Effects of a brassinosteroid and an ecdysone analogue on pollen germination of rice under heat stress

Introduction

Most of the rice cultivation on the central plain of Thailand occurs after the rainy season floods. Rice plants can become heat stressed and suffer yield reductions if exposed to excess radiation and high temperatures during the reproductive stages before harvesting. Since heat stress reduces pollen germination,¹⁾ new technologies that reduce damage from heat stress could be a key for food security.

Brassinosteroids (BRs) are naturally occurring steroidal plant hormones that regulate plant growth and development. These hormones can induce a broad spectrum of cellular responses including cell division, cell elongation, pollen tube growth, induction of ethylene biosynthesis, proton pump activation, xylem differentiation, and regulation of gene expression.^2–4) Moreover, BRs are also known to alleviate various biotic and abiotic stresses, such as cold stress, temperature stress, salt stress, and pathogen attacks.⁵⁾ Singh and Shono reported that 24-epibrassinolide (EBR) increased the percentage of pollen germination and decreased bursting of tomato pollen under high temperature.⁶⁾ Based on the results obtained in laboratories or greenhouses, BRs, such as 24-epibrassinolide and 28-homobrassinolide, have been field tested under stressed conditions to determine their influence on plant growth, development, and crop yield. These trials demonstrated that BRs have the potential to reduce damages caused by stress conditions.^7,8) However, the application of synthetic or natural BRs to plants is not a practical due to the high cost to synthesize or isolate natural BRs.

In this context, we applied two kinds of BRs to rice. The first type was EBR, which is a C-24 epimer of brassinolide.⁹⁾ It has have been reported that EBR can be extracted from several plants, such as Vicia faba and Gypsophyta perfoliata.^9,10) The second type was 7,8-dihydro-8α-20-hydroxyecdysone (DHECD), which is a chemical that mimics the functions of natural brassinosteroids. BR mimics can serve as alternative to natural BRs in promoting crop production.^11,12) DHECD or 7,8-dihydro-8α-20-hydroxyecdysone was synthesized by catalytic hydrogenation of 20-hydroxyecdysone, which is easily obtainable from Vitex glabrata stem bark.^13,14) Our preliminary experiments suggested that DHECD, which is an ecdysteroid analogue, possessed similar biological activity as BRs in a rice laminar inclination test, although it was less active than brassinolides, the most active BRs. Some reports have provided evidence that BRs can increase the pollen properties of plants exposed to heat stress conditions.^6,15,16) However, none of the studies have examined the role of exogenous BRs on reproductive development in rice when they are applied under conditions of heat stress. Here, we tested the effects of both EBR and DHECD on pollen viability of rice under heat stress.

DHECD, which mimics the functions of BRs in a rice inclination test, may improve the pollen properties and seed setting of rice. The aims of this study are: 1) to estimate the appropriate concentration of EBR and DHECD for improving the heat tolerance in rice and 2) to evaluate the effectiveness of the compounds based on changes in growth and physiological attributes.

Materials and Methods

The brassinosteroid (EBR, Fig. 1a) and the brassinosteroid mimic (DHECD, Fig. 1b) were used as chemical substances. EBR of the formula C₂₈H₄₈O₆ was purchased from Ruina International Co., Ltd., China. DHECD was chemically modified from 20-hydroxyecdysone, which in turn was obtained from the stem bark of Vitex glabrata.^13,14) To prepare the compounds, 1 mM of EBR and 1 mM of DHECD were each dissolved in 0.01% ethanol as the stock solutions and stored in a freezer at 4°C. Various concentrations were prepared from these stocks as and when required.

Fig. 1. Chemical structures of 24-epibrassinolide (a) and 7,8-dihydro-8α-20-hydroxyecdysone (b).

1. Rice seeds and seedlings

Seeds of rice (Oryza sativa L.) cv. Pathum Thani 1 were sown in 500 cm² plastic pots. The plants were grown in a greenhouse at the Department of Botany, Kasetsart University, Bangkok, Thailand (latitude 13°50′N, longitude 100°34′E). Healthy seedlings were selected for use in all experiments and were grown in a greenhouse that had an average day/night temperature regime of 30/25°C, an average natural daytime irradiance of 275 µmol PPF/m² s, and an average relative humidity of 72% between May and August 2011.

