.

2.4. ALS Activity Assay

2.4.1. Extraction of the ALS Enzyme

The ALS enzyme was extracted following the methodology of Yu et al. (2010) [36], with minor modifications. The seedlings were incubated in a nutrient solution containing 75% HRW alone for 24 h or sprayed with BS alone at 30 g a.i./hm², or their combination. Leaf tissues (about 1 g) were harvested from each treatment (or treatment combination) at 1, 2, 3, 4, and 5 days after treatment (DAT), immediately frozen in liquid nitrogen, and stored at −80 °C. Frozen materials were then placed into a precooled glass mortar and ground into a fine powder in liquid N₂ and homogenized in an 8 mL extraction buffer, which contained 0.1 M potassium phosphate buffer (pH 7.0), 10 mM sodium pyruvate, 0.5 mM MgCl₂, 10 µM flavin adenine dinucleotide (FAD), 0.5 mM TPP, 1 mM DTT, 10% (v/v) glycerol, 0.5% PVP, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The homogenate was centrifuged at 25,000× g for 30 min at 4 °C. The ensuing supernatant was then transferred to a new tube and slowly brought to 50% (NH₄)₂SO₄ saturation. After being allowed to precipitate for 2 h, the solution was centrifuged again at 25,000× g for 30 min at 4 °C. The pellet was re-dissolved in 2 mL of pH 7.5 solution buffer containing 50 mM HEPES (N-(2-hydroxyethyl) piperazine-N-(2-ethanesulfonic acid)), 20 mM MgCl₂, 200 mM sodium pyruvate, 20 µM FAD, and 2 mM TPP. The soluble protein concentration of the enzyme extract was determined by the Bradford method [37].

2.4.2. Measurement of ALS Enzyme Activity

ALS activity was determined colorimetrically (530 nm) by measuring the production of acetoin; this was performed according to the method described by Yu et al. (2010) [36]. First, 100 µL of enzyme extract was dark-reacted in a constant-temperature water bath at 37 °C for 1 h, at which point 40 µL of 3 M H₂SO₄ (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added to stop the reaction. Second, 190 µL of creatine solution (0.55% in deionized water) and 190 µL of α-naphthol solution (5.5% in 5 M NaOH) (Sangon Biotech Inc., Shanghai, China) were added, and the mixture was incubated at 60 °C for 15 min. Finally, the amount of acetoin formed in the reaction solution was determined by measuring the absorbance value at 530 nm with a microplate spectrophotometer (Epoch™, BioTek^® Instruments, Inc., Winooski, VT, USA). ALS activity was expressed in units of nmol acetoin formed per minute per milligram of protein.

2.5. Antioxidant Enzyme Assays

For antioxidant enzyme activity assays, the experimental design was the same as described above for ALS enzyme activity assays, except that the sampling was only carried out once, at 5 DAT. Frozen leaf segments (each 0.3 g) were ground in a mortar under liquid N₂ and homogenized in 5 mL of 50 mM cool phosphate buffer (pH 7.0) containing 1 mM EDTA and 1% (w/v) PVP, for the SOD (EC 1.15.1.1), POD (EC 1.11.1.7), and CAT (EC1.11.1.6) total-activity assays. The homogenates were centrifuged at 12,000× g for 20 min at 4 °C, and their supernatants were used for the corresponding assays of enzyme activity, as detailed below.

SOD activity was determined by measuring its capacity to reduce NBT by the superoxide anion generated by the riboflavin system under illumination [38]. The 3 mL reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 2 nM riboflavin, 75 nM NBT, 0.1 mM EDTA, and 100 µL of enzyme extract. The reaction mixtures were illuminated for 15 min at a light intensity of 1000 µmol m⁻² s⁻¹. One unit of SOD (U) was defined as the amount of crude enzyme extract required to cause 50% inhibition of the reduction of NBT as monitored at 560 nm.

POD activity was determined by measuring the oxidation of guaiacol according to the method of Han et al. (2008) [39]. The reaction mixture consisted of 50 mM potassium phosphate buffer (pH 7.0), 0.4% H₂O₂, 1% guaiacol, and 100 µL of enzyme extract. The increase in absorbance caused by guaiacol oxidation was measured at 470 nm for 1 min. The extinction coefficient was 26.6 mM⁻¹ cm⁻¹. One unit of POD was defined as an increase of 0.01 of A470 per min.

