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Flower Opening Time in – Difference and Effect of Weather Factors – Kazuhiro Kobayasi1, Hidetsugu Masui1, Yumi Atsuta1, Tsutomu Matsui2, Mayumi Yoshimoto3 and Toshihiro Hasegawa3 1Faculty of Life and Environmental Science, Shimane University, 1060 Nishi-Kawatsu-Cho, Matsue 690-8504 Japan; 2Faculty of Applied Biological Science, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan; 3National Institute for Agro-Environmental Sciences, 3-1-3, Kannondai, Tsukuba, Ibaraki 305-8604, Japan E-mail address [email protected]

Abstract: Flower opening in the early morning helps to avoid heat-stress-induced sterility in rice ( L.) at anthesis. Selection of with an early flower opening time (FOT) is thus an important method for reducing heat-induced sterility. However, cultivar differences are not well characterized. We discuss cultivar variation in FOT in the section “Cultivar differences in FOT.” We observed a wide diversity in FOT among rice cultivars. The earliest FOT (0901 h) was detected in indica rice, ‘Xiaomazhan.’ Although FOT is under genetic control, it is also affected by weather, and particularly by air temperature (Ta); however, the combined effects of Ta, solar radiation (Rs), and humidity (expressed as the vapor-pressure deficit, VPD) on the FOT of various rice genotypes remain unclear, particularly under field conditions. In the section “Effects of weather factors on FOT,” we compare the effects of Ta, Rs, and VPD at various time points before anthesis on rice FOT under field conditions. First, we analyzed the correlations between FOT and Ta, Rs, or VPD averaged hourly from 0000 to 1000 h. Second, to evaluate the individual effects of cultivar, Ta, Rs, and VPD on FOT, we constructed general linear models (GLMs), which revealed that the average Ta, Rs, and VPD between 0600 and 0900 h significantly affected FOT. The standardized partial regression coefficients of Ta and Rs were negative and those of VPD were positive, indicating that higher Ta, higher Rs, and lower VPD in the early morning result in earlier FOT. There are several artificial methods for advancing FOT in rice plants. Some artificial FOT advancements such as strong wind, physical stimuli, and 1-h dark treatment, however, are accompanied by undesirable effects such as anther indehiscence, poor pollination, and sterility. In “Effects of MeJA on FOT, fertility, pollination, and anther dehiscence,” we discuss the effects of methyl jasmonate (MeJA) on advancing FOT and on anther dehiscence, pollination, and sterility in standard ‘Hinohikari.’ 'Hinohikari' was subjected to application of 4 mmol L−1 MeJA solution (3 mL per panicle) and water, as a control, on panicles at three different time points (0900, 1000, and 1100 h). Application of MeJA at 0900 h caused FOT to occur about 2 h earlier than without treatment and 1.5 h earlier than after treatment with water. MeJA application at 1000 h also caused FOT to occur earlier. In mid-August, when MeJA application at 0900 h caused FOT to occur, the temperature at 1000 h in Matsue is >2 °C lower than at 1200 h, when most flowers of japonica rice open under natural conditions. These results suggest that MeJA application before flowering would allow flower opening in the early morning to mitigate potential heat-induced sterility. Although MeJA application at 0900 h caused FOT to occur earlier, it increased flower sterility and decreased pollination stability and anther dehiscence. This result suggests that these flowers opened too early to develop pollen grains. Keywords: Cultivar, Flower opening in the early morning, Flower opening time, Heat-induced sterility, Methyl jasmonate, Oryza sativa, Rice, Solar radiation, Temperature, Vapor-pressure deficit.

