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Article

Effects of Abscisic Acid on Rice Seed Dormancy: Antioxidant Response and Accumulations of Melatonin, Phenolics and Momilactones

by
Ramin Rayee
1,
La Hoang Anh
1,2 and
Tran Dang Xuan
1,2,3,*
1
Transdisciplinary Science and Engineering Program, Graduate School of Advanced Science and Engineering, Hiroshima University, Hiroshima 739-8529, Japan
2
Center for the Planetary Health and Innovation Science (PHIS), The IDEC Institute, Hiroshima University, Hiroshima 739-8529, Japan
3
Faculty of Smart Agriculture, Graduate School of Innovation and Practice for Smart Society, Hiroshima University, Hiroshima 739-8529, Japan
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(8), 1229; https://doi.org/10.3390/agriculture14081229 (registering DOI)
Submission received: 2 June 2024 / Revised: 16 July 2024 / Accepted: 17 July 2024 / Published: 26 July 2024
(This article belongs to the Special Issue Rice Ecophysiology and Production: Yield, Quality and Sustainability)
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Abstract

:
Abscisic acid (ABA) is a well-known phytohormone that initiates seed dormancy. This is the first study elucidating the variability and relationship in the accumulation of phenolics, melatonin, and momilactones A (MA) and B (MB) during a rice (Oryza sativa L. var. Koshihikari, Akisakari, and Akiroman) seed dormancy process treated by ABA with concentrations of 0 (control), 10, 50, and 100 µM over 8 days. Accordingly, increased concentrations of ABA resulted in an extended dormancy period of rice seeds, along with reducing fresh weight while maintaining stable dry weight in all varieties. ABA treatment elevated total phenolic (TPC) and flavonoid (TFC) contents. Particularly, quantities of ferulic and cinnamic acids were enhanced, in line with the promoted antioxidant capacities of ABA-treated rice seeds. Momilactone contents were increased (3.8% to 32.6% for MA and 16.3% to 31.3% for MB) during the extension of rice seed dormancy regulated by ABA. Notably, the accumulation tendency of MA and MB under ABA effects was consistent with that of melatonin, a phytohormone associated with seed dormancy prolongation. The finding implies that MA and MB may play a role alongside melatonin in signaling the extended dormancy of rice seeds through the ABA pathway. Future research should delve into the molecular mechanisms underlying these observations.

1. Introduction

More than half of the world’s population relies on rice as a staple food, emphasizing the vital role of rice production in securing global food stability. To enhance and maintain rice productivity, it is essential for rice seeds to have sufficient dormancy periods, ensuring they show strong germination characteristics after sowing. Rice production is challenged by both biotic and abiotic stress factors, including elevated humidity and wet conditions prior to harvest, which lead to the germination of grains on the spike, commonly known as preharvest sprouting [1]. The preharvest sprouting of rice seeds is closely related to the extent of dormancy, an adaptive trait that prevents seed germination in favorable environmental conditions [2]. A high level of dormancy in cereal seeds can lead to unfavorable outcomes, such as uneven and delayed postharvest germination. Maintaining an optimal level of seed dormancy is required for improving the yield and quality of cereal crops [3]. Previous studies have documented insights into the importance of the metabolic and signaling aspects of various plant hormones, as well as their potential interaction in regulating and releasing dormancy in cereal seeds [1,4,5]. Among the phytohormones, abscisic acid (ABA) and gibberellic acid (GA) are recognized as key contributors to the regulation of dormancy and germination. ABA plays a positive role in inducing and maintaining dormancy, while GA promotes seed germination [6,7]. However, there is currently no study addressing the duration for which ABA can maintain dormancy in rice seeds [8].
Phenolic compounds in rice play important roles in terms of physiological processes, including functions related to metabolism, biosynthesis of lignin and suberin molecules, and responses to the environment. It is believed that both phenolic compounds and ABA function as inhibitors of seed germination and development, playing roles in the regulation of plant growth [9]. Apart from their individual inhibitory effects, phenolic compounds have been found to antagonize some effects of ABA. For example, they can reverse ABA-induced abscission, hypocotyl growth, and seed germination [10,11,12]. Collectively, these studies indicate the significant involvement of phenolic compound metabolism in response to ABA. However, it is not clear if ABA is involved in regulating phenolic compound synthesis during rice seed dormancy. Therefore, there is a lack of direct biochemical evidence to support this idea.
Reactive oxygen species (ROS), specifically the superoxide anion radical (O2•−) and hydrogen peroxide (H2O2), play a crucial role in the regulation of seed germination [13,14,15]. These ROS are produced as metabolic by-products during aerobic reactions in both seed germination and dormancy [13,16]. Under optimal conditions, the levels of ROS in seeds, particularly H2O2, are increased during the initial imbibition period in numerous plant species such as rice [17], barley [18], and tobacco [19]. Accumulation of ROS is considered important for seed germination, and exogenous application of H2O2 is recommended to overcome seed dormancy and promote the germination process in various plant species [14,20,21]. Conversely, suppressing ROS production through diphenylene iodonium results in delayed seed germination and improved dormancy [20]. However, the ABA effect on inhibiting these ROS through promoting antioxidant activity during rice seed dormancy has not been studied yet.
Momilactones and melatonin are natural compounds that influence plant seed germination. Momilactones are diterpenoids that were initially discovered by Kato et al. [22]. The term “momilactone” is derived from the Japanese word “momi”, which means rice husk, and it essentially refers to any natural lactone originating from rice husks. MA and MB were reported to inhibit the growth and germination of some test plants [23]. MB may affect the initial growth of Arabidopsis seedlings by inhibiting the mobilization of protein storage reserves [23]. These compounds demonstrated inhibitory effects on the germination of Arabidopsis at concentrations exceeding 10 μM and 30 μM [24]. MB exhibited complete inhibition of germination in Leptochloa chinenesis (at 4 µg/mL), Amaranthus retroflexus (at 20 µg/mL), and Cyperus difformis (at 20 µg/mL) [25]. In another study, MA and MB inhibited the germination of Echinochloa crus-galli and Solidago altissima [26]. Melatonin (N-acetyl-5-methoxytryptamine) is a widespread indole hormone in plants. It possesses functionalities the same as auxin (IAA), effectively scavenging free radicals, fostering plant growth and development, and enhancing plant resilience against both abiotic and biotic stresses [27]. Also, melatonin is reported to affect diverse physiological processes, including germination in plants [28]. To date, the interaction of ABA with momilactones and melatonin during rice seed dormancy has not been explored yet. The application of exogenous ABA influences rice seed dormancy under normal and stressful conditions. However, many questions remain unknown. For instance, what is the duration for which ABA can maintain dormancy in rice seeds under normal environmental conditions? What is the physiological basis for the regulation of rice seed dormancy by ABA? Additionally, how does ABA interact with known seed germination signals, such as ROS, melatonin, and momilactones?