CAT activity was determined by monitoring the consumption of H₂O₂ (extinction coefficient: 39.4 mM⁻¹ cm⁻¹) at 240 nm for 3 min [40]. The 3 mL reaction mixture contained a 50 mM potassium phosphate buffer (pH 7.0), 10 mM H₂O₂, and 100 µL of enzyme extract.

2.6. Determination of the BS Content in Rice Leaves

Previous research showed that a greater percentage of absorbed BS was retained in treated leaves than in other plant sections, indicating that BS had limited translocation to other plant parts; thus, we paid special attention to the changes in BS content in leaves [9]. For the residue analysis of BS in rice leaves, 0.5 g of rice leaf samples first ground with liquid nitrogen were weighed into a 250 mL Erlenmeyer flask, to which 100 mL of a 4% NaHCO₃ aqueous solution was added. All the samples were shaken and extracted for 60 min, after which the extract was successively washed with 50 mL of petroleum ether and dichloromethane. The aqueous phase (containing the final residue) was acidified with 1 mol/L HCl, and then re-extracted twice using 40 mL and 30 mL ethyl acetate, respectively. The ethyl acetate phase combined by the two extractions was dehydrated with anhydrous sodium sulfate and concentrated on a rotating evaporator (40 °C) to a near-dry state. Next, the residual component was dissolved in 100 µL of methanol. The samples were analyzed by HPLC (high-performance liquid chromatography) coupled with an ultraviolet (UV) detector, equipped with a stainless column (250 mm × 4.0 mm ODS; Shimadzu Corporation, Kyoto, Japan) for chromatography. The mobile phase was methanol:water (75:25, v/v), and the flow rate was set to 0.8 mL/min. The detection wavelength was 250 nm and the injection volume was 20 µL [41].

2.7. Statistical Analysis

All experiments were conducted in a completely randomized design with three replications, and each experiment was conducted three times. Two-way ANOVA (SPSS 20.0 software, IBM Corporation, New York, NY, USA) was conducted to determine whether there was a significant difference in results between experiment repeats. The treatments were the fixed factor, the experimental run (run) was the random factor, and the relevant indicator was the dependent variable. The data were combined over the experiments for analysis after the run, and the treatment interaction effects were nonsignificant for all of the experiments. The data were the mean of three runs and were subjected to one-way ANOVA (single factor with X levels (~treatments)). The averages were compared by Duncan’s multiple-range test at 5% significance.

3. Results

3.1. Effects of HRW and BS on the Growth of Indica and Japonica Rice

No differences were detected in plant height, root length, or fresh weight of either cultivar when treated with 50% HRW, which differed little from the treatments with nutrient solution only (

Table 1. Effects of the herbicide BS (Bispyribac-sodium) and HRW (hydrogen-rich water) on root length, biomass, and plant height of indica rice (at five days post treatment).

Treatments	Response Variables
	Root Length (cm)	Biomass (g/FW)	Plant Height (cm)
CK	6.07 ± 0.25a	0.243 ± 0.010a	19.47 ± 0.54a
50% HRW alone	6.25 ± 0.40a	0.246 ± 0.021a	19.24 ± 0.25a
75% HRW alone	6.31 ± 0.23a	0.253 ± 0.013a	19.04 ± 0.07a
100% HRW alone	5.91 ± 0.22a	0.231 ± 0.004a	18.98 ± 0.62a
S1	6.31 ± 0.06a	0.200 ± 0.002b	17.04 ± 0.23b
S2	6.25 ± 0.08a	0.193 ± 0.003b	16.81 ± 0.20b

Values are the mean ± SE from three independent experiments. Different letters within columns indicate significant differences according to Duncan’s multiple-range test (p < 0.05). S1 and S2 denote BS treatments at concentrations of 30 and 45 g a.i./hm², respectively. CK: the control group. FW: fresh weight.

and

Table 2. Effects of the herbicide BS (Bispyribac-sodium) and HRW (hydrogen-rich water) on root length, biomass, and plant height of japonica rice (at five days post treatment).

Treatments	Response Variables
	Root Length (cm)	Biomass (g/FW)	Plant Height (cm)
CK	5.31 ± 0.23a	0.195 ± 0.013ab	16.74 ± 0.42ab
50% HRW alone	5.24 ± 0.17ab	0.179 ± 0.005b	17.00 ± 0.23a
75% HRW alone	5.26 ± 0.42ab	0.206 ± 0.016a	16.21 ± 0.19b
100% HRW alone	5.57 ± 0.14ab	0.193 ± 0.008ab	17.24 ± 0.14a
S1	4.95 ± 0.07ab	0.142 ± 0.001c	13.71 ± 0.17c
S2	4.62 ± 0.46b	0.138 ± 0.002c	13.11 ± 0.17c

Values are the mean ± SE from three independent experiments. Different letters within columns indicate significant differences according to Duncan’s multiple-range test (p < 0.05). S1 and S2 denote BS treatments at concentrations of 30 and 45 g a.i./hm², respectively. CK: the control group. FW: fresh weight.