1. Introduction The opening of rice flowers in the early morning is a beneficial response that can avoid heat-stress-induced sterility at anthesis. The sensitivity of rice flowers to high temperatures decreases during the 1-h period after flowering [1]; thus, a flower opening time (FOT) 1 h earlier than normal may lead to anthesis before the air −1 temperature (Ta) reaches a critical level. Ta can rise at a rate of >3 °C h starting around 1000 h [2]. A controlled-environment experiment has revealed that flowers of ‘Milyang 23’ that opened earlier in the morning and at a lower temperature had higher seed-set than those that opened later (near midday) and at higher temperatures [3]. Selection of cultivars with an early FOT is therefore an important technique to reduce heat-induced sterility [2, 4]. For example, the flowers of Steud. open earlier than those of O. sativa L. [2, 4], and the flowers of interspecific hybrids between O. glaberrima and O. sativa open earlier than those of O. sativa [5]. Among the cultivars of O. sativa, Imaki et al. [3, 6] reported cultivars with flowers that opened 1 to 2 h earlier than those of ‘,’ a standard Japanese cultivar. Under controlled conditions, 'Milyang 23' exhibited heat tolerance due to the earlier opening of its flowers [6]. Flowers of several indica cultivars open earlier than those of japonica cultivars [3]; however, cultivar differences have not been well characterized. We discuss cultivar variation in FOT later in the text. Although FOT is under genetic control, it is affected by weather, and particularly by Ta [4, 5, 6, 7, 8, 9, 10]. In a study of indica cultivars, FOT occurred about 45 min earlier at temperatures that were 7 C° above the normal temperature [8]. However, most of the studies of FOT [4, 6, 8,] have been conducted under controlled environments, and rice plants grown in a glass house or a growth chamber have been reported to open their flowers 1 to 2 h later than those grown outdoors [11]. The temperature response of flower opening differs among rice cultivars. FOT occurs earlier under high temperatures in 'Milyang 23', whereas it is delayed in 'Nipponbare' [6]. In addition to temperature, other weather factors such as solar radiation (Rs) and relative humidity appear to affect FOT. For example, Nakagawa and Nagata [9] found that Rs from 0400 to 0800 h influenced FOT in 'Koshihikari,' but not in EG0, heading-time tester line. Strong winds [12], as well as low atmospheric pressure and rain [7], also affect

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FOT. In addition, light conditions can influence FOT, because the duration of anthesis increases under continuous light or dark conditions [7]. This result suggests that both the light intensity and the cycle of light and dark affect FOT. However, the combined effects of Ta, Rs, and humidity variations on the FOT of various rice genotypes remain unclear, particularly under field conditions, and this limits our ability to predict FOT. In the second section “Effects of weather factors on FOT,” we compared the effects of Ta, Rs, and humidity (expressed as the vapor-pressure deficit, VPD) at various periods before anthesis on rice FOT under field conditions. First, we analyzed the correlations between FOT and Ta, Rs, or VPD averaged hourly from 0000 to 1000 h. Second, we used general linear models (GLMs) to separately evaluate the effects of cultivar, Ta, Rs, and VPD on FOT. In the second analysis, we used a 3-h time span based on Japan Standard Time (JST). There are several artificial methods used to advance FOT in rice plants. Dark treatment for 1 h before natural flower opening advances FOT by 2 h or more [13]. Light conditions can also influence FOT since the duration of anthesis increases under continuous light or dark conditions [7, 13]. Subjecting rice panicles to physical stimuli has been shown to advance FOT [12]. Applications of CO2 and methyl jasmonate (MeJA) on panicles also advance FOT [14, 15, 16]. In a report by Zeng et al. [14], MeJA induced flower opening in rice within 80 min after treatment; clear flower-opening effects were observed with 0.4 and 4 mmol L−1 MeJA treatment. Some artificial FOT advancements, however, are accompanied by undesirable effects such as anther indehiscence, poor pollination, and sterility [12, 13]. For example, advancement in FOT caused by wind or physical stimulation of rice panicles reportedly resulted in anther indehiscence and poor pollination [12]. Advancement in FOT caused by 1-h dark treatment has also been reported to result in anther indehiscence [13]. Tsuboi [12] speculated that such poor pollination may be due to flowering before pollen maturation and preparation of anther dehiscence. In the section “Effects of MeJA on FOT, fertility, pollination, and anther dehiscence,” we examine the effects of MeJA application on advancing FOT as well as on anther dehiscence, pollination, and sterility in standard japonica rice ‘Hinohikari’ in order to develop artificial techniques of FOT control in rice.

