|Phyton-International Journal of Experimental Botany|
Foliar Application of Brassinolide Induced Regulation of Grain Yield and Quality, Antioxidant Responses and Aroma in Fragrant Rice
1State Key Laboratory for Conservation and Utilization of Subtropical Agricultural Bioresources, South China Agricultural University, Guangzhou, 510642, China
2Scientific Observing and Experimental Station of Crop Cultivation in South China, Ministry of Agriculture, Guangzhou, 510642, China
3Guangzhou Key Laboratory for Science and Technology of Aromatic Rice, Guangzhou, 510642, China
4College of Agronomy, Hunan Agricultural University, Changsha, 410128, China
*Corresponding Author: Xiangru Tang. Email: email@example.com
#These authors contributed equally to the work
Received: 26 May 2021; Accepted: 09 October 2021
Abstract: Brassinolide (BR) is a new green plant growth regulator. The present field study was conducted on two fragrant rice cultivars (i.e., Meixiangzhan-2 and Xiangyaxiangzhan) to study the effects of foliar application of BR on fragrant rice performance. At the heading stage, BR solutions at 0.05, 0.15, 0.25 and 0.50 mg L-1 were sprayed on fragrant rice at 600 liters per hectare; these treatments were named as BR1, BR2, BR3 and BR4, respectively. The treatment sprayed with distilled water was taken as a control (CK). Compared with CK, the BR2 treatment significantly enhanced the chlorophyll concentration and net photosynthetic rate for Meixiangzhan-2, and Xiangyaxiangzhan. The BR3 and BR4 treatments increased the concentrations of chlorophyll a, chlorophyll b and carotenoid, and also enhanced net photosynthetic rate by 31.91% and 40.43%, respectively. Higher grain yields were recorded in the BR2 treatment than on CK on Meixiangzhan-2, while on Xiangyaxiangzhan, the BR3 and BR4 treatments increased grain yield compared with CK. In relation to CK, higher head rice rates were recorded in the BR2 treatment for Meixiangzhan-2, and in the BR3 and BR4 treatments for Xiangyaxiangzhan. BR treatments (BR2 for Meixiangzhan-2, BR3 and BR4 for Xiangyaxiangzhan) also significantly enhanced the activities of GPX, SOD and CAT by 10.22% to 23.00%, and reduced the malonaldehyde concentration. In addition, we observed that some BR treatments (BR2 for Meixiangzhan-2, BR3 and BR4 for Xiangyaxiangzhan) decreased the grain 2-acetyl-1-pyrroline concentration of fragrant rice.
Keywords: Brassinolide; chlorophyll; yield formation; fragrant rice; antioxidant enzyme
As a new green plant growth regulator, brassinolide (BR) can promote crop growth and development, improve crop quality, increase the yield of crops, and make the color of crops bright and the leaves thicker . Many studies have been conducted to investigate the effects of BR on the growth, development, quality and abiotic stress tolerance of crops. For example, an early study revealed that exogenous BR was able to alleviate the negative impacts from water logging stress on maize . The study of Dehghan et al.  showed that the application of BR could significantly improve the seed quality characters of wheat under different irrigation systems. Lv et al.  demonstrated the BR would alter Leymus chinensis performance at both morphology and physiology levels under drought stress. Furthermore, the study of Li et al.  showed that BR induced regulation of growth and development on adventitious roots which involved nitric oxide in cucumber.
Rice is the main food crop in China which could be significantly affected by plant growth regulators on grain yield and quality attributes . As a special kind of rice, fragrant rice is well known by people around the world for its good grain quality characters especially the unique aroma with 2-acetyl-1-pyrroline (2-AP) as the main component . A previous study revealed that foliar application of plant growth regulators such as paclobutrazol, indole acetic acid and gibberellic acid had significant effects on fragrant rice growth and development as well as on yield and quality . However, the effects of BR on fragrant rice performances (i.e., yield formation, grain quality attributes, aroma, antioxidant responses, and photosynthesis) have not been reported.