). No significant changes in root length, biomass or plant height with higher concentrations of HRW (75% and 100%) compared with the nutrient solution only indicates that HRW alone does not influence the growth of japonica or indica rice. After spraying two concentrations of BS (30 or 45 g a.i./hm²) on rice seedlings at the 2–3-leaf stage, their growth dramatically decreased relative to the control counterparts; this suggested that applying BS at either concentration inhibited the growth of both rice cultivars by impairing the plants’ physiology or causing damage to their tissues. Meanwhile, the growth inhibition of japonica and indica rice was similar between the two BS concentrations. Surprisingly, across the treatments, no significant differences in the root length of indica rice seedlings were found, showing that the HRW and BS used at the above concentrations had no obvious effect on the root length of indica rice seedlings.

3.2. Effect of HRW on BS-Induced Growth Inhibition

Treatment with HRW can alleviate the detrimental effects of various abiotic stresses [42]. For indica rice, the plant height as well as fresh weight of seedlings treated with 75% HRW increased by 12.0% and 22%, respectively, under 30 g a.i./hm² of BS, in comparison with those of the samples subjected to BS alone, indicating that the 75% HRW pretreatment had a significant mitigation effect against BS-induced growth reductions (Figure 1A,B). However, at an HRW concentration of 50% and 100%, the seedling growth of indica was markedly lower, being similar to that under the BS treatment alone. As shown in Figure 1C,D, the plant height and fresh weight of japonica rice seedlings pretreated with 50%, 75% or 100% HRW were always significantly higher than those pretreated with BS alone, and on par with the growth of the control group.

3.3. Effects of HRW and BS on ALS Enzyme Activity

Given that the mitigation effect of 75% HRW pretreatment was more pronounced, and the two concentrations of BS had similar effects on rice (Figure 1), in this experiment we applied 75% HRW alone or 30 g a.i./hm² of BS alone, or both together. The activity of ALS in indica rice seedlings treated with HRW alone was relatively stable, being similar to the control group, but it was significantly higher than in those seedlings treated with BS alone or with HRW and BS. The ALS enzyme activity in response to BS alone and co-treatment with HRW and BS sharply decreased at first, and then slowly increased with time; still, the reduced activity in the samples co-treated with HRW and BS was nonetheless lower than that under BS alone within five days post-treatment, indicating that the HRW pretreatment conferred to indica rice seedlings a protective effect against ALS enzyme activities (Figure 2A). The pattern of changed ALS activity in response to each treatment for japonica rice was similar to that found for indica rice. Among these treatments, the highest activity occurred under HRW, followed by HRW + BS and then BS (Figure 2B).

3.4. Changed Activity of Antioxidant Enzymes

In indica rice, SOD activity was greatest in the seedling leaves treated with HRW alone and least in the control group lacking HRW (Figure 3A). Both BS and HRW treatments seemed to stimulate SOD activity. Compared with the control group, no changes in POD activity were detected under the BS treatment alone, but the POD activity in response to the HRW treatment alone was significantly enhanced, indicating that HRW but not BS could augment the POD activity of indica rice. Furthermore, POD activity was highest under the co-treatment with HRW and BS (Figure 3B). The results revealed that both HRW and BS could improve CAT enzyme activity (Figure 3C). Similar to the changes in POD activity, the highest levels of CAT activity in indica rice occurred when rice seedlings were co-treated with HRW and BS. The post-treatment responses of SOD and CAT activity in japonica rice displayed similar tendencies. HRW and BS could both increase the enzyme activities of SOD and CAT. The enzyme activities of CAT peaked under the co-treatment HRW and BS (Figure 3F). POD activity of the HRW + BS treatments was higher than the control treatments and HRW alone, but there were no differences among HRW + BS and BS only treatments. (Figure 3E).