2. Cultivar differences in FOT The study was conducted at an experimental field of Shimane University in Matsue, Shimane Prefecture, Japan (35°29'N, 133°04'E, 4 m asl) in 2008. To cover wide range of genetic diversity, we used the National Institute of Agrobiological Sciences (NIAS) global rice core collection [17], the NIAS rice core collection of Japanese [18], and cultivars that were reported to flower in the early morning or demonstrated heat stress resistance, including O. glaberrima Steud. Cultivars used in this study are listed in Table 1. The planting density was 22.2 hills m−2 (one −2 −2 seedling per hill, 15-cm hill spacing, and 30-cm row spacing). A basal dressing of 4 gm N, 12.8 gm P2O5, and −2 13.6 gm K2O was applied. No top dressing was used.

1) Measurements of FOT Physical stimuli such as touch may promote flower opening in rice [12]. To avoid this phenomenon, we used digital photographs of the panicles instead of physically inspecting plants. The panicles were photographed automatically at 10-min intervals with waterproof digital cameras (Optio W30, Pentax, Tokyo, Japan) to determine the FOT of each cultivar. We used tripods for the cameras and a built-in electronic timer to control measurement intervals. We recorded the time of anther extrusion for all observable flowers (>30% flowers per panicle) on the obverse side of panicles. The median anther extrusion time for all observed flowers in each panicle was calculated and FOT was defined as the mean of the individual panicle medians per day. Weather data were obtained from the data presented on the website of the Matsue Local Meteorological Observatory, located near the experimental site.

2) Cultivar differences in FOT There is a wide diversity in FOT among rice cultivars examined in this study (Table 1). The earliest FOT (0901 h) was detected in indica rice, ‘Xiaomazhan.’ FOTs in most of indica cultivars occurred before 1100 h and those in most of the japonica cultivars occurred after 1100 h. FOTs in O. glaberrima occurred between 0904 and 1054 h, which is earlier than most of the O. sativa cultivars, but later than ‘Xiaomazhan.’ FOTs varied widely among tropical japonica cultivars. For example, the ‘Jaguary’ FOT was 0930 h, but the ‘Kahei’ FOT was 1220 h. However, FOTs in most cultivars that originated in Japan occurred after 1100 h while FOTs in some tropical japonica that originated in Japan occurred before 1100 h. Weak correlations were found between Ta during 0000 and 0600 h and FOTs in glaberrima (r = −0.504), indica (r = −0.326), japonica (r = −0.255), and tropical japonica (r = −0.337) cultivars. Average, maximum, and minimum Ta during the FOT-observing period (July 16–September24) were 24.1, 27.6, and 18.2°C, respectively. Average Ta during the FOT-observing period in ‘Xiaomazhan’ was relatively high at 26.1 °C. Advanced research assessing genetic diversity in FOT is needed regarding weather factors such as Ta and Rs.

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Table 1. , their types (G, Oryza glaberrima; I, indica; J, japonica; TJ, tropical japonica), their origins, and flower opening time (FOT, h). z) Classification of cultivar types is mainly according to http://www.gene.affrc.go.jp/databases-core_collections_wr_en.php and http://www.gene.affrc.go.jp/databases-core_collections_jr_en.php.