2. Determination of the most effective concentration of BRs on pollen viability

The aim of this experiment was to determine the most effective concentrations of BRs to improve pollen viability and reduce pollen bursting at high temperatures. Pollen grains were collected from freshly opened florets during 8:00 to 10:00. All collected pollen grains were mixed together thoroughly and then divided into small portions of equal quantities. Each portion was then inoculated into 2 mL of a medium containing 15% sucrose, 250 mg/L boric acid and 200 mg/L calcium nitrate with the addition of either EBR or DHECD of 0, 0.001, 0.01, 0.1, or 1 µM. The addition of 0 µM provided the control. The experimental design was factorial in a completely random design consisting of two factors, the BR types and BR concentrations. The pollen grains in each 2 mL suspendsoid were allowed to incubate for 6 hr at room temperature (29±1°C), 40°C, and 50°C. The viability of the pollen was examined by staining all of the incubated samples with 1% iodine in a potassium iodide (KI) solution, with the iodine-stained pollen being counted as viable. The percentages of viable pollen, nonviable pollen, and burst pollen were calculated from 10 counts of pollen grains in 10 different microscopic fields of view per slide mounted with 20 µL of pollen suspension and 5 slides per treatment.

3. Examination of the effectiveness of BRs on plant survival at high temperature

The most appropriate concentrations for pollen viability (0.001 µM EBR or 0.001 µM DHECD, each in 0.01% ethanol) were prepared, and 15 mL of each solution was separately sprayed onto individual plants at age 78-days-old plants (microsporogenesis, R₂ stage). The control plants were sprayed with 0.01% ethanol mixed with 0.025% Tween-20, a surfactant, and divided into two groups. One group (the unstressed control plants) was grown at ambient day/night temperatures of 30/25°C under natural irradiance, and the other group (the stressed plants) was grown at high day/night temperatures of 40/30°C. The BR solutions were mixed with 0.025% Tween-20 prior to use. Five days after treatment (heading stage), the plants treated with BRs and the stressed control plants were exposed to 40/30°C day/night temperatures for 7 days in the growth chamber under 300 µmol PPF/m² s light irradiance and 75% relative humidity for heat stress conditions and then transferred to normal greenhouse conditions for further growth. The climate in the greenhouse following the transfer of the plants up to harvesting had an average day/night temperature of 30/25°C, average natural daytime irradiance of 255 µmol PPF/m² s, and an average relative humidity of 74%. The pots were arranged in a completely random design for various independent experiments.

4. Experiment I: Pollen properties

In vitro pollen viability and in vivo pollen germination were studied. Measurements of the heat stress conditions were taken daily from days 1 through 7 and on day 8, which was the day of recovery (re-1). The pollen properties were observed from the florets of a whole panicle, which were collected from five florets each from the top, middle, and basal parts.

5. In vitro pollen viability

Pollen grains from the top, middle, and basal parts of panicles in each treatment were collected in the morning during 08:00 to 10:00 hr. The percentages of viable pollen, nonviable pollen, and burst pollen were calculated by staining with 1% I₂/KI solution and were analyzed using five readings per slide.

6. In vivo pollen germination

After natural fertilization, florets from the top, middle, and basal parts of the panicles in each treatment were collected in the afternoon during 13:00 to 15:00 hr. The pistils were fixed in a fixing solution and stained with aniline blue. Under a fluorescence microscope, the germinated pollen grains were observed as callose deposits in the pollen tube. The percentage of in vivo pollen germination was calculated by counting the number of pollen grains germinating on a stigma per total number of pollen.

7. Experiment II: Seed setting

After the 7-day recovery period from the heat stress conditions, one set each of the heat-stressed and BR-treated plants (EBR or DHECD), stressed control plants, and unstressed control plants were maintained at average day/night temperatures of 30/25°C with average natural daytime irradiance of 255 µmol PPF/m² s in a greenhouse until the final harvest, which is 124 days after sowing. Fifteen panicles per treatment were harvested, and their top, middle, and basal parts were separated. The percentage of seed setting was calculated by counting the number of filled seeds per the total seed.

8. Statistical analysis

The data were analyzed statistically. Analysis of variances and mean differences among BR treatments and the control were evaluated at the 5% level by Tukey–Kramer’s HSD (honestly significant difference) test.