3.5. HRW Enhances BS Metabolism in Rice Leaves

Recent findings suggest that the transcripts of glutathione S-transferase (GST) and glutathione reductase (GR), which are major detoxifying enzymes acting against exogenous toxic compounds, are increased after HRW pretreatment [42]. So, we posited that HRW pretreatment could enhance herbicide metabolism in rice. A pilot experiment confirmed that BS was fully absorbed by rice leaves after 6 h at 30 g a.i./hm² of BS; at this time, the content of BS in the leaves of indica rice and japonica rice was 1.278 mg/kg and 1.024 mg/kg, respectively (

Table 3. Effect of HRW (hydrogen-rich water) pretreatment on the metabolism of the herbicide BS (Bispyribac-sodium) in rice (at five days post treatment).

Treatments	9311 (Indica Rice)		Nipponbare (Japonica Rice)
	Residue (mg/kg)	Degradation (%)	Residue (mg/kg)	Degradation (%)
Control	1.278 ± 0.018	-	1.024 ± 0.191	-
BS	0.445 ± 0.072	65.18 ± 5.63b	0.557 ± 0.059	45.61 ± 5.71b
50% HRW + BS	0.271 ± 0.015	78.79 ± 1.18a	0.273 ± 0.035	73.34 ± 3.38a
75% HRW + BS	0.243 ± 0.032	80.99 ± 2.54a	0.294 ± 0.052	71.29 ± 5.05a
100% HRW + BS	0.304 ± 0.048	76.21 ± 3.74a	0.241 ± 0.028	76.46 ± 2.74a

Values are the mean ± SE from three independent experiments. Different letters within columns indicate significant differences according to Duncan’s multiple-range test (p < 0.05). The “Control” here is actually a baseline measure. BS indicates treatment with BS (Bispyribac-sodium) alone. 50% HRW + BS, 75%HRW + BS and 100%HRW + BS indicate the combination treatment with different concentration of HRW and BS.

). Therefore, the BS content in leaves at this time point served as the control (baseline). The amount of BS degraded in indica and japonica rice reached 65.18% and 45.61%, respectively, after treatment with BS alone. Pretreatment with HRW significantly enhanced the degradation of BS in both rice plants. However, the effect of different concentrations of HRW on the metabolic rate of BS in rice was not significant. Irrespective of the HRW pretreatment, the ability to break down BS seemed to be higher in the indica than japonica rice seedlings.

4. Discussion

The impact upon crop plants should not be underestimated when controlling weeds using herbicides. BS acts by inhibiting the synthesis of branched-chain amino acids, which are essential for normal cell division and growth. The mode of action of BS via the inhibition of this pathway is indicative of the fact that, directly or indirectly, the biochemical quality attributes of a crop may be adversely affected by BS application, leading to an inferior crop quality. Previous studies have shown that BS might cause morpho-physiological and biochemical changes, including the reduction of growth, chlorophyll content, total phenolic compounds and some anatomical leaf parameters [7,43,44]. For most paddy herbicides, rice may suffer certain initial adverse effects, and in a few cases this could persist until a crop’s harvest [45,46]. A few studies have shown that BS has weak adsorption, moderate to high mobility in soil, and is slightly persistent in aerobic soil, rendering it liable to cause phytotoxicity to sensitive crops and non-target organisms [47,48,49]. Here we demonstrated that the growth of both japonica and indica rice seedlings, in terms of their height and fresh weight, were slowed by BS, and their leaves also turned yellow, indicating that they are prone to impaired growth and tissue damage from exposure to BS. However, their root length was not affected by BS in this study (Table 1 and Table 2). The results also showed that the plant height and fresh weight of indica rice seedlings incurring BS stress decreased less in comparison with japonica rice seedlings. For example, after exposing them to a BS concentration of 30 g a.i./hm², while the plant height and fresh weight of indica rice seedlings fell by 12.48% and 17.70%, they were reduced by 18.1% and 27.18% in japonica rice seedlings (Table 1 and Table 2). This suggests that indica rice cultivars have a stronger innate tolerance to BS than do japonica rice cultivars. It was found that CYP72A31, which is a cytochrome P450 gene, confers tolerance to BS in rice of the indica variety, while japonica rice varieties are BS sensitive [13]. Beside this, Wang et al. found that increasing the tolerance to BS was able to allow glutathione homeostasis to recover in indica rice cultivars compared with japonica rice cultivars [12].