Cultivar Typez) Origin FOT Cultivar Typez) Origin FOT Xiaomazhan I China 0901 Kaneko TJ Japan 1059 85-352 G - 0904 Akamai (Kochi) J Japan 1100 GNF-7 G - 0911 Milky Queen J Japan 1101 GNF-2 G - 0919 Padi Perak TJ Indonesia 1101 Jaguary TJ Brazil 0930 Shinriki J Japan 1103 85-354 G - 0950 Sekiyama J Japan 1104 Hong Cheuh Zai I China 0952 Calotoc I Philippines 1105 Nepal 555 I India 0953 Bouzumochi TJ Japan 1106 IR58 I Philippines 0955 Hanaechizen J Japan 1106 Jinguoyin I China 1002 Lebed I Philippines 1108 Shuusoushu I China 1008 Pinulupot 1 I Philippines 1110 Karahoushi I Japan 1008 Hinode TJ Japan 1110 Lemont J USA 1010 Muha I India 1111 Keiboba I China 1010 Davao 1 I Philippines 1115 Basilanon I Philippines 1011 Co 13 I India 1116 Milyang 23 I South Korea 1012 Ginbouzu J Japan 1118 Arc 7047 I India 1015 Meguromochi TJ Japan 1118 Sanguicao I China 1016 Shichimenchomochi J Japan 1119 Oiran TJ Japan 1019 Bei Khe I Cambodia 1119 Asu I Bhutan 1020 Wataribune J Japan 1120 Local I India 1020 Hassokuho J Japan 1120 Deejiaohualuo I China 1020 Nipponbare J Japan 1120 G-21 G - 1021 Akitakomachi J Japan 1122 Senshou TJ Japan 1024 Fukoku J Japan 1124 Arc 5955 I India 1024 Omachi J Japan 1125 Arc 7291 I India 1025 Kabashiko J Japan 1129 Kaluheenati I Sri Lanka 1028 Badari Dhan I Nepal 1130 Kasalath I India 1030 Bingala I Myanmar 1131 Arc 11094 I India 1032 Kameji J Japan 1132 Dahonggu I China 1035 Irimanishiki TJ Japan 1137 85-351 G - 1035 Shinyamadaho 2 J Japan 1138 Ch86 I China 1037 Khau Mac Kho TJ Vietnam 1138 Shwe Nang Gyi I Myanmar 1038 M102 J USA 1142 Urasan 1 TJ Japan 1039 Akamai (Nagasaki) I Japan 1142 Hirayama TJ Japan 1039 Jiu 9304 J China 1144 Naba I India 1041 Raiden J Japan 1145 Qingyu I Taiwan 1042 Mansaku J Japan 1147 Ryou Suisan Koumai I China 1043 Shinrikimochi J Japan 1148 Padi Kuning I Indonesia 1044 Himenomochi J Japan 1150 Vary Futsi I Madagascar 1045 Moritawase J Japan 1150 Shoni I Bangladesh 1047 Shiroine J Japan 1151 Hunanxian I China 1047 Shinshuu J Japan 1151 Jena 035 I Nepal 1051 Rikutou Rikuu 2 J Japan 1151 Jhona 2 I India 1051 Dango J Japan 1153 Rexmont TJ USA 1053 Khao Nok TJ Laos 1156 IR24 I Philippines 1054 Puluik Arang I Indonesia 1200 M401 J USA 1054 Dianyu 1 J China 1202 G-8 G - 1054 Kahei TJ Japan 1220 Hosogara J Japan 1055 Aichiasahi J Japan 1234 Ratul I India 1057 Kyotoasahi J Japan 1235

3. Effects of weather factors on FOT The combined effects of Ta, Rs, and humidity variations on the FOT of various rice genotypes remain unclear, particularly under field conditions, and this limits our ability to predict FOT. We assessed the effects of Ta, Rs, and humidity (expressed as VPD) at various periods before anthesis on rice FOT under field conditions. First, we analyzed the correlations between FOT and Ta, Rs, or VPD averaged hourly from 0000 to 1000 h. Second, we used GLMs to evaluate the individual effects of cultivar, Ta, Rs, and VPD on FOT. We collected the FOT data of five rice cultivars at an experimental field of Shimane University in Matsue, Shimane Prefecture, Japan in 2007. Used cultivars were the indica cultivar ‘IR72,’ and the japonica cultivars ‘Hanaechizen,’ ‘Fujihikari,’ ‘Shinriki,’ and ‘Asahi.’ The planting density was 22.2 hills m−2. A basal dressing of 5 −2 −2 −2 gm N, 5.6 gm P2O5, and 4.4 gm K2O was applied. No top dressing was used. Measurement of FOT was similar