2. Materials and Methods

2.1. Materials and Treatments

Seeds of three Japonica rice varieties, namely Koshihikari, Akisakari, and Akiroman, were sourced from the Japan Agricultural (JA) Group in Hiroshima, Japan, in August 2023. These varieties are distinguished as elite cultivars with good performance not only in yield but also in quality. The solvents methanol and hexane were purchased from Junsei Chemical Co., Ltd., Tokyo, Japan. Other chemicals such as sodium hypochlorite (NaClO), Folin-Ciocalteu’s reagent, sodium carbonate (Na2CO3), aluminum chloride (AlCl3), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), formic acid, gallic acid, sinapic acid, cinnamic acid, salicylic acid, ferulic acid, and melatonin were procured from Kanto Chemical Co., Inc., Tokyo, Japan. Additionally, pure MA and MB were previously isolated from rice husk at our Laboratory of Plant Physiology and Biochemistry, Graduate School of Advanced Science and Engineering, Hiroshima University, Japan [29]. The identification and confirmation of MA and MB were reported in a prior study conducted by Hasan et al. [29].
The seeds were surface sterilized using 0.1% NaClO solution for 20 min, followed by three successive rinses in distilled water to remove any residual sterilant. After sterilization, the seeds were placed in Petri dishes (90 × 15 mm) and exposed to various ABA concentrations (0, 10, 50, and 100 µM) using 10 mL of solution for each treatment, with distilled water serving as the control group. The selection of these specific ABA concentrations was based on prior studies [8,30,31]. The seeds were then incubated under dark conditions at 35 °C for the initial 24 h, followed by a 30/28 °C (day/night), 12-h photoperiod, and 75–80% humidity in a growth chamber. The experiment was divided into two groups: (i) evaluating seed dormancy and (ii) biochemical analysis. Samples for biochemical analysis were collected on the fourth day (Figure 1), marking the end of the control group’s germination period, while dormancy evaluation extended to the eighth day of germination. The study structure was comprised of seven treatments (three rice varieties and four ABA concentration levels), with each treatment replicated thrice. In total, 72 Petri dishes were used in the experiment.

2.2. Dormancy Evaluation

Petri dishes were observed daily, and the number of germinated seeds was recorded through day 8. A seed was considered germinated when the radical was visible and the growth reached 2 mm. The data collected daily were used to calculate dormancy days’ fresh and dry weights [32,33,34].

2.3. Sample Extraction

The collected samples were first dried in an oven at 50 °C and ground into fine powder. Following that, 20 g of rice samples were subjected to extraction with 40 mL of pure methanol for 2 days using a shaker. The obtained extracts were combined with hexane in a separator, and the methanolic phase was then collected. Subsequently, the filtered methanolic extract was concentrated through a vacuum evaporator. The dried extract was then stored in a vial for the assessment of phenolic and momilactone contents, as well as antioxidant activity.

2.4. Total Phenolic Content (TPC) and Total Flavonoid Content (TFC)

The determination of TPC and TFC was performed following the method reported in our previous publication, with slight modifications [35]. TPC calculation relied on the absorbance recorded at 765 nm and was expressed in milligrams of gallic acid equivalent per gram of sample dry weight (mg GAE/g DW). TFC measurement was conducted at 430 nm and reported as milligrams of rutin equivalent per gram of sample dry weight (mg RE/g DW).