The application of herbicides, as an abiotic stress factor, can induce plants to form ROS that disturb their redox homeostasis [50]. For example, the diphenyl ethers, cyclic imides and lutidine derivatives act by inhibiting biosynthetic pathways, leading to the subsequent accumulation of reactive, radical-forming intermediates [51]. According to the latest research, auxin herbicides can increase the production of superoxide free radicals ($O_2^{•-}$) and hydrogen peroxide, accompanied by the up-regulation of ROS-scavenging enzymes in vivo [52]. The antioxidant activities of superoxide dismutase, catalase, glutathione reductase, and glutathione S-transferase were induced in the leaves of 2,4-D-treated Pisum sativum as well as in the leaves and roots of quinclorac-treated early watergrass (E. oryzicola Vasing.) plants [53,54]. These latter findings are consistent with the significant increase we found in the antioxidant activities of indica and japonica rice seedlings treated with BS alone (Figure 3).

The biological regulation function of H₂ in plants has drawn increasing interest in recent years [55,56]. Nearly 20 years ago, Dong et al. (2003) observed that soil treated with H₂ led to the improved growth of canola, and they were the first to propose the “H₂ fertilization” hypothesis [57]. Different from this, compared with the control, treatment with HRW (saturated forms of H₂) alone had no effect on the growth of rice plants in this study. H₂ plays a positive role in plant resistance responses to stresses; it can especially alleviate various stresses caused by drought, salinity, light, freezing, heavy metals, and so on. It has been deduced that the H₂-mediated enhancement of plant adaptive responses against abiotic stresses is a universal phenomenon [27,28,29,30,31,32,33,34,56,58]. Supporting that view, our results demonstrated that rice seedlings pretreated with HRW displayed a better tolerance to BS at two different concentrations (30 or 45 g a.i./hm²) when compared with their counterparts that were not pretreated. Nevertheless, only pretreatment with 75% HRW significantly alleviated the inhibition of growth (plant height and fresh weight) of indica rice seedlings vis-à-vis BS alone (neither the 50% nor 100% HRW pretreatment led to a similar alleviation role). For japonica rice, the impact of BS on the rice seedlings was similarly alleviated by all three pretreatments of HRW (50%, 75% and 100%). Previous studies revealed that pretreatment using exogenous HRW led to a concentration-dependent effect on the growth of plants [33,41], but the differences among various treatment concentrations were negligible (i.e., not significant), demonstrating that the HRW pretreatment can alleviate BS toxicity in a dose-independent manner. The discrepancy between japonica and indica rice seedlings in their response to the HRW pretreatment may be due to genetic differences (Figure 1). The ability of HRW to protect crop plants from herbicide damage is similar to that of herbicide safeners [59]. Yet, whether HRW likewise impacts the bio-efficacy of BS in controlling weeds must be investigated as well.

In the experiment, both HRW and BS could increase the activity of antioxidant enzymes (SOD and CAT), albeit via different ways (Figure 3). That of HRW might entail H₂ readily permeating the cell membrane, thereby increasing the gene expression of antioxidant genes encoding SOD and CAT [35], while that of BS is simply positive feedback to exogenous stress. Our results also showed that the antioxidant enzyme activity was the highest under the HRW and BS co-treatment (Figure 3A), a result that might be related to the superposition of the two effects. The HRW pretreatment could protect the enzyme activity of ALS in rice seedlings, which could be one reason for why HRW is able to alleviate BS toxicity in rice; supporting that, we did observe a higher activity of ALS in the co-treatment with HRW than in response to the treatment with BS alone (Figure 2). Additionally, the HRW pretreatment enhanced the capacity of herbicide metabolism in both rice cultivars, thus reducing the amount of herbicide reaching ALS and alleviating the inhibition of ALS (Table 3). In terms of the in vivo degradation of BS, this was clearly higher in the indica than japonica rice cultivar (Table 3), a finding consistent with previous work reported by Saika et al. (2014) showing that indica rice plants might harbor greater tolerance to BS due to their innately higher metabolic rate [13].

5. Conclusions

Altogether, the results obtained in this work prove that HRW is able to alleviate BS phytotoxicity. The protective role of HRW in rice plants in response to BS stress may be linked to the fact that HRW can increase the activity of antioxidant enzymes. Supplementary results revealed that HRW has a certain positive effect on both ALS activity and the degradation of BS, which lessens the impact of BS on rice plants, thus improving their tolerance to this herbicide. It is worth noting that this study was conducted under greenhouse conditions. Field trials would be needed to see whether the beneficial effects of HRW hold up under actual rice-growing conditions in paddy fields. The mechanisms identified here by which HRW contributes to BS-tolerant responses should be investigated by genetic and molecular techniques in future research.