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to the method described in the section “Cultivar differences in FOT.” We measured Ta, relative humidity, and Rs every 5 min. VPDs were calculated from Ta and relative humidity using the method of Buck [19]. In this analysis, we used average hourly Ta, Rs, and VPD values between 0000 and 1000 h. Since inherent relationships exist between weather factors, relatively high correlations among Ta, Rs, and VPD may exist. Therefore, we used GLMs to evaluate individual effects as well as the cultivar effects on FOT. In this analysis, Ta, Rs, and VPD values were averaged over four 3-h periods (0000–0300 h, 0300–0600 h, 0600–0900 h, 0900–1200 h) based on JST. Details of this analysis is to be described [20].

1) Correlations between weather factors and FOT In 'IR72,' we found significant negative correlations between FOT and mean hourly Ta for every hour between 0000 and 0900 h (Fig. 1). No significant correlations were detected between FOT and mean hourly Ta between 0000 and 0800 h for any of the other cultivars; however, for 'Shinriki,' a significant negative correlation between FOT and mean hourly Ta for the two 1-h periods between 0800 and 1000 h was found. 'Fujihikari' and 'Asahi' showed 1.0 IR72 significant negative correlations between FOT and mean T a 0.8 Hanaechizen only during the final hour (0900–1000 h). Fujihikari For 'Fujihikari,' significant negative correlations 0.6 Shinriki between FOT and mean Rs were observed for every hour 0.4 Asahi between 0500 and 1000 h (Fig. 2). In contrast, 'IR72' showed significant positive correlations between FOT and 0.2

mean R for the two hours between 0500 and 0700 h. coefficient s 0.0 'Shinriki' and 'Asahi' showed significant negative -0.2 correlations between FOT and mean Rs for the two hours between 0700 and 0900 h. -0.4

Few significant correlations were found between FOT Correlation -0.6 and the hourly averaged VPD values (Fig. 3). Significant negative correlations between FOT and the hourly -0.8 averaged VPD were detected in 'IR72' (0500–0900 h), -1.0 versus significant positive correlations for 'Hanaechizen' 012345678910 (0400–0700 h) and a significant negative correlation for Time of day (h) 'Asahi' (0900–1000 h). Simple correlation analysis showed Fig. 1. Time course of the correlation coefficients (r) only a limited number of significant correlations between between flower opening time (FOT) and average air temperature (Ta) of five Oryza sativa cultivars. FOT and hourly averaged Ta (Fig. 1), Rs (Fig. 2), and VPD Black and white symbols represent significant and (Fig. 3), and differences between cultivars in the direction nonsignificant correlation coefficients, respectively (positive or negative) of the correlation. (p < 0.05). IR72 Hanaechizen 1.0 IR72 1.0 Fujihikari

0.8 Hanaechizen Shinriki Fujihikari 0.8 Asahi 0.6 Shinriki 0.6 0.4 Asahi 0.4 0.2 0.2 coefficient coefficient

0.0 0.0 -0.2 -0.2 -0.4 -0.4 Correlation Correlation -0.6 -0.6

-0.8 -0.8 -1.0 -1.0 012345678910 012345678910 Time of day (h) Time of day (h) Fig. 2. Time course of the correlation coefficients (r) between Fig. 3. Time course of the correlation coefficients (r)

flower opening time (FOT) and average solar radiation (Rs) between flower opening time (FOT) and average for each hour from 0400 to 1000 h in 'Fujihikari' and from vapor-pressure deficit (VPD) for each hour from 0000 0500 to 1000 h in the other four Oryza sativa cultivars. to 1000 h for the five Oryza sativa cultivars. Black and white symbols represent significant and Black and white symbols represent significant and nonsignificant correlation coefficients, respectively (p nonsignificant correlation coefficients, respectively (p < 0.05). < 0.05).