2.5. Antioxidant Activity

The antioxidant activity of rice seeds in response to ABA exogenous application was assessed through ABTS cation decolorization and DPPH radical scavenging assays. The procedures with three replications were outlined in the study by Xuan et al. [36] and Quan et al. [37], respectively. The radical inhibition percentage was determined by the discoloration of the final solution containing samples, measured as a reduction in absorbance compared to the negative control (MeOH) at 517 nm and 734 nm for DPPH and ABTS assays, respectively.

2.6. Quantification of Phenolics by High-Performance Liquid Chromatography (HPLC)

Sample extracts were subjected to analysis using an HPLC system consisting of a PU-4180 RHPLC pump, LC-Net II/ADC controller, and UV-4075 UV/VIS detector (Jasco, Tokyo, Japan). The analytical parameters were set as follows for the HPLC: column, XBridge® Shield RP18 (5 μm, 2.1 × 100 mm, Waters Corporation, Milford, MA, USA). The mobile phase included 0.1% formic acid in water and acetonitrile (pure) as solvents A and B, respectively. The gradient program was A:B (95:5, v/v) at 0–2 min, A:B (30:70, v/v) at 2–12 min, and A:B (0:100, v/v) at 12–22 min. The mobile phase was returned to the initial condition from 22 to 34 min. The flow rate was 0.40 mL/min. The injection volume was 5 μL. Phenolic compounds were detected at 280 nm, and the calibration curve was established using various concentrations of phenolic standards (5, 10, 25, 50, and 100 μg/mL).

2.7. Quantification of Momilactones by Ultra-Performance Liquid Chromatography-Electrospray Ionization-Mass Spectrometry (UPLC-ESI-MS)

Momilactones A (MA) and B (MB) were quantified following the method reported by Quan et al. [38]. The UPLC-ESI-MS system consisted of an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) and an electrospray ionization (ESI) source. The analytical parameters were set as follows: UPLC column, ZORBAX Eclipse Plus C18 (1.8 μm, 2.1 × 50 mm) (Agilent Technologies, Santa Clara, CA, USA); solvent A, 0.1% trifluoroacetic acid in water; solvent B, 0.1% trifluoroacetic in acetonitrile; gradient program, 50% B at 0–5 min and 100% B at 5–10 min. The mobile phase was returned to the initial condition from 10 to 15 min. The flow rate was 0.30 mL/min and the injection volume was 3 μL. The presence of MA and MB in rice samples was confirmed by comparing their extracted ion chromatograms (EIC) and mass spectra (MS) with those of standard momilactones (Figure 2). Momilactone contents were measured from the calibration curve that was established using various concentrations of momilactone standards (0.1, 0.25, 0.5, 1, and 2.5 μg/mL).

2.8. Extraction and Quantification of Melatonin

Melatonin was extracted following the method reported by Zheng et al. [39]. Briefly, 3 g of freeze-dried samples were transferred into 50-mL centrifuge tubes. Subsequently, 40 mL of methanol (98%) was added to each tube and vortexed for 24 h at room temperature. To enhance melatonin extraction, ultrasonication in a water bath (180 W) was followed by 30 min of cooling on ice. After centrifugation at 10,000× g for 5 min at 4 °C, the supernatants were collected, filtered through a 0.2-μm syringe filter, and stored in amber vials suitable for HPLC (PU-4180 RHPLC pump, LC-Net II/ADC controller, and UV-4075 UV/V detector) (Jasco, Tokyo, Japan). Each sample was tested using a C18 column (XBridge Shield PR, 5 μm, 2.1 × 100 mm, Waters Corporation, Wexford, Ireland). The mobile phases consisted of 0.1% formic acid in water (A) and pure MeOH (B). The gradient elution program used was as follows: 0–2 min, 10% (B); 2–4 min, 90% (B); 4–8 min, 10% (B); 8–15 min, 5% (B). The flow rate was set to 0.40 mL/min, with an injection volume of 5.0 μL. Melatonin was detected at 280 nm, and a calibration curve was established using different concentrations of melatonin (5, 10, 25, 50, and 100 μg/mL).

2.9. Statistical Analysis

The data were analyzed using Minitab 16.0 (Minitab Inc., State College, PA, USA). Results were reported as means ± standard error (SE). Differences among varieties and treatments were found using ANOVA (analysis of variance), followed by Tukey multiple comparison tests. The level of significance was 5% (p < 0.05).

3. Results

3.1. Seed Dormancy Duration in Response to ABA Treatment

The study revealed that both the Koshihikari and Akiroman varieties demonstrated a significant prolongation of seed dormancy with increasing concentrations of ABA (Figure 3). Specifically, at the highest concentration of 100 µM ABA, these varieties experienced the maximum dormancy duration of 8 days. In contrast, Akisakari displayed a markedly shorter dormancy period, only extending to 3.3 days at 100 µM ABA concentration, which suggests a varietal distinction in dormancy response. The study indicates that the influence of ABA on rice seed dormancy varies among different rice varieties, highlighting the importance of considering varietal differences in responses to hormonal treatments.