Results

The most effective concentrations of EBR and DHECD for reducing the damage of rice plants exposed to high temperature were determined by treating rice with various concentrations of EBR and DHECD at temperatures of 40°C and 50°C for 6 hr. In this test, the effects of both chemicals on pollen viability and pollen bursting were examined. At room temperature, neither treatment of EBR nor DHECD improved pollen viability of rice. Furthermore, at 1 µM concentrations, EBR and DHECD greatly reduced the percentage of pollen viability, possibly due to a significant increase in pollen bursting (Table 1). In contrast, when pollen grains were incubated at 40°C, pollen viability increased and both nonviable pollen and pollen bursting decreased at all treatment concentrations (Table 2). When pollen grains were incubated at 50°C, treatment of EBR or DHECD induced significantly higher pollen viability compared to the pollen viability of the stressed control at 0.001 µM or 0.01 µM concentrations. However, pollen bursting increased at concentrations higher than 0.001 µM (Table 3). Thus, the results demonstrated that the 0.001 µM concentration was best for EBR and DHECD to improve pollen properties during heat stress. At 40°C, DHECD was more effective for pollen viability than EBR (Table 2). On the other hand, EBR treatment could induce greater pollen viability than DHECD treatment at 50°C (Table 3).

Table 1. Effects of varied brassinosteroids concentrations under room temperature on in vitro pollen viability, nonviable pollen and pollen bursting in rice cv. Pathum Thani 1

Concentration (µM)	Room temperature
	EBRa)	DHECDa)	Meanb)
	Pollen viability (%)
0	88.63abc±0.75	90.48ab±1.46	89.56YZ±1.11
0.001	92.18a±0.60	91.98a±0.74	92.08Z±0.67
0.01	76.62c±4.10	90.95ab±1.03	83.78XY±2.57
0.1	78.04bc±4.32	88.93abc±1.60	83.49XY±2.96
1	76.76c±4.39	76.16c±3.96	76.46X±4.18
Meanc)	82.45B±2.83	87.70A±1.76
	Nonviable pollen (%)
0	2.51b±0.37	1.06b±0.44	1.78Y±0.41
0.001	2.37b±0.39	1.62b±0.24	2.00Y±0.31
0.01	2.83b±0.44	2.47b±0.78	2.65YZ±0.61
0.1	2.87b±0.40	2.29b±1.36	2.58YZ±0.88
1	1.54b±0.32	7.30a±1.32	4.42Z±0.82
Meanc)	2.42±0.38	2.95±0.83
	Pollen bursting (%)
0	4.43bc±0.30	4.23bc±0.53	4.33XY±0.41
0.001	2.73c±0.16	3.19c±0.44	2.96X±0.30
0.01	10.27ab±2.00	3.29c±0.50	6.78YZ±1.25
0.1	9.54ab±2.08	4.39bc±0.52	6.97YZ±1.30
1	10.85a±2.25	8.27abc±1.63	9.56Z±1.94
Meanc)	7.56A±1.36	4.67B±0.72

^{a), b), c)} The means followed by the same letter in the same order (a, b, ···; Z, Y, ···; A, B, ···) are not significantly different at the 5% level by Tukey–Kramer’s HSD test. Each value is the mean±SE of five replicates and ten reading of microscopic field per replicate.

Table 2. Effects of varied brassinosteroids concentrations under 40°C on in vitro pollen viability, nonviable pollen and pollen bursting in rice cv. Pathum Thani 1