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2) Evaluation of the effects of cultivar, Ta, Rs, and VPD on FOT Among the four 3-h time spans based on JST, the adjusted R2 value determined by the GLMs (Table 2) was 2 highest (adjusted R = 0.399, p < 0.001) from 0600 to 0900 h. During this period, all four factors (cultivar, Ta, Rs, and VPD) were significant (p < 0.05). The standardized partial regression coefficient for Ta was –0.480, indicating that a higher Ta during this period resulted in an earlier FOT. Similarly, the standardized partial regression coefficient of Rs was –0.540, indicating that a higher Rs during this period also resulted in an earlier FOT. However, the standardized partial regression coefficient of VPD was +0.346, indicating that a lower VPD (i.e., more humid conditions) resulted in an earlier FOT. As suggested by their standard partial regression coefficients, the relative contributions of these three factors to FOT were similar. These cultivar-specific differences indicate that all three weather factors (Ta, Rs, and VPD) should be considered while analyzing the effects of weather on FOT. Air temperature showed negative standardized partial regression coefficients for all time periods (Table 2), thus higher Ta resulted in an earlier FOT. This result suggests that rice plants may open their flowers under cooler conditions in the early morning by detecting and responding to high night temperatures. Typically, average Ta at 0900 h in mid-August in Matsue, Japan is more than 2 °C lower than the temperature at 1100 h. The combination of genetic control of FOT with the dependence of FOT on night temperatures may allow earlier flower opening in the morning to mitigate or avoid potential heat-induced sterility in rice. These results suggest that higher Ta, higher Rs, lower VPD, or a combination of these conditions during the early morning resulted in an earlier FOT.

Table 2. Results of multiple-regression analysis using general linear models (GLMs) for the correlations among three weather factors (Ta, air temperature; Rs, daily total solar radiation; VPD, daily mean vapor-pressure deficit) and cultivars, and flower opening time (FOT) during 3-h periods based on Japan Standard Time (JST), and the significance.

Standardized partial regression F values in the GLMs Period (JST) Adjusted R2 coefficient

Cultivar Ta Rs VPD Ta Rs VPD 0000–0300 0.160* 4.156** 1.529 ns — 4.023* −0.164 — 0.282 0300–0600 0.351*** 9.454*** 13.124*** 19.743*** 14.653*** −0.488 −0.666 0.475 0600–0900 0.399*** 2.580* 9.408** 27.204*** 4.850* −0.480 −0.540 0.346 0900–1200 0.392*** 2.526* 16.368*** 9.603** 8.986** −1.009 −0.531 0.935

* ** *** ns, not significant; significant at p < 0.05, p < 0.01, and p < 0.001, respectively.