3.2. Effect of Exogenous ABA Application on Fresh Weight (FW) and Dry Weight (DW) of Rice Seeds

The application of exogenous ABA led to a notable reduction in the FW of all varieties (Koshihikari, Akisakari, and Akiroman) compared to the control (Figure 4). For instance, the FW of Akisakari in the control group was 8.5 g, but it decreased to 3.8 g at 100 µM ABA. Conversely, the DW of seeds from all varieties in the ABA-treated groups exhibited a marginal increase relative to the control. Nonetheless, these variations in DW across the different treatments did not reach statistical significance. This modest rise in DWs, despite lacking statistical significance, might indicate a nuanced effect of ABA on seed mass conservation.

3.3. Effect of Exogenous ABA Application on Total Phenolic Content (TPC), Total Flavonoid Content (TFC), and Momilcatone Contents

Significant variations in the quantities of TPC, TFC, MA, and MB were observed between different ABA concentrations and the control group (Table 1). Specifically, ABA application significantly enhanced the TPC in Akiroman, with the most notable increase observed at the 100 µM concentration, where TPC surged from 26.5 mg GAE/g DW in the control to 120.3 mg GAE/g DW. At a 10 µM dose of ABA, Akiroman also recorded the highest TFC level (73.8 mg GAE/g DW) when compared to the control group. Conversely, the control group exhibited the lowest TPC (26.4 mg GAE/g DW) in Akiroman and the lowest TFC (16.5 mg GAE/g DW) in Akisakari. ABA significantly enhanced the MA concentration in Koshihikari, particularly at 100 µM ABA (21.5 µg/g DW), compared to the control (14.5 µg/g DW). For Akisakari, a 50 µM dose of ABA elevated the MA level to 11.8 µg/g DW; however, this increase did not reach statistical significance when compared to the control. MB levels were consistently lower than those of MA across all samples. The minimum MB concentration (7.7 µg/g DW) was recorded in Akisakari’s control group. The effect of ABA on MA levels demonstrated a dose-responsive pattern, with the highest dose (100 µM) eliciting the most significant increase in Koshihikari. The increase in TPC and TFC with ABA treatment suggests that ABA plays a role in the regulation of these phytochemicals during rice seed dormancy. The observed differences in responses among varieties highlight the importance of considering the specific characteristics of each rice variety in studies involving ABA and phytochemical content. The findings suggest that the application of ABA differentially impacts the concentrations of MA and MB in rice seeds throughout dormancy.

3.4. Effect of Exogenous ABA Application on Phenolic Compounds

Table 2 presents the change in the phenolic composition of rice seeds during dormancy in response to exogenous ABA application. Among the samples, four out of five targeted phenolic compounds, including sinapic acid, ferulic acid, salicylic acid, and cinnamic acid, were identified. The accumulation of ferulic and cinnamic acids in rice seeds was elevated under increasing contents of ABA application. The maximal quantity of ferulic acid, reaching 631.9 µg/g dry weight (DW), was found in Akiroman with a 100 µM ABA treatment, whereas the minimal amount, 118.7 µg/g DW, was detected in Akisakari within the control group. Koshihikari displayed a significant increase in cinnamic acid content, particularly at the 50 µM ABA concentration. While gallic acid was not detected, variations were observed in the concentrations of sinapic acid and salicylic acid across the different treatments and control groups for all varieties examined. The findings suggest that ABA treatments and rice variety contribute to the observed changes in phenolic compounds, with potential implications for understanding the regulatory role of ABA in the phenolic metabolism of rice seeds during dormancy.

3.5. Effect of Exogenous ABA Application on Antioxidant Activity

This research focused on the antioxidant activity of rice seeds throughout their dormancy period (Figure 5). Antioxidant activity was assessed using two antiradical assays, DPPH and ABTS. The findings indicate a marked increase in antioxidant activity in rice seeds during dormancy with increasing ABA concentrations when compared to the control group. Specifically, in the DPPH assay, Akisakari showed the most pronounced antioxidant activity (80%) with 100 µM ABA, followed by Akiroman (70%) and Koshihikari (50%) at the same concentration of ABA. Akiroman’s antioxidant activity did not significantly vary across different treatments compared to the control within the DPPH assay. In the ABTS assay, both Akisakari and Akiroman displayed the highest antioxidant activity (90%) at 100 µM ABA. Although Koshihikari’s antioxidant activity did not significantly change across treatments, it was enhanced compared to the control in the ABTS assay. Overall, observations provide valuable insights into the antioxidant properties of rice seeds during dormancy and their reaction to ABA treatments, emphasizing the importance of considering both ABA concentration and rice variety in understanding antioxidant behavior.