Concentration (µM)	40°C
	EBR	DHECD	Meana)
	Pollen viability (%)
0	83.11±0.76	86.08±1.50	84.60Y±1.13
0.001	93.19±1.00	94.86±0.96	94.02Z±0.98
0.01	89.97±2.10	93.06±1.08	91.52Z±1.59
0.1	89.94±1.08	93.14±1.44	91.54Z±1.26
1	90.60±1.80	92.56±0.94	91.58Z±1.37
Meanb)	89.36B±1.35	91.94A±1.19
	Nonviable pollen (%)
0	4.05±0.56	3.46±0.68	3.76Z±0.62
0.001	2.30±0.70	1.87±0.71	2.09Y±0.70
0.01	3.18±0.42	1.75±0.44	2.46YZ±0.43
0.1	1.38±0.22	2.40±0.31	1.89Y±0.27
1	2.13±.072	2.06±0.47	2.10Y±0.59
Meanb)	2.61±0.52	2.31±0.52
	Pollen bursting (%)
0	12.84±0.88	10.46±2.13	11.65Z±1.51
0.001	4.51±0.70	3.27±0.29	3.89Y±0.49
0.01	6.85±1.79	5.19±0.83	6.02Y±1.31
0.1	8.68±1.21	4.46±1.18	6.57Y±1.19
1	7.26±1.56	5.38±0.56	6.32Y±1.06
Meanb)	8.03A±1.23	5.75B±1.00

^a), b) The means followed by the same letter in the same order (Z, Y, ···; A, B, ···) are not significantly different at the 5% level by Tukey–Kramer’s HSD test. Each value is the mean±SE of five replicates and ten reading of microscopic field per replicate.

Table 3. Effects of varied brassinosteroids concentrations under 50°C on in vitro pollen viability, nonviable pollen and pollen bursting in rice cv. Pathum Thani 1

Concentration (µM)	50°C
	EBRa)	DHECDa)	Meanb)
	Pollen viability (%)
0	72.66±1.78	73.20±3.22	72.93Y±2.50
0.001	83.45±1.44	83.84±0.64	83.65Z±1.04
0.01	83.83±0.74	74.23±4.83	79.03YZ±2.78
0.1	80.17±3.35	68.39±4.87	74.28Y±4.11
1	74.63±1.22	72.94±3.74	73.78Y±2.48
Meanc)	78.95A±1.71	74.52B±3.46
	Nonviable pollen (%)
0	9.89a±2.27	8.43ab±1.97	9.16Z±2.12
0.001	4.68abc±0.96	4.06bc±0.65	4.37Y±0.81
0.01	0.20c±0.20	3.36bc±0.85	1.78Y±0.52
0.1	0.32c±0.20	4.38bc±1.14	2.35Y±0.67
1	5.77ab±0.72	3.48bc±0.27	4.62Y±0.49
Meanc)	4.17±0.87	4.74±0.98
	Pollen bursting (%)
0	17.45±1.49	18.37±2.25	17.91YZ±1.87
0.001	11.87±1.01	12.09±0.70	11.98Y±0.86
0.01	15.98±0.74	22.41±4.06	19.19YZ±2.40
0.1	19.51±3.34	27.22±4.13	23.37Z±3.73
1	19.60±1.60	23.59±3.66	21.60Z±2.63
Meanc)	16.88B±1.64	20.74A±2.96

^{a), b), c)} The means followed by the same letter in the same order (a, b, ···; Z, Y, ···; A, B, ···) are not significantly different at the 5% level by Tukey–Kramer’s HSD test. Each value is the mean±SE of five replicates and ten reading of microscopic field per replicate.

Next, we tested the effects of EBR and DHECD on pollen grains located in each part of the panicle. In general, EBR and DHECD significantly increased pollen viability in the whole panicle when compared with the stressed control plants during the first day after heat stress. DHECD increased pollen viability more than EBR in the top part of the panicle; however, EBR and DHECD had the same effect in the middle and basal parts of the panicle (Fig. 2a). The increase in pollen viability in the first day after heat stress was related to a decrease in nonviable pollen. DHECD application significantly reduced the percentage of nonviable pollen when compared with EBR at the top part of the panicle; however, EBR and DHECD had the same effect of decreasing nonviable pollen in the middle and basal parts of the panicle (Fig. 2b). Moreover, both compounds significantly reduced pollen bursting in all parts of the panicle compared with the unstressed control plants (Fig. 2c). During the second day after heat stress, EBR and DHECD significantly increased pollen viability in the top and middle parts of the panicle and slightly increased pollen viability in the basal part of the panicle compared with the stressed control plants (Fig. 2a). Furthermore, EBR and DHECD significantly increased pollen viability in all parts of the panicle during the third day after heat stress (Fig. 2a). The increase in pollen viability resulting from EBR or DHECD use is consistent with the reduction in nonviable pollen and pollen bursting observed during the second day after heat stress and the decrease in nonviable pollen observed during the third day after heat stress (Figs. 2b and 2c). Four days following exposure of plants to high temperatures, the pollen property results were similar to those 3 days after heat stress. EBR and DHECD treatment increased the percentage of pollen viability compared with the stressed control plants, and the application of both compounds decreased only the percentage of nonviable pollen. Moreover, the percentage of pollen viability of the whole panicle of the stressed plants tended to decrease according to the number of days the plants received long-term heat. All of the high-temperature treatments severely reduced the percentage of pollen viability at 7 days after heat stress. The stressed control, EBR-, and DHECD-treated plants had pollen viability percentages in the amounts of 34.08%, 50.71%, and 43.99%, respectively, and the unstressed control plants retained pollen viability of 93.02%. In addition, the pollen viability of all treatments could not recover during the first day at normal temperatures (re-1).