4. Effects of MeJA on FOT, fertility, pollination, and anther dehiscence Several artificial methods exist for advancing FOT in rice plants, including applications of MeJA on panicles [14, 16]. Zeng et al. [14] observed a response within 80 min after treatment and found clear flower-opening effects with 0.4 and 4 mmol L−1 MeJA. Some artificial FOT advancements such as strong wind, physical stimuli, and 1-h dark treatment, however, are accompanied by undesirable effects such as anther indehiscence, poor pollination, and sterility [12, 13]. In this section, we discuss the effects of MeJA application on advancing FOT as well as on anther dehiscence, pollination, and sterility in standard japonica rice ‘Hinohikari’ in order to develop artificial techniques of FOT control in rice. In 2008, a pot experiment was conducted outdoors at Shimane University, Matsue, Japan, using the japonica type rice cultivar 'Hinohikari'. Selected seeds were sown for uniformity on June 15, 2008. Twenty germinated seeds were planted in each Wagner pot (l/5000 a) containing 3.6 kg equivalent of oven-dry soil (air-dried andosol and a granitic saprolite cambisol mixture, 1:1 volume) using the circular dense-culture method [21]. To obtain uniform plants, tillers were removed as they appeared. MeJA applications were performed on September 9, 2008. Panicles judged to have flowers nearing anthesis, in which several flowers had flowered before the experiment date, were selected for tests. ‘Hinohikari’ was subjected to applications of 4 mmol L−1 MeJA, or water as a control (referred to as “control water” hereafter), at three different hours (0900, 1000, and 1100 h). MeJA was dissolved in a small amount of ethanol and then diluted to 4 mmol L−1 [14]; the same volume of ethanol was also added to control water. Approximately 3 mL MeJA solution or control water were sprayed on each panicle. A no-spraying group was also used as a control. Measurement of FOT was similar to the method described in the section “Cultivar differences in FOT.” We gathered and analyzed data on sterility, pollination, and anther dehiscence. The percentage of flowers having more than 80 pollen grains on the stigmata (referred to as “>80-pollen-grain flowers” hereafter) was calculated for each treatment. The percentage of >80-pollen-grain flowers strongly correlated with the number of pollen grains deposited on the stigmata and was thus used as a measure of the reliability of pollination under normal conditions [22]. Details of this method will be described [23].

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1) Effect of MeJA on FOT FOT of ‘Hinohikari’ was about 1 h after the application of 4 Table 3. Effect of methyl jasmonate treatments on −1 flower opening time in rice (cv. ‘Hinohikari’) at mmol L MeJA solution when applied at 0900 and 1000 h three different time points. (Table 3). Application of MeJA solution at 0900 h caused FOT to occur about 2 hr and 1.5 hr earlier, respectively, than plants Flower opening time receiving no application and application of control water at (JST) 0900 h. MeJA application at 1000 h also caused FOT to occur MeJA9 z) 1016 a w) earlier, but at 1100 h did not bring the change. Applications of MeJA10 1108 b MeJA11 1244 d control water at 0900, 1000, and 1100 h caused FOT to occur y) slightly earlier than no application (p > 0.05). In mid-August in CW9 1214 c,d CW10 1147 b,c Matsue, when MeJA application at 0900 h induced earlier FOT, CW11 1148 b,c the temperature at 1000 h is >2 °C lower than that at 1200 h, No treatment x) 1212 c,d the time when most flowers of japonica rice open under natural conditions. These results suggest that MeJA application before z)MeJA9, MeJA10, and MeJA11 indicate treatments flowering would allow flower opening in the early morning to of 4 mmol L−1 methyl jasmonate solution at 0900, 1000, and 1100 h, respectively. mitigate potential heat-induced sterility. y) CW9, CW10, and CW11 indicate control water treatments at 0900, 1000, and 1100 h, respectively. 2) Effects of MeJA on fertility, pollination, and anther x)No treatment indicates that neither methyl dehiscence jasmonate solution nor control water were Flower sterility in the rice plants was increased by the sprayed on rice panicles. application of MeJA solution at 0900 h compared to control w)Values with the same letters were not water at 0900 h (p > 0.05; Table 4). MeJA application at 1000 significantly different at the level of p = 0.05 with h also increased flower sterility. Applications of control water Tukey’s HSD test (n= 6–10). at 0900, 1000, and 1100 h also increased flower sterility slightly (p > 0.05). The percentage of >80-pollen-grain flowers was significantly decreased by applications of MeJA solution at three different time points. Applications of control water at these time points did not affect the percentage of >80-pollen-grain flowers. Pollen germination was not affected by applications of either MeJA solution or control water. Application of MeJA solution at 0900 and 1100 h significantly decreased thecae with both basal and apical dehiscence. While application of MeJA solution at 1000 h significantly decreased thecae with apical dehiscence, it decreased thecae with basal dehiscence, but not significantly (p > 0.05). Applications of control water at 0900, 1000, and 1100 h slightly decreased apically and basally dehisced thecae (p > 0.05). There was a significant negative correlation (r = −0.874, p = 0.01) between FOT and the percentage of flower sterility. A correlation between FOT and the percentage of thecae with basal dehiscence was not detected. However, excluding the result of MeJA application at 1100 h, a significant positive correlation (r = 0.988, p < 0.001) between FOT and the percentage of thecae with basal dehiscence was found. Although MeJA application at 0900 h induced earlier FOT, it also increased flower sterility. MeJA application decreased the percentage of >80-pollen-grain flowers, which is used as a measure of reliability of pollination under normal conditions [22]. Such poor pollination was caused by anther indehiscence at the basal and apical parts of the thecae. This result suggests that these flowers opened too early to develop pollen grains.