3.6. Effect of Exogenous ABA Application on Melatonin

Melatonin levels in rice seeds were observed to rise following ABA treatment during the dormancy period (Figure 6). Koshihikari and Akisakari experienced a notable increase in melatonin concentrations, in contrast to Akiroman, which demonstrated negligible changes. Koshihikari showed a pronounced elevation in melatonin at the 100 µM ABA concentration, achieving 154.7 µg/g FW, marking an 84% increase from the control’s 23.4 µg/g FW. Meanwhile, Akisakari reached its peak melatonin level at a 10 µM ABA dosage. Conversely, Akiroman’s melatonin content did not exhibit significant differences, remaining comparable across the control and 100 µM ABA treatments. The observed increase in melatonin content in response to ABA treatment implies a regulatory role of ABA in melatonin biosynthesis in rice seeds during their dormancy. Understanding these variations is crucial for unraveling the complex interactions between ABA and melatonin in different rice varieties.

4. Discussion

Seed dormancy is a vital and crucial factor that confirms the initiation of the next crop generation. Seed dormancy is achieved after seed maturation when molecular dependence on the mother plant disappears. It involves the synthesis of storage products, dehydration, and storage of de novo ABA [40,41]. ABA is the primary internal physiological factor in inducing seed dormancy by influencing various physiological pathways, including storage proteins and lipids in seeds [42,43]. A study on rice seeds revealed that ABA accumulation, linked to dormancy induction, occurs early in seed development [44]. Maintaining an optimal level of seed dormancy is required for improving the yield and quality of cereal crops [3]. Our study highlights that different rice varieties have distinct responses to ABA treatments regarding seed dormancy (Figure 3). Specifically, Koshihikari and Akiroman varieties showed a significant extension of seed dormancy as ABA concentrations increased, indicating a strong sensitivity to the hormone. On the other hand, the Akisakari variety exhibited a considerably shorter dormancy period, implying a lower sensitivity to ABA. The varying perception and physiological responses of the tested rice varieties to ABA application may be due to differences in their genetic factors. For instance, various rice varieties may activate distinct signaling pathways, leading to variations in the expression of genes related to seed dormancy under ABA influence [44]. This may also result in differences in the biosynthesis and degradation processes of enzymes related to metabolism and energy storage in the seeds of these rice varieties [1]. These nonuniform changes in the dormancy period among the varieties are crucial for breeding programs like genetic selection and utilizing molecular markers to select genes associated with dormancy with the aim of modifying seed dormancy characteristics to suit agricultural needs. Our research, for the first time, indicates ABA effect on extending rice seed dormancy in terms of duration (Figure 3). This information can be leveraged to manipulate dormancy through ABA, ensuring that seeds sprout when environmental conditions are favorable for subsequent growth.
The reduction in FW of rice seeds upon ABA treatment in this study suggests that ABA plays a significant role in regulating water content within the seed (Figure 4). This is consistent with ABA’s known function in promoting seed maturation and desiccation tolerance, processes that are closely linked to the establishment of seed dormancy [45]. The increase in dry seed weight from ABA-treated groups compared to the control aligns with the findings reported by Wang et al. [46], suggesting that ABA treatment enhances seed filling or maturation processes, leading to a higher accumulation of storage reserves relative to water content. This is beneficial for seed quality, as a higher ratio of DW to FW can be indicative of seeds that are better equipped for storage and subsequent germination [47]. Knowledge about how ABA affects seed weight characteristics can be used to select or engineer rice varieties with desired storage and germination traits. This is particularly relevant for improving the resilience of rice crops to changing environmental conditions and for optimizing seed handling and storage practices.
Various phytochemical compounds are increased during the germination of seeds. The germination periods of mung bean sprouts were correlated with their TPC enhancement [48]. Phenolic contents in Chinese wild rice were reported to decline at the initial germination (36 h) and then increase [49]. Hasan et al. [29] reported that TPC and TFC increased in response to salinity stress at the germination stage. Phenolic compounds, including phenolic acids, have been extensively researched as bioactive substances during seed germination and dormancy [50,51,52]. The germination process is inhibited by phenolic acids like caffeic, ferulic, and cinnamic acids present in the seed, as well as phenolic substances including polyphenols (tannins) and flavanols (quercetin) [53,54]. Momilactones A (MA) and B (MB) are natural diterpenoids that also impact the germination of plant seeds. MA and MB exhibited inhibitory effects on Arabidopsis germination, with concentrations exceeding 30 μM and 10 μM [24,55]. The application of exogenous phytohormone auxin partially enhanced the inhibitory effects of MB on rice root growth [56]. A previous study by Jamalian et al. [30] reported an increased level of TFC in response to 40 µM of ABA in strawberries. ABA led to an elevation in the concentrations of three phenolic acids, including ferulic acid, caffeic acid, and p-coumaric acid, under saline conditions [30]. The effect of ABA on phenolic acid accumulation is dose-dependent. The highest level of ferulic acid was observed with the application of 40 μM ABA on strawberries, whereas the concentrations of caffeic acid and p-coumaric acid increased with the application of 10 μM ABA [30]. In this study, the variations in the accumulations of 4 phenolic