The present study was conducted in the field to study the effects of the foliar application of BR on grain yield, grain quality and rice aroma in fragrant rice cultivars. We hypothesized that foliar application of BR will enhance antioxidant enzymatic activities and improve photosynthesis and grain yield. This study would provide new information about the application of BR in agricultural production.
2 Materials and Methods
2.1 Plant Materials and Growing Conditions
In 2019, a field experiment was conducted at the Experimental Research Farm, College of Agriculture, South China Agricultural University, Zengcheng (23°13′N, 113°81′E, altitude 11 m.a.s.l.), China. The experimental site enjoys a subtropical-monsoon climate. The experimental soil was sandy loam consisting of 14.60 g kg–1 organic matter, 1.23 g kg–1 total nitrogen, 53.49 mg kg–1 available nitrogen, 0.88 g kg–1 total phosphorus, 15.95 mg kg–1 available phosphorus, 12.02 mg kg–1 total potassium, and 122.76 mg kg–1 available potassium, and the pH was 6.57. Seeds of two fragrant rice cultivars, “Meixiangzhan-2” (Lemont × Fengaozhan) and “Xiangyaxiangzhan” (Xiangsimiao126 × Xiangyaruanzhan), well known and widely cultivated in South China, were provided by the College of Agriculture, South China Agricultural University, Guangzhou, China. Detailed information about the cultivars can be found at https://www.ricedata.cn/. The seeds were sowed in July, transplanted in August, and harvested in November. A special biological organic fertilizer (Jing Lv) (manufactured by Dongguan Foota, Ltd., China; comprised of (N-P2O5-K2O, 15%–4%–6%) was applied at 900 kg ha−1. Sixty percent of it (540 kg ha−1) was applied as a basal dose and 40% (360 kg ha−1) at tillering. Water management practices were followed as adopted by local farmers. After the rice seedlings were transplanted and turned green, the paddy field kept a 3 cm water layer until the end of tillering. Drain water for 7 days at the end of tilling to control the production of non-productive/infertile tillers. At the following stages, a water of 5–7 cm was kept to the grain-filling stage. All other agronomic practices (i.e., pest and diseases management, and weed control) were the same in all treatments and followed the guidelines and standards recommended by the province.
2.2 Treatment Description and Sampling
At the heading stage of fragrant rice plants, 0.05, 0.15, 0.25 and 0.50 mg L–1 BR solutions were sprayed on aromatic rice at 600 liters per hectare; treatments were named BR1, BR2, BR3 and BR4, respectively. The foliar application was carried out once during the whole experiment. The treatment sprayed with distilled water was taken as a control (CK). At the grain filling phase (10 days after treatment application), fresh leaves were collected and stored at –80°C for the estimation of chlorophyll and antioxidant responses. Meanwhile, the net photosynthetic rate was measured using a portable photosynthesis system (LI-6400, LI-COR, USA). At harvest, fresh grains were collected and stored at –80°C for estimation of 2-AP.
2.3 Estimation of the Grain 2-AP Concentration
The grain samples were finely ground and used for measurements of 2-AP via the simultaneous distillation-extraction method (SDE) to make a molar solution, which was then analyzed by a GCMS-QP 2010 Plus instrument (Shimadzu Corporation, Japan) following Luo et al. .
2.4 Estimation of Net Photosynthetic Rate
At the grain filling stage (10 days after spraying with the BR solution), the net photosynthetic rate was determined with the portable photosynthesis system (LI-6400, LI-COR, USA) with the following adjustments: the photosynthetically active radiation at the leaf surface was between 1100 and 1200 μmol m−2 s−1, and the ambient CO2 concentration was between 385.5 and 399.7 μmol mol−1.
2.5 Estimation of Grain Yield and Related Traits
At the maturity stage, the rice grains were harvested from three sampling areas (1 m2 each) in each plot, and they were threshed by a machine. The average panicle number per unit surface area (1 m2) was calculated from the three 1 m2 replicates at each plot. The effective panicle number of 3 m2 was manually cleared in each plot. Then six hills of rice plants from each plot were taken to determine the other yield-related traits including seed-setting rate, 1000-grain weight and grain number per panicle.