Fig. 2. Effects of EBR and DHECD applications on in vivo pollen viability (a), nonviable pollen (b), and pollen bursting (c) under heat stress conditions. The means of the whole panicle superscripted with the same letter are not significantly different at the 5% level by Tukey–Kramer’s HSD test. Each value±SE. Data are shown as the means of five florets of each part of the panicle and fifteen florets of the whole panicle.

In vivo pollen germination after heat stress was determined by counting the number of pollen grains germinating on the stigma. The results showed that EBR and DHECD application could improve in vivo pollen germination in the whole panicle in 1 to 3 days after heat stress, especially in the basal part (Fig. 3). Pollen germination occurring in the top and middle parts of panicles was not significantly different compared with the stressed control plants, but EBR and DHECD treatments tended to increase the germination of pollen from these two parts of the panicles compared with the stressed control plants (Fig. 3). However, among the BR-treated plants, there were no significant differences in the pollen germination percentages after 4 days at high temperature (data not shown). In the present study, EBR and DHECD had similar levels of effectiveness on pollen properties and pollen germination (Figs. 2 and 3).

Fig. 3. Effects of EBR and DHECD applications on pollen germination under heat stress conditions. The means of the whole panicle superscripted with the same letter are not significantly different at the 5% level by Tukey–Kramer’s HSD test. Each value±SE. Data are shown as the means of five florets of each part of the panicle and fifteen florets of the whole panicle.

The seed setting of BR-treated plants significantly increased during heat stress. Furthermore, the BR-treated plants had the same number of filled seeds as the unstressed control plants in all panicle positions (Table 4). Consequently, EBR and DHECD could alleviate heat stress when the percentage of seed setting was considered. Moreover, EBR and DHECD were not significantly different in increasing seed setting in Pathum Thani 1 rice cultivars.

Table 4. Effects of EBR and DHECD applications on the percentage of filled seed

Treatment	Filled seed (%)
	Whole paniclea)	Top part of paniclea)	Middle part of paniclea)	Basal part of paniclea)
Unstressed control	85.49a±1.87	77.56a±5.91	89.52a±6.10	93.00a±4.90
Stressed control	1.00 b ±1.00	0.00b±0.00	2.50b±2.50	0.00b±0.00
EBR 0.001 µM	72.77a±10.01	63.44a±10.94	80.56a±6.81	78.48a±11.64
DHECD 0.001 µM	74.04a±12.52	66.48a±13.59	77.86a±13.19	80.44a±12.36

^a) The means followed by the same letter are not significantly different at the 5% level by Tukey–Kramer’s HSD test. Each value is the mean±SE of fifteen panicles for each treatment.