compounds, including sinapic, ferulic, salicylic, and cinnamic acids, are shown in Table 2. Among them, the quantities of ferulic and cinnamic acids were elevated, corresponding with the enhanced concentration of ABA application. In contrast, sinapic and salicylic acids did not consistently increase with higher ABA application. Accordingly, different concentrations of ABA application may have varying effects on the activation or inhibition of biosynthetic pathways of specific compounds, leading to diverse changes in chemical composition [57]. On the other hand, the inconsistent changes in phytochemical contents when applying ABA at different concentrations (e.g., reduction at 50 μM ABA treatment compared to 10 and 100 μM) (Table 1 and Table 2), may be due to the complex interactions between ABA and other phytohormones within the hormonal crosstalk network [58], resulting in nonlinear and unpredictable physiological and biochemical responses in plants [58]. The significant increase in TPC, TFC, ferulic acid, and cinnamic acid (Table 2) in response to ABA application, especially at the 100 µM concentration, suggests that these germination-inhibitor signaling molecules may have the potential to improve rice seed dormancy. Phenolics, flavonoids, and momilactones are reported to have antioxidant activity [26,59]. Therefore, the enhancement of these compounds during rice seed dormancy in response to ABA may contribute to the maintenance of seed dormancy by protecting seeds from damage that could prematurely trigger germination [15]. Following these results, questions arise about whether momilactones act as phytohormones in rice seed dormancy. Further genetic exploration is needed to investigate this question and uncover the relationship between momilactones and ABA associated with seed dormancy. In our study, the changes in the accumulation of phytochemicals, including phenolics, flavonoids, melatonin, and momilactones, during the seed dormancy process of three typical Japonica rice varieties (Koshihikari, Akisakari, and Akiroman) treated with ABA were clarified. Koshihikari is considered a model Japonica rice cultivar that has been used in numerous studies [60]. The results showed that the contents of ferulic and cinnamic acids increased, aligning with the prolonged rice seed dormancy under ABA effects. Notably, the accumulation of momilactones was consistent with that of melatonin, a phytohormone related to extended seed dormancy [61]. These findings imply the contributions of ferulic acid, cinnamic acid, and momilactones to prolonging rice seed dormancy. To confirm their roles as phenotypic markers, further research is needed to determine their biosynthesis and accumulation during the dormancy process of various rice varieties (Indica and Japonica subspecies) with different origins and characteristics.
In another consideration, the crucial role of ROS in the regulation of seed dormancy has been extensively documented [62,63]. The high level of ROS contributes to physiological seed dormancy [63,64]. Furthermore, an excess of ROS is often associated with the deterioration of orthodox seeds in both natural and artificial environments, impacting lipid peroxidation, membrane permeability, and the antioxidant system [62]. Hence, maintaining a balance in ROS levels is important for the proper regulation of seed dormancy. The marked increase in antioxidant activity in dormant seeds suggests that antioxidants might be crucial for protecting the seeds from oxidative damage during dormancy (Figure 5). This is particularly relevant because dormant seeds are metabolically active and need to mitigate damage from ROS. The findings imply that antioxidant systems are upregulated in response to ABA treatment, which may help to stabilize and extend the dormancy period by minimizing oxidative stress. Understanding how ABA concentrations and antioxidant activity interact to affect seed dormancy has practical implications for seed storage and germination strategies.
Melatonin is a signaling molecule linked to the germination process [65,66]. According to Lv et al. [61], melatonin significantly prolonged Arabidopsis dormancy at concentrations of 500 and 1000 µM, and gene analysis revealed that melatonin’s impact on seed germination was associated with ABA regulation. Additionally, another study found that ABA and melatonin acted synergistically to inhibit seed germination and prolonged dormancy [61]. Koshihikari and Akisakari varieties experienced a significant increase in melatonin levels, suggesting a heightened response to the conditions that promote melatonin synthesis, whilst the Akiroman variety exhibited negligible changes in melatonin concentrations (Figure 6). The significant elevation of melatonin levels in response to ABA, especially in Koshihikari, highlights melatonin’s potential role as a marker for dormancy regulation (Figure 6). An 84% increase in melatonin concentration at 100 µM ABA in Koshihikari indicates a potential biochemical mechanism for enhancing seed dormancy and protection through antioxidant upregulation. This relationship between melatonin and ABA provides insights into how seeds may modulate dormancy duration through endogenous hormone levels and antioxidant capacity.
The insights into the effects of exogenous ABA application on specific phenolic compounds, melatonin, and momilactones and their relationships set the stage for investigating the functional roles and interactions of these compounds in dormancy maintenance and potential applications in crop improvement. The identification of ABA as a regulator of antioxidant activity underscores the complex interplay between hormonal signaling and the antioxidative defense mechanisms in seeds, providing a basis for future studies on improving crop resilience and nutritional value. Future research may also focus on elucidating the regulatory mechanisms linking ABA signaling to the biosynthesis of specific phenolic acids, melatonin, and momilactones. Understanding these interactions could pave the way for targeted interventions to manipulate phytochemical profiles in crops.