2.6 Estimation of Photosynthetic Pigments Concentrations
The concentrations of total chlorophyll (total Chl), chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoids were measured according to the methods described by He et al. . A ground leaf sample was placed within 95% absolute ethyl alcohol until the sample turned white. Then, the absorbance was read at 645, 652 and 663 nm, respectively, on an ultraviolet-visible spectrophotometer. The formulas of the calculations were as follows:
CT: Total chlorophyll concentration; Ca: Chlorophyll a concentration; Cb: Chlorophyll b concentration; Cx.c: Carotenoid concentration.
2.7 Estimation of Antioxidant Enzyme Activities and Malonaldehyde (MDA) Concentration
The activities of the guaiacol peroxidase (GPX, EC 18.104.22.168), superoxide dismutase (SOD, EC 22.214.171.124) and (CAT, EC 126.96.36.199) were estimated following Luo et al.  as well as the MDA concentration. The absorbances of GPX and SOD and CAT were read at 470, 560 and 240 nm, MDA concentration was determined after reacting with thiobarbituric acid and the absorbance was read at 532, 600, and 450 nm, respectively, and the activities were expressed as U g–1 min–1 FW. The concentration of MDA was expressed as μmol g–1 FW.
2.8 Estimation of Grain Quality Attributes
After air drying, the brown rice rate was estimated using a rice huller (Jiangsu, China) and milled rice and head rice rate were determined and calculated by using a Jingmi testing rice grader (Zhejiang, China). Grains with chalkiness and chalkiness degree were estimated by using an SDE-A light box (Guangzhou, China) while an Infratec-1241 grain analyzer (FOSS–TECATOR) was used to determine the grain amylose and protein concentrations .
2.9 Statistical Analysis
Data were analyzed using Statistix 8.1 (Analytical Software, Tallahassee, FL, USA) while differences among means were separated by using the least significant difference (LSD) test at a 5% probability level. Graphical representation was conducted via Sigma Plot 14.0 (Systat Software Inc., California, USA).
3.1 Grain Yield and Yield Related Attributes
Exogenous BR application significantly affected the grain yield of fragrant rice as shown in Table 1. For Meixiangzhan-2, BR2 treatment significantly increased grain yield and seed-setting rate by 11.67% and 17.46%, respectively compared with CK whilst there was no significant difference between CK and other BR treatments in grain yield. For Xiangyaxiangzhan, compared with CK, both BR1 and BR2 treatment significantly increased grain yield; compared with CK, BR2 treatment significantly increased Seed-setting rate. On the other hand, there were no significant differences among all treatments in panicle number per unit surface area, grain number per panicle and 1000-grain weight.
Within each cultivar, means in the same column followed by different lowercase letters differ significantly at P ≤ 0.05 according to the t-test. CK: water was sprayed at 600 liters per hectare. BR1: 0.05 mg L–1 BR solution was sprayed at 600 liters per hectare. BR2: 0.15 mg L–1 BR solution was sprayed at 600 liters per hectare. BR3: 0.25 mg L–1 BR solution was sprayed at 600 liters per hectare. BR4: 0.5 mg L–1 BR solution was sprayed at 600 liters per hectare.
3.2 Photosynthetic Pigments
As shown in Fig. 1, exogenous BR application significantly affected the concentrations of total chlorophyll, chorophyll a, chorophyll b and carotenoid. For Meixiangzhan-2, the highest photosynthetic pigment concentrations were recorded in the BR2 treatment when compared with the CK and the remaining BR treatments (Fig. 1). The BR2 treatment significantly increased the concentrations of total chlorophyll, chorophyll a, chorophyll b and carotenoids by 13.78, 13.94, 11.98 and 12.26%, respectively. For Xiangyaxiangzhan, concentrations of total chlorophyll, chorophyll a, chorophyll b and carotenoids were higher on the BR1, BR2, BR3 and BR4 treatments than on CK. The highest concentrations of total chlorophyll, chorophyll a, chorophyll b and carotenoids were recorded in BR4.