Discussion

Heat stress impaired the rice pollen properties associated with pollen viability and pollen bursting. The addition of EBR or DHECD to the pollen medium could alleviate these attributes at 40°C and 50°C. Both EBR and DEHCD showed the highest activity at 0.001 µM for pollen viability associated with the least amount of nonviable pollen and pollen bursting at 40°C and 50°C (Tables 2, 3). Heat stress negatively affects pollen development and pollen quality and results in nonviable pollen, which is caused by a decrease in starch concentration in developing pollen.^17–19) In addition to nonviable pollen, heat stress also increased pollen bursting. Sakata and Higashitani reported that heat stress arrested the function of secondary parietal cells of the pollen walls.²⁰⁾ Therefore, the middle layer and tapetum of pollen walls do not develop well, which may cause the pollen to burst under heat stress conditions. Our study indicated that EBR and DHECD increased the percentage of viable pollen under heat stress conditions and improved pollen properties. Moreover, EBR and DHECD increased pollen viability in all parts of the panicle following 3 days of heat stress compared with the pollen viability of the stressed control plants (Fig. 3a). The increase in pollen viability associated with the decrease in nonviable pollen and pollen bursting occurred within 2 days after heat stress, and the reduction in nonviable pollen was observed after 3 days of heat stress (Figs. 2b and 2c). Cao et al. reported that high temperatures during the heading stage of rice development mainly affected development of the male gametophyte (pollen) and had little effect on the development of the pistil or female gametophyte.²¹⁾ BRs may also affect fertilization by stimulating filament and pollen growth and modifying pollen properties.²²⁾

Sheoran and Saini reported that normal starch accumulation during pollen development was strongly inhibited in stressful environments and caused nonviable pollen.²³⁾ The inhibition of starch accumulation in pollen grains could seriously affect pollen fertility because starch is the major energy source for pollen development.^23,24) In this study, it was observed that BR and BR-mimic substances counteract heat stress by increasing pollen viability (Fig. 2a). Therefore, EBR and DHECD may alleviate the starch decrease that occurs in pollen grains subjected to heat stress.

EBR and DHECD application could improve in vivo pollen germination of the whole panicle in 1 to 3 days after heat stress, especially in the basal part, and tended to increase the germination of pollen from the top and middle parts of the panicles compared with the germination results from the stressed control plants (Fig. 3). It is well known that fertilization in rice is consistently associated with the number of germinated pollen on the stigma.²⁵⁾ Pollen grains at the middle part of the panicle usually have the most germination, followed by the top part and, finally, the basal part. The number of anthesed spikelets increases progressively from the top part of the panicle and reaches a maximum in the middle part before declining in the basal part.²⁶⁾ There is indication that the basal part of the panicle is indicated to have a higher incidence of unfilled seed. In this study, EBR and DHECD application could increase in vivo pollen germination at the basal part of the panicle (Fig. 3). Therefore, EBR and DHECD may increase the percentage of fertilization and lead to increases in seed setting in the basal part of the panicle.

The beneficial effects of EBR or DHECD application on seed setting were also observed under heat stress conditions (Table 4). This study showed that heat stress severely reduced pollen germination and seed setting in the stressed control plants in all parts of the panicle (Fig. 3 and Table 4). High temperature affects pollen properties and seed setting in various ways. These ways are likely linked to panicle position because the development of caryopses in rice panicles is asynchronous. The anthesis within the panicle is in a basipetal sequence. Therefore, spikelets located in the upper part of the panicle develop faster compared with those in the lower part of the panicle. Moreover, spikelets in the lower branch exhibit poor assimilation.²⁷⁾ Rice grains in the upper part of the panicle are normally filled first, while large numbers of blanks occur in the basal part of the panicle.²⁸⁾ Under heat stress conditions, high temperatures affect the grain filling of spikelets in the base of the panicle that flower later than the spikelets at the top of the panicle.^29,30) Mohammed and Tarpley reported that a high night temperature decreased rice yield by increasing spikelet sterility, which was reflected in unfilled grains in all positions of the rice panicle.³⁰⁾ When rice plants are exposed to heat stress, larger numbers of unfilled seed occur mostly at the basal parts of the panicle because seed setting at the top part of the panicle is normally filled first.^28,30) In our study, application of EBR or DHECD significantly increased the percentage of pollen germination at the basal part of the panicle and increased seed setting at all parts of the panicle under heat stress (Fig. 3 and Table 4). The results in this report strongly suggest that the application of a BR or a BR mimic should be useful for increasing rice yields under high temperatures in field conditions.

In conclusion, this study demonstrated that EBR and DHECD were effective in increasing pollen viability and reducing pollen bursting under heat stress conditions. Furthermore, the BR and BR mimic improved in vivo pollen germination and seed setting of rice. DHECD, a brassinosteroid mimic that shows similar biological activities as EBR, was a good candidate for improving rice seed setting under heat stress. Further study on the mode of action of DHECD will reveal whether DHECD functions as a BR.

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