Table 4. Effect of methyl jasmonate treatments on flower fertility, percentage of flowers with more than 80 pollen grains deposited on the stigmata after anthesis (>80-pollen-grain flowers), pollen grain germination, and dehisced anthers at the basal and apical parts of thecae in rice (cv. ‘Hinohikari’) at three different time points.

Flower sterility >80-pollen-grain Pollen grain Basally dehisced Apically dehisced (%) flowers (%) germination (%) thecae (%) thecae (%) MeJA9 z) 49.6 a w) 41.7 a 22.8 a 19.8 a 15.3 a MeJA10 20.4 a,b 41.7 a 13.8 a 63.1 b 57.3 b MeJA11 8.7 b 45.8 a 22.3 a 21.8 a 21.1 a y) CW9 16.6 a,b 95.8 b 20.9 a 96.9 b 96.5 c CW10 11.3 b 100.0 b 20.3 a 86.6 b 94.2 c CW11 9.8 b 100.0 b 23.0 a 90.7 b 90.3 c No treatment x) 5.3 b 95.8 b 30.2 a 100.0 b 99.2 c

z)MeJA9, MeJA10, and MeJA11 indicate treatments of 4 mmol L−1 methyl jasmonate solution at 0900, 1000, and 1100 h, respectively. y)CW9, CW10, and CW11 indicate control water treatments at 0900, 1000, and 1100 h, respectively. x) No treatment indicates that neither methyl jasmonate solution nor control water were sprayed on rice panicles. w)Values with the same letters were not significantly different at the level of p = 0.05 with Tukey’s HSD test (n= 4).

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5. Conclusion 1) There was a wide diversity in FOT among rice cultivars. The earliest FOT (0901 h) was detected in indica rice, ‘Xiaomazhan.’ 2) The GLMs revealed that the average Ta, Rs, and VPD between 0600 and 0900 h significantly affected FOT. The standardized partial regression coefficients of Ta and Rs were negative and those of VPD were positive, indicating that higher Ta, higher Rs and lower VPD in the early morning resulted in earlier FOT. 3) Application of 4 mmol L−1 MeJA solution at 0900 h caused FOT to occur about 2 h earlier than without application and 1.5 h earlier than after application of control water at 0900 h. However, MeJA application at 0900 h also increased flower sterility and decreased pollination stability and anther dehiscence.

6. Acknowledgment We thank Dr. Cailin Wang, Ms. Ling Zhao, and staff (Jiangsu Academy of Agricultural Sciences, China) for providing seeds (Xiaomazhan, Hunanxian, and Jiu 9304). We thank Dr. Fumihiko Adachi (Shimane University, Japan) for scientific support in obtaining the weather data from the Shimane University site. We thank Dr. Yasuo Kamuro (BAL Planning Co. Ltd., Japan) for providing scientific advice about methyl jasmonate.

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