3.3 Net Photosynthetic Rate
As shown in Fig. 2, foliar application of BR affected the net photosynthetic rate of fragrant rice. For Meixiangzhan-2, there were no statistical differences among the CK, BR1, BR3 and BR4 treatments on the net photosynthetic rate. The highest net photosynthetic rate was recorded on the BR2 treatment. For Xiangyaxiangzhan, compared with CK, the BR3 and BR4 treatments significantly increased the net photosynthetic rate by 31.91% and 40.43%, respectively. Also, there were no significant differences (P > 0.05) among the CK, BR1 and BR2 treatments.
3.4 Antioxidant Enzymes and MDA Concentration
As shown in Fig. 3, exogenous BR treatments significantly influenced the antioxidant enzymes activities as well as the MDA concentrations. For Meixiangzhan-2, the highest activities of SOD, GPX and CAT were all recorded in BR2 treatment. Compared with CK, BR2 treatment significantly enhanced SOD, GPX and CAT activities by 10.22, 12.17 and 24.54% while 23.00% lower MDA concentration was recorded in BR2 than CK; BR4 treatment significantly reduced CAT and GPX activities. BR3 treatment significantly reduced GPX activities. For Xiangyaxiangzhan, higher activities of SOD, GPX and CAT and lower MDA concentrations were recorded in BR4. Compared with CK, BR3 treatment significantly enhanced SOD, GPX and CAT activities, significantly reduced MDA concentration. Whilst there was no remarkable difference among CK, BR1 and BR2 (except GPX activity in BR1).
3.5 Grain Quality Attributes
As shown in Table 2, foliar application of BR had no significant influences on fragrant rice grain quality attributes except head rice rate. For Meixiangzhan-2, BR2 treatment significantly increased head rice rate by 4.23% compared with CK, whilst there was no remarkable difference among CK, BR1, BR3 and BR4; For Xiangyaxiangzhan, BR3 and BR4 treatments significantly increased head rice rate than CK. Whilst there was no remarkable difference among CK, BR1 and BR2.
Within each cultivar, means in the same column followed by different lowercase letters differ significantly at P ≤ 0.05 according to the LSD-test. Akali: Alkali dissipation value. CK: water was sprayed at 600 liters per hectare. BR1: 0.05 mg L–1 BR solution was sprayed at 600 liters per hectare. BR2: 0.15 mg L–1 BR solution was sprayed at 600 liters per hectare. BR3: 0.25 mg L–1 BR solution was sprayed at 600 liters per hectare. BR4: 0.5 mg L–1 BR solution was sprayed at 600 liters per hectare.
3.6 Grain 2-AP Concentration
As shown in Fig. 4, exogenous BR influenced the grain 2-AP concentration of fragrant rice. For Meixiangzhan-2, there were no significant differences among CK, BR1 and BR2, in the 2-AP concentration whilst BR3 and BR4 significantly decreased the 2-AP concentrations by 22.22% and 44.09%, respectively. Compared with CK, for Xiangyaxiangzhan, BR1, BR2, BR3 and BR4 showed a similar (P > 0.05) 2-AP concentration. Also, the concentration of 2-AP was similar (P > 0.05) among CK, BR1 and BR3 (Fig. 4).