5. Conclusions

This study highlights that exogenous application of ABA extended rice seed dormancy period, with the longest span of 8 days observed at 100 µM ABA. The study also provides insights into the accumulation of phenolics, melatonin, and momilactones and their relationships during the dormancy process under ABA treatments. Significantly, the accumulation of MA and MB in ABA-treated rice seeds aligned with that of melatonin, a phytohormone known to prolong seed dormancy. Based on this, MA and MB are indicated, for the first time, to play a potential role in a signaling pathway through which they may contribute to regulating the seed dormancy process. These findings may pave the way for future research exploring the functional roles and interactions of these compounds in rice seed dormancy, as well as their promising applications in crop improvement. In addition, ABA treatment markedly enhanced antioxidant activity during seed dormancy, indicating its possible role in suppressing ROS production, which is involved in regulating seed germination. This synergy could potentially offer a strategic approach to mitigate the adverse effects of preharvest sprouting and safeguard against the negative effects of unsuitable storage conditions that lead to germination prior to cultivation.

Author Contributions

Conceptualization, R.R., T.D.X. and L.H.A.; methodology, R.R.; investigation, R.R. and L.H.A.; writing original manuscript, R.R.; review and editing, R.R., L.H.A. and T.D.X.; supervision, T.D.X.; project administration, T.D.X. and L.H.A. The authors have read and agreed to publish the article. All authors have read and agreed to the published version of the manuscript.

Funding

This work received no external funding.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data are available from the corresponding author upon a reasonable request.

Acknowledgments

The authors thank the Japanese government (Monbukagakusho) for awarding Ramin Rayee a scholarship. Additionally, thanks to Mohmmad Zaman Muzamil for his partial support in collecting germination data and extracting samples.

Conflicts of Interest

There is no conflict of interest among the authors for publishing this research.