The present study revealed the effects of exogenous BR on fragrant rice performances, i.e., yield formation, photosynthesis, antioxidant response and grain quality. Compared with CK, BR treatments (BR2 for Meixiangzhan-2, BR3 and BR4 for Xiangyaxiangzhan) significantly increased grain yield of fragrant rice, which could be explained by increased seed-setting rate. The improvement in yield formation of fragrant rice cultivars was attributed to the enhancement in photosynthesis including photosynthetic pigments concentrations and net photosynthetic rate. In our study, higher concentrations of chlorophyll a, chlorophyll b and carotenoid were recorded in BR treatments (BR2 for Meixiangzhan-2, BR3 and BR4 for Xiangyaxiangzhan) than CK. A similar trend was also recorded in the net photosynthetic rate. Chlorophyll concentration is an important factor in affecting fragrant rice photosynthesis which is a key bioprocess for yield formation . A previous study showed that agronomy measures such as water management and microelement were used to increase chlorophyll concentrations and photosynthesis so the goal of higher grain yield would be achieved in fragrant rice production [13,14]. In our study, BR had similar positive effects on the yield formation of fragrant rice and the possible reason might be exogenous BR enhancing biosynthesis of chlorophyll. Moreover, the nitrogen fertilizer was not applied at the later growth stage, which possibly cause premature senility. Our results suggest that BR may be able to reduce the phenomenon of premature aging by improving photosynthesis. In addition, we observed that exogenous BR induced regulations in antioxidant responses in terms of GPX, SOD, CAT and MDA. The activities of GPX, SOD and CAT all be enhanced due to exogenous BR treatments for both Meixiangzhan-2 and Xiangyaxiangzhan (BR2 for Meixiangzhan-2, BR3 and BR4 for Xiangyaxiangzhan) while BR treatments significantly decreased MDA concentrations. The microclimate and environment of the paddy field are very complex, sometimes it will cause stress (transient extreme temperature, pest, water stress) to the growing rice and induce oxidative damage and membrane peroxidation which could be judged by MDA concentration [14–17]. As the key enzymes on cleaning reactive oxygen, GPX, SOD and CAT play important roles in an antioxidant system in fragrant rice . The enhancement in GPX, SOD and CAT activities indicated that foliar application of BR at a suitable concentration is able to promote the stress resistance of fragrant rice. Our results agreed with the research of Wang et al.  who demonstrated BR application was able to improve the resistance of rice at the booting stage to cold stress. The study of Su et al.  also showed that BR could mediate various physiological processes and improve crop resistance to abiotic stresses.
Besides grain yield, grain quality attributes such as milling, nutrients, aroma, texture and appearance are the factors that influenced the income of rice production [21–23]. The results of the present study showed that exogenous BR application (BR2 for Meixiangzhan-2, BR3 and BR4 for Xiangyaxiangzhan) improved the head rice rate of fragrant rice although other quality characters such as amylose concentration and protein concentration had no remarkable response to BR treatments. The biosynthesis and accumulation of 2-AP in aromatic rice is a complicated phenomenon which is affected by many factors [24,25]. In our study, for Meixiangzhan-2, BR4 treatment significantly decreased grain 2-AP concentration but other treatments had no significant effect. For Xiangyaxiangzhan, BR4 treatment significantly increased grain 2-AP concentration but other treatments had no significant effect. The different results on aroma in two fragrant rice cultivars induced by BR indicated that 2-AP biosynthesis in different fragrant rice cultivars responds differently to exogenous BR, and more studies are required to be conducted to understand the mechanism of the regulation of BR on 2-AP formation in fragrant rice.
In our study, we observed that different fragrant rice cultivars’ responses applied concentration of BR differently. For Meixiangzhan-2, the optimum applied concentration of BR was 0.15 mg L–1 because the highest grain yield was recorded in BR2 treatment while BR3 and BR4 treatments inhibited chlorophyll biosynthesis and net photosynthetic rate, and consequently decreased grain yield. For Xiangyaxiangzhan, the highest grain yields were recorded in BR4 treatment, which indicated that the optimum applied concentration of BR was 0.50 mg L–1. Our finding indicated the effects of different applied concentrations of BR on different rice cultivars could vary largely and thus, people should pay more attention to cultivar characteristics when applying BR for rice production.
Our study indicated that foliar application has substantial effects on yield formation, grain quality attributes, photosynthesis, antioxidants and 2-AP concentration of fragrant rice. The trend of chlorophyll concentration in two fragrant rice varieties treated with different BR concentrations was not consistent. For Meixiangzhan-2, BR2 was the best-applied treatment whilst for Xiangyaxiangzhan, BR4 was the best-applied treatment. The application of BR at a suitable concentration could increase chlorophyll concentration, enhance photosynthetic rate, improve antioxidant enzymatic activities, and lead to a higher grain yield of fragrant rice.
Funding Statement: This study was financially supported by National Natural Science Foundation of China (31971843), Technology System of Modern Agricultural Industry in Guangdong (2020KJ105) and Guangzhou Science and Technology Project (202103000075).
Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study.
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