References

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Agriculture 14 01229 g001
Figure 1. Rice seed samples (var. Akisakari) on day 4 of the germination with ABA treatments (0, 10, 50, and 100 µM).
Figure 1. Rice seed samples (var. Akisakari) on day 4 of the germination with ABA treatments (0, 10, 50, and 100 µM).
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Figure 2. UPLC-ESI-MS chromatogram of (a) standard momilactones A (MA) and B (MB) (1 μg/mL), and (b) MA and MB detected in rice seed (var. Koshihikari) treated with 100 µM ABA.
Figure 2. UPLC-ESI-MS chromatogram of (a) standard momilactones A (MA) and B (MB) (1 μg/mL), and (b) MA and MB detected in rice seed (var. Koshihikari) treated with 100 µM ABA.
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Figure 3. Effect of different ABA treatments on seed dormancy of rice (var. Koshihikari, Akisakari, and Akiroman). The means that do not share the same letter are significantly different according to the Tukey multiple range test (5%).
Figure 3. Effect of different ABA treatments on seed dormancy of rice (var. Koshihikari, Akisakari, and Akiroman). The means that do not share the same letter are significantly different according to the Tukey multiple range test (5%).
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Figure 4. Effects of different ABA treatments on (a) fresh weight and (b) dry weight of rice seeds (var. Koshihikari, Akisakari, and Akiroman). The means that do not share the same letter are significantly different according to the Tukey multiple range test (5%).
Figure 4. Effects of different ABA treatments on (a) fresh weight and (b) dry weight of rice seeds (var. Koshihikari, Akisakari, and Akiroman). The means that do not share the same letter are significantly different according to the Tukey multiple range test (5%).
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Figure 5. Effects of different ABA treatments on antioxidant activities of rice seeds (var. Koshihikari, Akisakari, and Akiroman) via (a) DPPH assay and (b) ABTS assay. The means in each bar that do not share a same letter are significantly different.
Figure 5. Effects of different ABA treatments on antioxidant activities of rice seeds (var. Koshihikari, Akisakari, and Akiroman) via (a) DPPH assay and (b) ABTS assay. The means in each bar that do not share a same letter are significantly different.
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Figure 6. Effect of different ABA treatments on melatonin content of rice seeds (var. Koshihikari, Akisakari, and Akiroman). FW: fresh weight. The means that do not share a same letter are significantly different.
Figure 6. Effect of different ABA treatments on melatonin content of rice seeds (var. Koshihikari, Akisakari, and Akiroman). FW: fresh weight. The means that do not share a same letter are significantly different.
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Table 1. Effects of ABA on TPC, TFC, MA, and MB accumulation.
Table 1. Effects of ABA on TPC, TFC, MA, and MB accumulation.
VarietyABA ConcentrationTPC
(mg GAE/g DW)		TFC
(mg RE/g DW)		MA
(µg/g DW)		MB
(µg/g DW)
Koshihikari0 µM30.6 ± 2.2 c29.4 ± 2.4 ab14.5 ± 0.2 b11.2 ± 0.4 bc
10 µM82.2 ± 5.8 ab42.4 ± 13.6 ab18.6 ± 1.0 a15.4 ± 1.4 a
50 µM105.8 ± 0.8 a47.6 ± 19.9 ab18.2 ± 1.0 a13.8 ± 0.2 ab
100 µM99.1 ± 10.5 a58.0 ± 10.3 ab21.5 ± 1.3 a16.3 ± 0.6 a
Akisakari0 µM28.1 ± 1.9 c16.5 ± 4.8 b8.9 ± 0.1 c7.7 ± 0.3 c
10 µM49.3 ± 2.0 bc23.8 ± 2.6 ab9.6 ± 0.5 c9.2 ± 0.7 c
50 µM80.3 ± 6.6 ab17.4 ± 4.2 b11.8 ± 0.5 bc10.3 ± 0.1 bc
100 µM99.5 ± 19.6 a17.5 ± 2.6 b10.6 ± 0.3 c10.2 ± 0.5 bc
Akiroman0 µM26.4 ± 3.6 c23.9 ± 2.3 ab10.1 ± 0.4 c9.1 ± 1.4 c
10 µM91.7 ± 8.0 a73.9 ± 4.9 a10.5 ± 0.7 c8.6 ± 0.8 c
50 µM117.2 ± 7.3 a59.3 ± 18.3 ab10.7 ± 0.3 c9.1 ± 0.1 c
100 µM120.3 ± 10.5 a39.8 ± 8.6 ab11.8 ± 0.2 c8.3 ± 0.8 c
ANOVA
Variety ****
Treatment ****
Variety * Treatment nsns**
Means with the same letter in each column are not significantly different (ns) according to the Tukey multiple range test (5%). ABA: abscisic acid; TPC: total phenolic content; TFC: total flavonoid content; MA: momilactone A; MB: momilactone B; GAE: gallic acid equivalent; DW: dry weight; RE: rutin equivalent; * indicates significant difference at p < 0.05.
Table 2. Effects of ABA on the accumulation of phenolic acids.
Table 2. Effects of ABA on the accumulation of phenolic acids.
VarietyABA ConcentrationSinapic Acid
(µg/g DW)		Ferulic Acid
(µg/g DW)		Salicylic Acid (µg/g DW)Cinnamic Acid (µg/g DW)
Koshihikari0 µM29.8 ± 12 ef132.3 ± 9.6 d137.5 ± 4.3 bcd122.1 ± 17.3 b
10 µM177.1 ± 7 cde244.4 ± 9.4 ab255.6 ± 21.8 ab163.2 ± 41.1 ab
50 µM208.5 ± 3.1 bcd235.7 ± 30.1 bcd170.5 ± 20.9 bcd433 ± 167 a
100 µM278.5 ± 23.4 bc379.3 ± 32.6 bc275.8 ± 27.8 a223.5 ± 5.0 ab
Akisakari0 µM444.4 ± 33.7 a118.7 ± 22.5 d129.8 ± 8 cd145.3 ± 26.0 ab
10 µM351.3 ± 22.8 ab166.1 ± 77.1 cd94.2 ± 8.6 d183.3 ± 74.9 ab
50 µM20.3 ± 0.6 f373.6 ± 5.3 bc74.7 ± 19.9 d47.66 ± 2.5 b
100 µM116.3 ± 93.2 def235.8 ± 34.0 cd145.3 ± 28.6 bcd198.8 ± 44.3 ab
Akiroman0 µM58.1 ± 10.35 def328.5 ± 25.9 bcd224 ± 60.2 abc114.3 ± 22.5 ab
10 µM24.8 ± 1.4 ef365.7 ± 71.8 bc136.6 ± 12.5 bcd229.5 ± 15.5 ab
50 µM24.6 ± 0.6 ef385.5 ± 7 b269.5 ± 5 a180.9 ± 5.7 ab
100 µM27.7 ± 3.7 ef631.9 ± 73.7 a153.9 ± 6.8 bcd261.2 ± 16.9 ab
ANOVA
Variety ***ns
Treatment ns*nsns
Variety * Treatment ****
Means with the same letter in each column are not significantly different (ns) according to the Tukey multiple range test (5%). ABA: abscisic acid; DW: dry weight; ns denotes not significant; * indicates significant difference at p < 0.05.
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Rayee, R.; Anh, L.H.; Xuan, T.D. Effects of Abscisic Acid on Rice Seed Dormancy: Antioxidant Response and Accumulations of Melatonin, Phenolics and Momilactones. Agriculture 2024, 14, 1229. https://doi.org/10.3390/agriculture14081229

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Rayee R, Anh LH, Xuan TD. Effects of Abscisic Acid on Rice Seed Dormancy: Antioxidant Response and Accumulations of Melatonin, Phenolics and Momilactones. Agriculture. 2024; 14(8):1229. https://doi.org/10.3390/agriculture14081229

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Rayee, Ramin, La Hoang Anh, and Tran Dang Xuan. 2024. "Effects of Abscisic Acid on Rice Seed Dormancy: Antioxidant Response and Accumulations of Melatonin, Phenolics and Momilactones" Agriculture 14, no. 8: 1229. https://doi.org/10.3390/agriculture14081229

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